U.S. patent application number 15/321893 was filed with the patent office on 2017-05-18 for use of cell-permeable peptide inhibitors of the jnk signal transduction pathway for the treatment of various diseases.
The applicant listed for this patent is Xigen Inflammation Ltd.. Invention is credited to Jean-Marc Combette, Catherine Deloche.
Application Number | 20170137481 15/321893 |
Document ID | / |
Family ID | 48703406 |
Filed Date | 2017-05-18 |
United States Patent
Application |
20170137481 |
Kind Code |
A1 |
Combette; Jean-Marc ; et
al. |
May 18, 2017 |
Use of Cell-Permeable Peptide Inhibitors of the JNK Signal
Transduction Pathway for the Treatment of Various Diseases
Abstract
The present invention refers to the use of protein kinase
inhibitors and more specifically to the use of inhibitors of the
protein kinase c-Jun amino terminal kinase, JNK inhibitor
sequences, chimeric peptides, or of nucleic acids encoding same as
well as pharmaceutical compositions containing same, for the
treatment of various diseases or disorders strongly related to JNK
signaling.
Inventors: |
Combette; Jean-Marc; (Saint
Cergues, FR) ; Deloche; Catherine; (Genf,
CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Xigen Inflammation Ltd. |
Limassol |
|
CY |
|
|
Family ID: |
48703406 |
Appl. No.: |
15/321893 |
Filed: |
June 26, 2015 |
PCT Filed: |
June 26, 2015 |
PCT NO: |
PCT/EP2015/001294 |
371 Date: |
December 23, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 3/10 20180101; A61P 41/00 20180101; A61K 38/16 20130101; C07K
2319/09 20130101; A61K 38/1709 20130101; A61P 43/00 20180101; C07K
2319/10 20130101; A61P 13/12 20180101; Y02A 50/414 20180101; A61K
38/005 20130101; A61P 25/28 20180101; C07K 14/4703 20130101; A61K
9/0048 20130101; C12N 2710/16711 20130101; Y02A 50/30 20180101;
A61K 9/0019 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 26, 2014 |
EP |
PCT/EP2014/001736 |
Oct 8, 2014 |
EP |
PCT/EP2014/002724 |
Claims
1. A method of treating a disease or disorder comprising
administering to a subject in need of treatment thereof a
pharmaceutical composition comprising a JNK inhibitor sequence
comprising less than 150 amino acids in length, wherein the disease
or disorder is selected from the group consisting of (a) Mild
Cognitive Impairment, (b) intraocular inflammation following
anterior and/or posterior segment surgery; (c) wet or dry
age-related macular degeneration and cataracts, (d) eye
inflammatory diseases, (e) cancer and tumor diseases, (f) diseases
of the mouth and/or the jaw bone, (g) Addison's disease,
Agammagiobulinemia, Alopecia areata, Amytrophic lateral sclerosis,
Antiphospholipid syndrome, Atopic allergy, Autoimmune aplastic
anemia, Autoimmune cardiomyopathy, Autoimmune enteropathy,
Autoimmune hemolytic anemia, Autoimmune inner ear, disease,
Autoimmune lymphoproliferative syndrome, Autoimmune polyendocrine
syndrome, Autoimmune progesterone dermatitis, Idiopathic
thrombocytopenic purpura, Autoimmune urticaria, Balo concentric
sclerosis, Bullous pemphigoid, Castleman's disease, Cicatricial
pemphigoid, Cold agglutinin disease, Complement component 2
deficiency associated disease, Cushing's syndrome, Dagos disease,
Adiposis dolorosa, Eosinophilic pneumonia, Epidermolysis bullosa
acquisita, Hemolytic disease of the newborn, Cryoglobulinemia,
Evans syndrome, Fibrodysplasia ossificans progressive,
Gastrointestinal pemphigoid, Goodpasture's syndrome, Hashimoto's
encephalopathy, Gestational pemphigoid, Hughes-stovin syndrome,
Hypogammaglobulinemia, Lambert-eaton myasthenic syndrome, Lichen
sclerosus, Morphea, Pityriasis lichenoides et varioliformis acuta,
Myasthenia gravis, Narcolepsy, Neuromyotonia, Opsoclonus myoclonus
syndrome, Paraneoplastic cerebellar degeneration, Paroxysmal
nocturnal hemoglobinuria, Parry-romberg syndrome, Pernicious
anemia, POEMS syndrome, Pyoderma gangrenosum, Pure red cell
aplasia, Raynaud's phenomenon, Restless legs syndrome,
Retroperitoneal fibrosis, Autoimmune polyendocrine syndrome type 2,
Stiff person syndrome, Susac's syndrome, Febrile neutrophilic
dermatosis, Sydenham's chorea, Thrombocytopenia, vitiligo, (h)
arthritis, (i) skin diseases, (j) tauopathies, amyloidoses and
prion diseases, (k) polypes, (l) inflammatory diseases of the mouth
or the jaw bone, (m) osteonecrosis, (n) encephalomyelitis, (o)
fibrotic diseases and/or disorders, (p) kidney diseases and/or
disorders, (q) sympathetic ophthalmia, (r) transplant rejection
reaction, (s) Corticobasal degeneration, progressive supranuclear
palsy, schizophrenia, inherited Kreutzfeld Jacob, motor neurone
disease, spinocerebellar ataxia/atrophie, dementia, (t) and a
hereditary or non-hereditary metabolic disease.
2. The method according to claim 1, wherein the disorder/disease is
intraocular inflammation following anterior and/or posterior
segment surgery, wherein the surgery is selected from the group
consisting of cataract surgery, laser eye surgery, glaucoma
surgery, refractive surgery, corneal surgery, vitreo-retinal
surgery, eye muscle surgery, oculoplastic surgery, ocular oncology
surgery, conjunctival surgery including pterygium, and/or surgery
involving the lacrimal apparatus.
3. The method according to claim 2, wherein the JNK inhibitor is
injected at a dose range selected from the Croup consisting of in
the range of 0.01 .mu.g/eye to 10 mg/eye, 0.1 .mu.g/eye to 5
mg/eye, 1 .mu.g/eye to 2 mg/eye, 100 .mu.g/eye to 1.5 mg/eye, and
500 .mu.g/eye to 1 mg/eye.
4. The method according to claim 3, wherein the JNK inhibitor is
injected is by instillation, intravitreally or
subconjunctivally.
5. The method according to claim 4, wherein the JNK inhibitor is
applied is by a single injection within three hours after
surgery.
6. The method according to claim 1, wherein the disease is
retinopathy.
7. The method according to claim 1, wherein the disease is
psoriasis.
8. The method according to claim 1, wherein the disease is
periodontitis.
9. The method according to claim 1, wherein the disease is a graft
rejection or transplant rejection reaction.
10. The method according to claim 1, wherein the disease is
glomerulonephritis.
11. The method according to claim 1, wherein the disease liver
cancer, prostate cancer, colon cancer.
12. A method of reducing the likelihood of transplant rejection,
comprising applying to the tissue being transplanted a
pharmaceutical composition comprising a JNK inhibitor sequence
comprising less than 150 amino acids in length prior its
transplantation.
13. The method according to claim 12, wherein the tissue being
transplanted is a kidney, heart, lung, pancreas, liver, blood cell,
bone marrow, cornea, accidental severed limb, face, nose, bone,
cardiac valve, blood vessel or intestine transplant.
14. The method of claim 1, wherein the JNK inhibitor sequence
comprises a range of 5 to 150 amino acid residues.
15. The method of claim 1, wherein the JNK inhibitor sequence binds
c-jun amino terminal kinase (JNK).
16. The method of claim 1, wherein the JNK inhibitor sequence
inhibits the activation of at least one JNK targeted transcription
factor when the JNK inhibitor sequence is present in a JNK
expressing cell.
17. The method of claim 16, wherein the JNK targeted transcription
factor is selected from the group consisting of c-Jun, ATF2, and
Elkl.
18. The method of claim 1, wherein the JNK inhibitor sequence
alters a JNK effect when the peptide is present in a JNK expressing
cell.
19. The method of claim 1, wherein the JNK inhibitor sequence is
composed of L-amino acids, D-amino acids, or a combination of
both.
20. The method of claim 1, wherein the JNK inhibitor sequence
comprises a fragment the amino acid sequence of SEQ ID NO: 102, SEQ
ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105.
21. The method of claim 1, wherein the JNK inhibitor sequence
comprises or consists of at least one amino acid sequence selected
from the group consisting of SEQ ID NOs: 1 to 4, 13 to 20 and 33 to
100, or a fragment, derivative or variant thereof.
22. The method of claim 1, wherein the JNK inhibitor sequence is
covalently linked to a trafficking sequence.
23.-37. (canceled)
Description
[0001] The present invention refers to the use of protein kinase
inhibitors and more specifically to the use of inhibitors of the
protein kinase c-Jun amino terminal kinase, JNK inhibitor
sequences, chimeric peptides, or of nucleic acids encoding same as
well as pharmaceutical compositions containing same, for the
treatment of various novel diseases or disorders strongly related
to JNK signaling.
[0002] The c-Jun amino terminal kinase (JNK) is a member of the
stress-activated group of mitogen-activated protein (MAP) kinases.
These kinases have been implicated in the control of cell growth
and differentiation, and, more generally, in the response of cells
to environmental stimuli. The JNK signal transduction pathway is
activated in response to environmental stress and by the engagement
of several classes of cell surface receptors. These receptors can
include cytokine receptors, serpentine receptors and receptor
tyrosine kinases. In mammalian cells, JNK has been implicated in
biological processes such as oncogenic transformation and mediating
adaptive responses to environmental stress. JNK has also been
associated with modulating immune responses, including maturation
and differentiation of immune cells, as well as effecting
programmed cell death in cells identified for destruction by the
immune system. This unique property makes JNK signaling a promising
target for developing pharmacological intervention. Among several
neurological disorders, JNK signaling is particularly implicated in
ischemic stroke and Parkinson's disease, but also in other diseases
as mentioned further below. Furthermore, the mitogen-activated
protein kinase (MAPK) p38alpha was shown to negatively regulate the
cell proliferation by antagonizing the JNK-cJun-pathway. The
mitogen-activated protein kinase (MAPK) p38alpha therefore appears
to be active in suppression of normal and cancer cell proliferation
and, as a further, demonstrates the involvement of JNK in cancer
diseases (see e.g. Hui et al., Nature Genetics, Vol 39, No. 6, June
2007). It was also shown, that c-Jun N-terminal Kinase (JNK) is
involved in neuropathic pain produced by spinal nerve ligation
(SNL), wherein SNL induced a slow and persistent activation of JNK,
in particular JNK1, wheras p38 mitogen-activated protein kinase
activation was found in spinal microglia after SNL, which had
fallen to near basal lavel by 21 days (Zhuang et al., The journal
of Neuroscience, Mar. 29, 2006, 26(13):3551-3560)).
[0003] Inhibition or interruption of JNK signaling pathway,
particularly the provision of inhibitors of the JNK signaling
pathway, thus appears to be a promising approach in combating
disorders strongly related to JNK signaling. However, there are
only a few inhibitors of the JNK signaling pathway known so
far.
[0004] Inhibitors of the JNK signaling pathway as already known in
the prior art, particularly include e.g. upstream kinase inhibitors
(for example, CEP-1347), small chemical inhibitors of JNK (SP600125
and AS601245), which directly affect kinase activity e.g. by
competing with the ATP-binding site of the protein kinase, and
peptide inhibitors of the interaction between JNK and its
substrates (D-JNKI and I-JIP) (see e.g. Kuan et al., Current Drug
Targets--CNS & Neurological Disorders, February 2005, vol. 4,
no. 1, pp. 63-67(5)).
[0005] The upstream kinase inhibitor CEP-1347 (KT7515) is a
semisynthetic inhibitor of the mixed lineage kinase family.
CEP-1347 (KT7515) promotes neuronal survival at dosages that
inhibit activation of the c-Jun amino-terminal kinases (JNKs) in
primary embryonic cultures and differentiated PC12 cells after
trophic withdrawal and in mice treated with 1-methyl-4-phenyl
tetrahydropyridine. Further, CEP-1347 (KT7515) can promote long
term-survival of cultured chick embryonic dorsal root ganglion,
sympathetic, ciliary and motor neurons (see e.g. Borasio et al.,
Neuroreport. 9(7): 1435-1439, May 11, 1998).
[0006] The small chemical JNK inhibitor SP600125 was found to
reduce the levels of c-Jun phosphorylation, to protect dopaminergic
neurons from apoptosis, and to partly restore the level of dopamine
in MPTP-induced PD in C57BL/6N mice (Wang et al., Neurosci Res.
2004 February; 48(2); 195-202). These results furthermore indicate
that JNK pathway is the major mediator of the neurotoxic effects of
MPTP in vivo and inhibiting JNK activity may represent a new and
effective strategy to treat PD.
[0007] A further example of small chemical inhibitors is the
aforementioned JNK-Inhibitor AS601245. AS601245 inhibits the JNK
signalling pathway and promotes cell survival after cerebral
ischemia. In vivo, AS601245 provided significant protection against
the delayed loss of hippocampal CA1 neurons in a gerbil model of
transient global ischemia. This effect is mediated by JNK
inhibition and therefore by c-Jun expression and phosphorylation
(see e.g. Carboni et al., J Pharmacol Exp Ther. 2004 July;
310(1):25-32. Epub 2004 Feb. 26).
[0008] A third class of inhibitors of the JNK signaling pathway
represent peptide inhibitors of the interaction between JNK and its
substrates, as mentioned above. As a starting point for
construction of such JNK inhibitor peptides a sequence alignment of
naturally occurring JNK proteins may be used. Typically, these
proteins comprise JNK binding domains UBDs) and occur in various
insulin binding (IB) proteins, such as IB1 or IB2. The results of
such an exemplary sequence alignment is e.g. a sequence alignment
between the JNK binding domains of IB1 [SEQ ID NO: 13], IB2 [SEQ ID
NO: 14], c-Jun [SEQ ID NO: 15] and ATF2 [SEQ ID NO: 16] (see e.g.
FIGS. 1A-1Q. Such an alignment reveals a partially conserved 8
amino acid sequence (see e.g. FIG. 1A). A comparison of the JBDs of
IB1 and IB2 further reveals two blocks of seven and three amino
acids that are highly conserved between the two sequences.
[0009] Sequences constructed on basis of such an alignment are e.g.
disclosed in WO 01/27268 or in WO 2007/031280. WO 2007/031280 and
WO 01/27268 disclose small cell permeable fusion peptides,
comprising a so-called TAT cell permeation sequence derived from
the basic trafficking sequence of the HIV-TAT protein and a minimum
20 amino acid inhibitory sequence of IB1. Both components are
covalently linked to each other. Exemplary (and at present the
only) inhibitors of the MAPK-JNK signaling pathway disclosed in
both WO 2007/031280 and WO 01/27268, are e.g. L-JNKI1
(JNK-inhibitor peptide composed of L amino acids) or the protease
resistant D-JNKI1 peptides (JNK-inhibitor peptide composed of
non-native D amino acids). These JNK-inhibitor (JNKI) peptides are
specific for JNK (JNK1, JNK2 and JNK3). In contrast to those small
compound inhibitors as discussed above, the inhibitor sequences in
WO 2007/031280 or WO 01/27268, e.g. JNKI1, rather inhibit the
interaction between JNK and its substrate. By its trafficking
sequence derived from TAT, the fusion peptide is efficiently
transported into cells. Due to the novel properties obtained by the
trafficking component the fusion peptides are actively transported
into cells, where they remain effective until proteolytic
degradation.
[0010] However, peptides according to WO 2007/031280 or WO 01/27268
have only shown to be active in a particularly limited number of
diseases, particularly non-malignant or immunological-related cell
proliferative diseases.
[0011] One object of the present invention is thus, to identify
further diseases, which can be combated with JNK inhibitor
peptides. Another object of the present invention is to provide
(the use of) new JNK inhibitor peptides and derivatives thereof for
the treatment and/or prevention of those diseases and of diseases
not yet or already known to be strongly related to JNK
signaling.
[0012] This object is solved by the use of a JNK inhibitor
sequence, preferably as defined herein, typically comprising less
than 150 amino acids in length for the preparation of a
pharmaceutical composition for treating and/or preventing various
inflammatory or non-inflammatory diseases strongly related to JNK
signaling in a subject, wherein the diseases or disorders are
selected from the following groups: [0013] (a) encephalomyelitis,
in particular acute disseminated encephalomyelitis, spondylitis, in
particular ankylosing spondylitis, antisynthetase syndrome,
dermatitis, in particular atopic dermatitis or contact dermatitis,
hepatitis, in particular autoimmune hepatitis, autoimmune
peripheral neuropathy, pancreatitis, in particular autoimmune
pancreatitis, Behcet's disease, Bickerstaff's, encephalitis, Blau
syndrome, Coeliac disease, Chagas disease, polyneuropathy, in
particular chronic inflammatory demyelinating polyneuropathy,
osteomyelitis, in particular chronic recurrent multifocal
osteomyelitis, Churg-Strauss syndrome, Cogan syndrome, giant-cell
arteritis, CREST syndrome, vasculitis, in particular cutaneous
small-vessel vasculitis and urticarial vasculitis, dermatitis
herpetiformis, dermatomyositis, systemic scleroderma, Dressler's
syndrome, drug-induced lupus erythematosus, discoid lupus
erythematosus, enthesitis, eosinophilic fasciitis, eosinophilic
gastroenteritis, erythema nodosum, Idiopathic pulmonary fibrosis,
gastritis, Grave's disease, Guillain-barre syndrome, Hashimoto's
thyroiditis, Henoch-Schonlein purpura, Hidradenitis suppurativa,
Idiopathic inflammatory demyelinating diseases, myositis, in
particular inclusion body myositis, cystitis, in particular
interstitial cystitis, Kawasaki disease, Lichen planus, lupoid
hepatitis, Majeed syndrome, Meniere's disease, microscopic
polyangiitis, mixed connective tissue disease, myelitis, in
particular neuromyelitis optica, thyroiditis, in particular Ord's
thyroiditis, rheumatism, in particular palindromic rheumatism,
Parsonage-Turner syndrome, pemphigus vulgaris, perivenous
encephalomyelitis, polyarteritis nodosa, polymyalgia, in particular
polymyalgia rheumatica, polymyositis, cirrhosis, in particular
primary biliary cirrhosis, cholangitis, in particular primary
sclerosing cholangitis, progressive inflammatory neuropathy,
Rasmussen's encephalitis, relapsing polychondritis, arthritis, in
particular reactive arthritis (Reiter disease) and rheumatoid
arthritis, rheumatic fever, sarcoidosis, Schnitzler syndrome, serum
sickness, spondyloarthropathy, Takayasu's arteritis, Tolosa-Hunt
syndrome, transverse myelitis, and Wegener's granulomatosis, [0014]
(b) inflammatory and non-inflammatory diseases of the eye, in
particular selected from uveitis, in particular anterior,
intermediate and/or posterior uveitis, sympathetic uveitis and/or
panuveitis; scleritis in general, in particular anterior scleritis,
brawny scleritis, posterior scleritis, and scleritis with corneal
involvement; episcleritis in general, in particular episcleritis
periodica fugax and nodular episcleritis; retinitis; corneal
surgery; conjunctivitis in general, in particular acute
conjunctivitis, mucopurulent conjunctivitis, atopic conjunctivitis,
toxic conjunctivitis, pseudomembraneous conjunctivitis, serous
conjunctivitis, chronic conjunctivitis, giant pupillary
conjunctivitis, follicular conjunctivitis vernal conjunctivitis,
blepharoconjunctivitis, and/or pingueculitis; non-infectious
keratitis in general, in particular corneal ulcer, superficial
keratitis, macular keratitis, filamentary keratitis,
photokeratitis, punctate keratitis, keratoconjunctivitis, for
example exposure keratoconjunctivitis, dry eye syndrome
(keratoconjunctivitis sicca), neurotrophic keratoconjunctivitis,
ophthalmia nodosa, phlyctenular keratoconjunctivitis, vernal
keratoconjunctivitis and other keratoconjunctivitis, interstitial
and deep keratitis, sclerosing keratitis, corneal
neovascularization and other keratitis; iridocyclitis in general,
in particular acute iridocyclitis, subacute iridocyclitis and
chronic iridocyclitis, primary iridocyclitis, recurrent
iridocyclitis and secondary iridocyclitis, lens-induced
iridocyclitis, Fuchs' heterochromic cyclitis, Vogt-Koyanagi
syndrome; iritis; chorioretinal inflammation in general, in
particular focal and disseminated chorioretinal inflammation,
chorioretinitis, chorioditis, retinitis, retinochoroiditis,
posterior cyclitis, Harada's disease, chorioretinal inflammation in
infectious and parasitic diseases; post-surgery inflammation of the
eye, preferably intraocular inflammation following anterior and/or
posterior segment surgery, for example after cataract surgery,
laser eye surgery (e.g. Laser-in-situ-Keratomileusis (LASIK)),
glaucoma surgery, refractive surgery, corneal surgery,
vitreo-retinal surgery, eye muscle surgery, oculoplastic surgery,
ocular oncology surgery, conjunctival surgery including pterygium,
and surgery involving the lacrimal apparatus, in particular
post-surgery intraocular inflammation, preferably post-surgery
intraocular inflammation after complex eye surgery and/or after
uncomplicated eye surgery, for example inflammation of
postprocedural bleb; inflammatory diseases damaging the retina of
the eye; retinal vasculitis, in particular Eales disease and
retinal perivasculitis; retinopathy in general, in particular
diabetic retinopathy, (arterial hypertension induced) hypertensive
retinopathy, exudative retinopathy, radiation induced retinopathy,
sun-induced solar retinopathy, trauma-induced retinopathy, e.g.
Purtscher's retinopathy, retinopathy of prematurity (ROP) and/or
hyperviscosity-related retinopathy, non-diabetic proliferative
retinopathy, and/or proliferative vitreo-retinopathy; blebitis;
endophthalmitis; sympathetic ophthalmia; hordeolum; chalazion;
blepharitis; dermatitis and other inflammations of the eyelid;
dacryoadenititis; canaliculitis, in particular acute and chronic
lacrimal canaliculitis; dacryocystitis; inflammation of the orbit,
in particular cellulitis of orbit, periostitis of orbit, tenonitis
of orbit, granuloma of orbit and orbital myositis; purulent and
parasitic endophthalmitis; [0015] (c) Addison's disease,
Agammaglobulinemia, Alopecia areata, Amytrophic lateral sclerosis,
Antiphospholipid syndrome, Atopic allergy, Autoimmune aplastic
anemia, Autoimmune cardiomyopathy, Autoimmune enteropathy,
Autoimmune hemolytic anemia, Autoimmune inner ear, disease,
Autoimmune lymphoproliferative syndrome, Autoimmune polyendocrine
syndrome, Autoimmune progesterone dermatitis, Idiopathic
thrombocytopenic purpura, Autoimmune urticaria, Balo concentric
sclerosis, Bullous pemphigoid, Castleman's disease, Cicatricial
pemphigoid, Cold agglutinin disease, Complement component 2
deficiency associated disease, Cushing's syndrome, Dagos disease,
Adiposis dolorosa, Eosinophilic pneumonia, Epidermolysis bullosa
acquisita, Hemolytic disease of the newborn, Cryoglobulinemia,
Evans syndrome, Fibrodysplasia ossificans progressive,
Gastrointestinal pemphigoid, Goodpasture's syndrome, Hashimoto's
encephalopathy, Gestational pemphigoid, Hughes-stovin syndrome,
Hypogammaglobulinemia, Lambert-eaton myasthenic syndrome, Lichen
sclerosus, Morphea, Pityriasis lichenoides et varioliformis acuta,
Myasthenia gravis, Narcolepsy, Neuromyotonia, Opsoclonus myoclonus
syndrome, Paraneoplastic cerebellar degeneration, Paroxysmal
nocturnal hemoglobinuria, Parry-romberg syndrome, Pernicious
anemia, POEMS syndrome, Pyoderma gangrenosum, Pure red cell
aplasia, Raynaud's phenomenon, Restless legs syndrome,
Retroperitoneal fibrosis, Autoimmune polyendocrine syndrome type 2,
Stiff person syndrome, Susac's syndrome, Febrile neutrophilic
dermatosis, Sydenham's chorea, Thrombocytopenia, and vitiligo,
[0016] (d) arthritis, in particular juvenile idiopathic arthritis,
psoriastic arthritis and rheumatoid arthritis, and arthrosis, and
osteoarthritis, [0017] (e) skin diseases and diseases of the
subcutaneous tissue, in particular selected from papulosquamous
disorders in general, in particular psoriasis in general, for
example psoriasis vulgaris, nummular psoriasis, plaque psoriasis,
generalized pustular psoriasis, impetigo herpetiformis, Von
Zumbusch's disease, acrodermatitis continua, guttate psoriasis,
arthropathis psoriasis, distal interphalangeal psoriatic
arthropathy, psoriatic arthritis mutilans, psoriatic spondylitis,
psoriatic juvenile arthropathy, psoriatic arthropathy in general,
and/or flexural psoriasis, parapsoriasis in general, for example
large-plaque parapsoriasis, small-plaque parapsoriasis, retiform
parapsoriasis, pityriasis lichenoides and lymphomatoid papulosis;
pityriasis rosea; lichen planus and other papulosquamous disorders
for example pityriasis rubra pilaris, lichen nitidus, lichen
striatus, lichen ruber moniliformis, and infantile popular
acrodermatitis; eczema; dermatitis in general, in particular atopic
dermatitis for example Besnier's prurigo, atopic or diffuse
neurodermatitis, flexural eczema, infantile eczema, intrinsic
eczema, allergic eczema, other atopic dermatitis, seborrheic
dermatitis for example seborrhea capitis, seborrheic infantile
dermatitis, other seborrheic dermatitis, diaper dermatitis for
example diaper erythema, diaper rash and psoriasiform diaper rash,
allergic contact dermatitis, in particular due to metals, due to
adhesives, due to cosmetics, due to drugs in contact with skin, due
to dyes, due to other chemical products, due to food in contact
with skin, due to plants except food, due to animal dander, and/or
due to other agents, irritant contact dermatitis, in particular due
to detergents, due to oils and greases, due to solvents, due to
cosmetics, due to drugs in contact with skin, due to other chemical
products, due to food in contact with skin, due to plants except
food, due to metal, and/or due to other agents, unspecified contact
dermatitis, exfoliative dermatitis, dermatitis for example general
and localized skin eruption due to substances taken internally, in
particular due to drugs and medicaments, due to ingested food, due
to other substances, nummular dermatitis, dermatitis gangrenosa,
dermatitis herpetiformis, dry skin dermatitis, factitial
dermatitis, perioral dermatitis, radiation-related disorders of the
skin and subcutaneous tissue, stasis dermatitis, Lichen simplex
chronicus and prurigo, pruritus, dyshidrosis, cutaneous
autosensitization, infective dermatitis, erythema intertrigo and/or
pityriasis alba; cellulitis (bacterial infection involving the
skin); lymphangitis, in particular acute or chronic lymphangitis;
panniculitis in general, in particular lobular panniculitis without
vasculitis, for example acute panniculitis, previously termed
Weber-Christian disease and systemic nodular panniculitis, lobular
panniculitis with vasculitis, septal panniculitis without
vasculitis and/or septal panniculitis with vasculitis;
lymphadenitis, in particular acute lymphadenitis; pilonidal cyst
and sinus; pyoderma in general, in particular pyoderma gangrenosum,
pyoderma vegetans, dermatitis gangrenosa, purulent dermatitis,
septic dermatitis and suppurative dermatitis; erythrasma;
omphalitis; pemphigus, in particular pemphigus vulgaris, pemphigus
vegetans, pemphigus foliaceous, Brazilian pemphigus, pemphigus
erythematosus, drug-induced pemphigus, IgA pemphigus, for example
subcorneal pustular dermatosis and intraepidermal neutrophilic IgA
dermatosis, and/or paraneoplastic pemphigus; acne in general, in
particular acne vulgaris, acne conglobata, acne varioliformis, acne
necrotica miliaris, acne tropica, infantile acne acne excoriee des
jeunes filles, Picker's acne, and/or acne keloid; mouth and other
skin ulcers; urticaria in general, in particular allergic
urticaria, idiopathic urticarial, urticarial due to cold and heat,
dermatographic urticarial, vibratory urticarial, cholinergic
urticarial, and/or contact urticarial; erythema in general, in
particular erythema multiforme for example nonbullous erythema
multiforme, Stevens-Johnson syndrome, toxic epidermal necrolysis
(Lyell), and Stevens-Johnson syndrome-toxic epidermal necrolysis
overlap syndrome, erythema nodosum, toxic erythema, erythema
annulare centrifugum, erythema marginatum and/or other chronic
figurate erythema; sunburn and other acute skin changes due to
ultraviolet radiation; skin changes due to chronic exposure to
nonionizing radiation; radiodermatitis; folliculitis;
perifolliculitis; pseudofolliculitis barbae; hidradenititis
suppurativa; sarcoidose; vascularitis; adult linear IgA disease;
rosacea, in particular perioral dermatitis, rhinophyma, and other
rosacea; and/or follicular cysts of skin and subcutaneous tissue,
in particular epidermal cyst, pilar cyst, trichodermal cyst,
steatocystoma multiplex, sebaceous cyst and/or other follicular
cysts; [0018] (f) tauopathies, amyloidoses and prion diseases,
[0019] (g) polypes, [0020] (h) inflammatory diseases of the mouth
or the jaw bone, in particular selected from pulpitis in general,
in particular acute pulpitis, chronic pulpitis, hyperplastic
pulpitis, ulcerative pulpitis, irreversible pulpitis and/or
reversible pulpitis; periimplantitis; periodontitis in general, in
particular chronic periodontitis, complex periodontitis, simplex
periodontitis, aggressive periodontitis, and/or apical
periodontitis, e.g. of pulpal origin; periodontosis, in particular
juvenile periodontosis; gingivitis in general, in particular acute
gingivitis, chronic gingivitis, plaque-induced gingivitis, and/or
non-plaque-induced gingivitis; pericoronitis, in particular acute
and chronic pericoronitis; sialadenitis (sialoadenitis); parotitis,
in particular infectious parotitis and autoimmune parotitis;
stomatitis in general, in particular aphthous stomatitis (e.g.,
minor or major), Bednar's aphthae, periadenitis mucosa necrotica
recurrens, recurrent aphthous ulcer, stomatitis herpetiformis,
gangrenous stomatitis, denture stomatitis, ulcerative stomatitis,
vesicular stomatitis and/or gingivostomatitis; mucositis, in
particular mucositis due to antineoplastic therapy, due to (other)
drugs, or due to radiation, ulcerative mucositis and/or oral
mucositis; cheilitis in general, in particular chapped lips,
actinic cheilitis, angular cheilitis, eczematous cheilitis,
infectious cheilitis, granulomatous cheilitis, drug-related
cheilitis, exfoliative cheilitis, cheilitis glandularis, and/or
plasma cell cheilitis; cellulitis (bacterial infection), in
particular of mouth and/or lips; desquamative disorders, in
particular desquamative gingivitis; and/or temporomandibular joint
disorder; [0021] (i) osteonecrosis, [0022] (j) diseases and/or
disorders relating to degeneration of macula and/or posterior pole
in general, in particular age-related macular degeneration (AMD),
in particular the wet or the dry form of age-related macular
degeneration, exudative and/or non-exudative age-related macular
degeneration, and cataract, [0023] (k) fibrotic diseases and/or
disorders particularly selected from lung, heart, liver, bone
marrow, mediastinum, retroperitoneum, skin, intestine, joint, and
shoulder fibrosis,
[0024] (l) kidney diseases and/or disorders in particular selected
from glomerulonephritis in general, for example nonproliferative
glomerulonephritis, in particular minimal change disease, focal
segmental glomerulosclerosis, focal segmental glomerular hyalinosis
and/or sclerosis, focal glomerulonephritis, membranous
glomerulonephritis, and/or thin basement membrane disease, and
proliferative glomerulonephritis, in particular
membrano-proliferative glomerulonephritis, mesangio-proliferative
glomerulonephritis, endocapillary proliferative glomerulonephritis,
mesangiocapillary proliferative glomerulonephritis, dense deposit
disease (membranoproliferative glomerulonephritis type II),
extracapillary glomerulonephritis (crescentic glomerulonephritis),
rapidly progressive glomerulonephritis (RPGN), in particular Type I
RPGN, Type II RPGN, Type III RPGN, and Type IV RPGN, acute
proliferate glomerulonephritis, post-infectious glomerulonephritis,
and/or IgA nephropathy (Berger's disease); acute nephritic
syndrome; rapidly progressive nephritic syndrome; recurrent and
persistent hematuria; chronic nephritic syndrome; nephrotic
syndrome; proteinuria with specified morphological lesion;
glomerulitis; glomerulopathy; glomerulosclerosis; acute kidney
injury ("AKI", also called "acute renal failure" or "acute kidney
failure") in general, in particular prerenal AKI, intrinsic AKI,
postrenal AKI, AKI with tubular necrosis for example acute tubular
necrosis, renal tubular necrosis, AKI with cortical necrosis for
example acute cortical necrosis and renal cortical necrosis, AKI
with medullary necrosis, for example medullary (papillary)
necrosis, acute medullary (papillary) necrosis and chronic
medullary (papillary) necrosis, or other AKI; chronic kidney
disease; nephropathies in general, in particular membranous
nephropathy, diabetic nephropathy, IgA nephropathy, hereditary
nephropathy, analgesic nephropathy, CFHR5 nephropathy,
contrast-induced nephropathy, amyloid nephropathy, reflux
nephropathy and/or Mesoamerican nephropathy; nephritis in general,
in particular lupus nephritis, pyelonephritis, interstitial
nephritis, tubulointerstitial nephritis, chronic nephritis or acute
nephritis, diffuse proliferative nephritis, and/or focal
proloferative nephritis, tubulo-interstitial nephritis, infectious
interstitial nephritis, pyelitis, pyelonephritits, interstitial
nephritis; tubulopathy, tubulitis, in particular RTA (RTA1 and
RTA2), Fanconi syndrome, Bartter syndrome, Gitelman syndrome,
Liddle's syndrome, nephrogenic diabetes insipidus, renal papillary
necrosis, hydronephrosis, pyonephrosis and/or acute tubular
necrosis chronic kidney disease (CKD); Goodpasture syndrome
(anti-glomerular basement antibody disease); granulomatosis with
polyangiitis; microscopic polyangiitis; and/or Churg-Strauss
syndrome; [0025] (m) diseases and/or disorders of the urinary
system in particular selected from ureteritis; urinary tract
infection (bladder infection, acute cystitis); cystitis in general,
in particular interstitial cystitis, Hunner's ulcer, trigonitis
and/or hemorrhagic cystitis; urethritis, in particular
nongonococcal urethritis or gonococcal urethritis; urethral
syndrome; and/or retroperitoneal fibrosis; [0026] (n) transplant
rejection reactions in particular selected from kidney, heart,
lung, pancreas, liver, blood cell, bone marrow, cornea, accidental
severed limb, in particular fingers, hand, foot, face, nose, bone,
cardiac valve, blood vessel or intestine transplant rejection
reaction, [0027] (o) Corticobasal degeneration, progresive
supranuclear palsy, schizophrenia, inherited Kreutzfeld Jacob,
motor neurone disease, spinocerebellar ataxia/atrophie, dementia,
in particular frontotemporal dementia, dementia with lewy bodies,
multiple system atrophy, hereditary spastic paraparesis,
Friedreich's ataxiea, Charcot Marie toot, [0028] (p) hereditary or
non-heriditary metabolic diseases, in particular selected from the
group of metabolic disorders of the carbohydrate metabolism, e.g.,
glycogen storage disease, disorders of amino acid metabolism, e.g.,
phenylketonuria, maple syrup urine disease, glutaric acidemia type
1, urea Cycle Disorder or urea Cycle Defects, e.g., carbamoyl
phosphate synthetase I deficiency, disorders of organic acid
metabolism (organic acidurias), e.g., alcaptonuria, disorders of
fatty acid oxidation and mitochondrial metabolism, e.g.,
medium-chain acyl-coenzyme A dehydrogenase deficiency (often
shortened to MCADD.), disorders of porphyrin metabolism, e.g. acute
intermittent porphyria, disorders of purine or pyrimidine
metabolism, e.g., Lesch-Nyhan syndrome, Disorders of steroid
metabolism, e.g., lipoid congenital adrenal hyperplasia, or
congenital adrenal hyperplasia, disorders of mitochondrial
function, e.g., Kearns-Sayre syndrome, disorders of peroxisomal
function. e.g., Zellweger syndrome, or lysosomal storage disorders,
e.g., Gaucher's disease or Niemann Pick disease, [0029] (q) cancer
and/or tumor diseases, in particular selected from solid tumors in
general; hematologic tumors in general, in particular leukemia, for
example acute lymphocytic leukemia (L1, L2, L3), acute lymphoid
leukaemia (ALL), acute myelogenous leukemia (AML), chronic
lymphocytic leukaemia (CLL), chronic myeloid leukaemia (CML),
promyelocytic leukemia (M3), monocytic leukemia (MS), myeloblastic
leukemia (M1), myeloblastic leukemia (M2), megakaryoblastic
leukemia (M7) and myelomonocytic leukemia (M4); myeloma, for
example multiple myeloma; lymphomas, for example non-Hodgkin's
lymphomas, mycosis fungoides, Burkitt's lymphoma, and Hodgkin's
syndrome; pancreatic cancer, in particular pancreatic carcinoma;
ovarian cancer, in particular ovarian carcinoma; liver cancer and
liver carcinoma in general, in particular liver metastases, liver
cell carcinoma, hepatocellular carcinoma, hepatoma, intrahepatic
bile duct carcinoma, cholangiocarcinoma, hepatoblastoma,
angiosarcoma (of liver), and other specified or unspecified
sarcomas and carcinomas of the liver; skin cancer; melanoma, in
particular malignant melanoma; squamous cell carcinoma;
glioblastoma; colon cancer and colon carcinoma in general, in
particular cecum carcinoma, appendix carcinoma, ascending colon
carcinoma, hepatic flexure carcinoma, transverse colon carcinoma,
splenic flexure carcinoma, descending colon carcinoma, sigmoid
colon carcinoma, carcinoma of overlapping sites of colon and/or
malignant carcinoid tumors of the colon; prostate cancer and
prostate tumours, in particular prostate carcinoma; [0030] (r)
further cancer and/or tumor diseases, in particular selected from
acusticus neurinoma lung carcinomas; adenocarcinomas; anal
carcinoma; bronchial carcinoma; cervix carcinoma; cervical cancer;
astrocytoma; basalioma; cancer with Bcr-Abl transformation; bladder
cancer; blastomas; bone cancer; brain metastases; brain tumours;
breast cancer; carcinoids; cervical cancer; corpus carcinoma;
craniopharyngeomas; CUP syndrome; virus-induced tumours;
EBV-induced B cell lymphoma; endometrium carcinoma; erytholeukemia
(M6); esophagus cancer; gallbladder cancer; gastrointestinal
cancer; gastrointestinal stromal tumors; gastrointestinal tumours;
genitourinary cancer; glaucoma; gliomas; head/neck tumours;
hepatitis B-induced tumours; hepatocell or hepatocellular
carcinomas; hepatocarcinomas; hepatomas; herpes virus-induced
tumours; HTLV-1-induced lymphomas; HTLV-2-induced lymphomas;
insulinomas; intestinal cancer; Kaposi's sarcoma; kidney cancer;
kidney carcinomas; laryngeal cancer; leukemia; lid tumour; lung
cancer; lymphoid cancer; mammary carcinomas; mantle cell lymphoma;
neurinoma; medulloblastoma; meningioma; mesothelioma; non-small
cell carcinoma; non-small cell carcinoma of the lung; oesophageal
cancer; oesophageal carcinoma; oligodendroglioma; papilloma
virus-induced carcinomas; penis cancer; pituitary tumour;
plasmocytoma; rectal tumours; rectum carcinoma; renal-cell
carcinoma; retinoblastoma; sarcomas; Schneeberger's disease; small
cell lung carcinomas; small intestine cancer; small intestine
tumours; soft tissue tumours; spinalioma; squamous cell carcinoma;
stomach cancer; testicular cancer; throat cancer; thymoma; thyroid
cancer; thyroid carcinoma; tongue cancer; undifferentiated AML
(MO); urethral cancer; uterine cancer; vaginal cancer; Von Hippel
Lindau disease; vulval cancer; Wilms' Tumor; Xeroderma pigmentosum;
[0031] (s) neural, neuronal and/or neurodegenerative diseases,
respectively, in particular selected from Alexander disease;
tauopathies, in particular Alzheimer's disease in general, for
example Alzheimer's disease with early onset, Alzheimer's disease
with late onset, Alzheimer's dementia senile and presenile forms;
Mild Cognitive Impairment, in particular Mild Cognitive Impairment
due to Alzheimer's Disease; amyotrophic lateral sclerosis (ALS),
apoplexy, Ataxia Telangiectasia, cut or otherwise disrupted axons,
axotomy, brain lesions, CMT (Charcot-Marie-Tooth), corticobasal
degeneration, dementia, diseases or disorders of the nervous
system, dystonia, epilepsy, Farber's disease, Friedreich ataxia
(SCA), gangliosidoses, Guillain-Barre syndrome, hereditary spastic
paraplegia, Hirschsprung's disease, human immunodeficiency virus
dementia, Huntington's disease, infarct of the brain, ischemic
stroke, Krabbe disease, Lennox Gastaut Syndrome, lissencephaly,
multiple sclerosis, myelodysplastic syndromes, myelopathy,
AIDS-related neurodegenerative diseases, neurofibromatosis type 2
(NF-2), neurolatyerism, neuronal apoptosis, neuronal death,
neuropathic pain, neuropathy, chemotherapy induced neuropathy,
diabetes induced neuropathy, NMDA-induced neurotoxicity, pain,
Parkinson's disease, parkinsonism, Pick's Disease, polyneuropathy,
progressive supranuclear palsy, Sandhoff disease, spina bifida,
stroke, Tay Sachs, TBI (diffuse axonal injury), treatment of dark
neurone induced for example by an inflammatory pain, West Syndrome,
spinal muscular atrophy, [0032] (t) diseases resulting from
bacterial or viral infection, in particular selected from
inflammatory reactions caused by said infections, for example viral
encephalitis, viral induced cancers (e.g. as mentioned above),
human immunodeficiency virus dementia, meningitis,
meningoencephalitis, encephalomyelitis, tonsillitis, varicella
zoster virus infections, [0033] (u) diseases of the respiratory
system and in particular lung diseases, in particular selected from
acute respiratory distress syndrome (ARDS); asthma; chronic
illnesses involving the respiratory system; chronic obstructive
pulmonary disease (COPD); cystic fibrosis; inflammatory lung
diseases; pneumonia; pulmonary fibrosis, and [0034] (v) metabolic
disorders in particular selected from diabetes mellitus in general,
in particular type 1 diabetes mellitus, type 2 diabetes mellitus,
diabetes mellitus due to underlying condition, for example due to
congenital rubella, Cushing's syndrome, cystic fibrosis, malignant
neoplasm, malnutrition, or pancreatitis and other diseases of the
pancreas, drug or chemical induced diabetes mellitus, and/or other
diabetes mellitus, Fabry disease, Gaucher disease, hypothermia,
hyperthermia hypoxia, lipid histiocytosis, lipidoses, metachromatic
leukodystrophy, mucopolysaccharidosis, Niemann Pick disease,
obesity, and Wolman's disease.
[0035] According to one preferred embodiment, the disorder/disease
to be prevented and/or treated is a disease and/or disorder
relating to the degeneration of the macula, in particular selected
from age-related macular degeneration (AMD), in particular the wet
or the dry form of age-related macular degeneration, exudative
and/or non-exudative age-related macular degeneration, and
cataract.
[0036] The "dry" form of advanced AMD, results from atrophy of the
retinal pigment epithelial layer below the retina, which causes
vision loss through loss of photoreceptors (rods and cones) in the
central part of the eye. Neovascular, the "wet" form of advanced
AMD, causes vision loss due to abnormal blood vessel growth
(choroidal neovascularization) in the choriocapillaris, through
Bruch's membrane, ultimately leading to blood and protein leakage
below the macula. Bleeding, leaking, and scarring from these blood
vessels eventually cause irreversible damage to the photoreceptors
and rapid vision loss, if left untreated. The inventive molecules
are suitable for treating both forms of AMD.
[0037] According to another preferred embodiment, the
disorder/disease to be prevented and/or treated is retinopathy, in
particular selected from diabetic retinopathy, (arterial
hypertension induced) hypertensive retinopathy, exudative
retinopathy, radiation induced retinopathy, sun-induced solar
retinopathy, trauma-induced retinopathy, e.g. Purtscher's
retinopathy, retinopathy of prematurity (ROP) and/or
hyperviscosity-related retinopathy, non-diabetic proliferative
retinopathy, and/or proliferative vitreo-retinopathy, whereby
diabetic retinopathy and retinopathy of prematurity (ROP) are
preferred and diabetic retinopathy is particularly preferred.
[0038] Retinopathy of prematurity (ROP), previously known as
retrolental fibroplasia (RLF), is a disease of the eye affecting
prematurely-born babies generally having received intensive
neonatal care. It is thought to be caused by disorganized growth of
retinal blood vessels which may result in scarring and retinal
detachment. ROP can be mild and may resolve spontaneously, but it
may lead to blindness in serious cases. As such, all preterm babies
are at risk for ROP, and very low birth weight is an additional
risk factor. Both oxygen toxicity and relative hypoxia can
contribute to the development of ROP. The inventive molecules are
suitable for treating ROP.
[0039] Furthermore, the inventive molecules are particularly
suitable to treat all forms of retinopathy, in particular diabetes
mellitus induced retinopathy, arterial hypertension induced
hypertensive retinopathy, radiation induced retinopathy (due to
exposure to ionizing radiation), sun-induced solar retinopathy
(exposure to sunlight), trauma-induced retinopathy (e.g.
Purtscher's retinopathy) and hyperviscosity-related retinopathy as
seen in disorders which cause paraproteinemia).
[0040] According to another preferred embodiment, the
disorder/disease to be prevented and/or treated is post-surgery or
post-trauma inflammation of the eye, in particular post-surgery
intraocular inflammation, preferably intraocular inflammation
following anterior and/or posterior segment surgery. While the
inner of the eye is usually not very prone to infection and (e.g.
subsequent) inflammation due to its self-contained and isolated
structure, inflammation is increasingly likely after surgical
treatment of eye tissue and/or after other (e.g. mechanical)
injuries (trauma). Despite technical advances in ocular surgery,
the physical trauma of this procedure continues to induce
post-operative (i.e. post-surgery) ocular inflammation warranting
treatment. In ocular tissue, arachidonic acid is metabolized by
cyclooxygenase (COX) to prostaglandins (PG) which are the most
important lipid-derived mediators of inflammation. Surgical trauma
causes a trigger of the arachidonic acid cascade which in turn
generates PGs by activation of COX-1 and COX-2. Phospholipids in
the cell membrane are the substrate for phospholipase A to generate
arachidonic acid from which a family of chemically distinct PGs and
leukotriens are produced. The conventional `golden standard` for
the treatment of ocular inflammation are topical corticosteroids
and/or Non-Steroidal Anti-inflammatory Drugs (NSAIDs). Side effects
reported with (short-term) corticosteroid use include cataract
formation, increased Intra Ocular Pressure (IOP), increased
susceptibility to viral infections and retardation of the corneal
epithelial and stromal wound healing. In addition, prolonged
treatment with corticosteroids is known to induce systemic side
effects such as glucose impairment, hypertension, development of
glaucoma, visual acuity defects, loss of visual field, and
posterior subcapsular cataract formation. Therefore, the compounds
for use in the present invention may in particular be used for the
treatment of intraocular inflammation after eye surgery or trauma
and in particular of inflamed wounds and wound edges.
[0041] Thereby, the ocular surgery may preferably concern the
anterior and/or the posterior segment (of the eyeball). In general,
the "anterior segment" refers to the front third of the eye. It
includes structures in front of the vitreous humour, e.g. the
cornea, iris, ciliary body, and lens, whereby within the anterior
segment there are two fluid-filled spaces: (i) the anterior chamber
between the posterior surface of the cornea (i.e. the corneal
endothelium) and the iris, and (ii) the posterior chamber between
the iris and the front face of the vitreous. The "posterior
segment" in general refers to the back two thirds of the eye. It
includes the anterior hyaloid membrane and all of the structures,
in particular optical structures, behind it: the vitreous humor,
retina, choroid, and optic nerve.
[0042] Examples of ocular surgery regarding post-surgery
intraocular inflammation include (i) anterior and posterior
combined surgery, which may include surgery for: cataract and
retinal detachment, cataract and epimacular membrane and/or
cataract and macular hole; (ii) glaucoma surgery; (iii) posterior
segment surgery, in particular complex posterior segment surgery;
(iv) complicated intraocular surgery which may include cataract
surgery associated with diabetic retinopathy and/or complicated
retinal detachment ocular surgery. Moreover, the JNK inhibitors of
the present invention can be used to treat and/or prevent
post-surgery intraocular inflammation, whereby the ocular surgery
is for example performed due to an indication selected from the
following group including cataract, epimacular membrane, epiretinal
membrane, foveoschisis, intravitreous haemorrhage, macular hole,
neovascular glaucoma, relief of intraocluar, subluxation of lens,
in particular of intraocular lens, and vitreomacular traction.
Further examples of eye surgeries include cataract surgery, laser
eye surgery (e.g. Laser-in-situ-Keratomileusis (LASIK)), glaucoma
surgery, refractive surgery, corneal surgery, vitreo-retinal
surgery, eye muscle surgery, oculoplastic surgery, ocular oncology
surgery, conjunctival surgery including pterygium, and/or surgery
involving the lacrimal apparatus. Preferably the disorder/disease
to be prevented and/or treated by the JNK inhibitor according to
the present invention is intraocular inflammation following
anterior and/or posterior segment surgery, preferably post-surgery
intraocular inflammation after complex eye surgery and/or after
uncomplicated eye surgery, e.g. inflammation of postprocedural
bleb, or post-traumatic intraocular inflammation (preferably by
subconjunctival injection).
[0043] According to another preferred embodiment, the
disorder/disease to be prevented and/or treated is uveitis, in
particular anterior, intermediate and/or posterior uveitis,
sympathetic uveitis and/or panuveitis, preferably anterior and/or
posterior uveitis.
[0044] According to another preferred embodiment, the
disorder/disease to be prevented and/or treated is Dry Eye
Syndrome. Dry eye syndrome (DES), also called keratitis sicca,
xerophthalmia, keratoconjunctivitis sicca (KCS) or cornea sicca, is
an eye disease caused by eye dryness, which, in turn, is caused by
either decreased tear production or increased tear film
evaporation. Typical symptoms of dry eye syndrome are dryness,
burning and a sandy-gritty eye irritation. Dry eye syndrome is
often associated with ocular surface inflammation. If dry eye
syndrome is left untreated or becomes severe, it can produce
complications that can cause eye damage, resulting in impaired
vision or even in the loss of vision. Untreated dry eye syndrome
can in particular lead to pathological cases in the eye epithelium,
squamous metaplasia, loss of goblet cells, thickening of the
corneal surface, corneal erosion, punctate keratopathy, epithelial
defects, corneal ulceration, corneal neovascularization, corneal
scarring, corneal thinning, and even corneal perforation. The JNK
inhibitors according to the present invention may be utilized in
treatment and/or prevention of dry eye syndrome, e.g. due to aging,
diabetes, contact lenses or other causes and/or after eye surgery
or trauma, in particular after Lasik (laser-assisted in situ
keratomileusis), commonly referred to simply as laser eye surgery,
in particular of Sjorgren or non-Sjorgren syndrome dry eye.
[0045] The standard treatment of dry eye may involve the
administration of artificial tears, cyclosporine (in particular
cyclosporine A; e.g. Restasis.RTM.); autologous serum eye drops;
lubricating tear ointments and/or the administration of
(cortico-)steroids, for example in the form of drops or eye
ointments. Therefore, the present invention also relates to the use
of the JNK inhibitor as described herein in a method of treatment
of dry eye syndrome, wherein the method comprises the combined
administration of the JNK inhibitor as defined herein together with
a standard treatment for dry eye, in particular with any one of the
above mentioned treatments. Particularly preferred is the
combination with cyclosporine A and most preferably with artificial
tears. Combined administration comprises the parallel
administration and/or subsequent administration (either first the
JNK inhibitor described herein and then the (cortico)steroids or
vice versa). Certainly, subsequent and parallel administration may
also be combined, e.g. the treatment is started with JNK inhibitors
described herein and at a later point in time in the course of the
treatment (cortico)steroids are given in parallel, or vice
versa.
[0046] According to another preferred embodiment, the
disorder/disease to be prevented and/or treated is a skin disease,
in particular papulosquamous disorders, in particular selected from
psoriasis in general, for example psoriasis vulgaris, nummular
psoriasis, plaque psoriasis, generalized pustular psoriasis,
impetigo herpetiformis, Von Zumbusch's disease, acrodermatitis
continua, guttate psoriasis, arthropathis psoriasis, distal
interphalangeal psoriatic arthropathy, psoriatic arthritis
mutilans, psoriatic spondylitis, psoriatic juvenile arthropathy,
psoriatic arthropathy in general, and/or flexural psoriasis;
parapsoriasis in general, for example large-plaque parapsoriasis,
small-plaque parapsoriasis, retiform parapsoriasis, pityriasis
lichenoides and lymphomatoid papulosis; pityriasis rosea; lichen
planus and other papulosquamous disorders for example pityriasis
rubra pilaris, lichen nitidus, lichen striatus, lichen ruber
moniliformis, and infantile popular acrodermatitis. Preferably, the
disorder/disease to be prevented and/or treated is psoriasis, for
example psoriasis vulgaris, nummular psoriasis, plaque psoriasis,
generalized pustular psoriasis, impetigo herpetiformis, Von
Zumbusch's disease, acrodermatitis continua, guttate psoriasis,
arthropathis psoriasis, distal interphalangeal psoriatic
arthropathy, psoriatic arthritis mutilans, psoriatic spondylitis,
psoriatic juvenile arthropathy, psoriatic arthropathy in general,
and/or flexural psoriasis.
[0047] According to another preferred embodiment, the
disorder/disease to be prevented and/or treated is a
neurodegenerative disease, in particular tauopathies, preferably
Alzheimer's disease, for example Alzheimer's disease with early
onset, Alzheimer's disease with late onset, Alzheimer's dementia
senile and presenile forms.
[0048] Alzheimer's disease (AD) is a devastating neurodegenerative
disorder that leads to progressive cognitive decline with memory
loss and dementia. Neuropathological lesions are characterized by
extracellular deposition of senile plaques, formed by
.beta.-amyloid (A.beta.) peptide, and intracellular neurofibrillary
tangles (NFTs), composed of hyperphosphorylated tau proteins
(Duyckaerts et al., 2009, Acta Neuropathol 118: 5-36). According to
the amyloid cascade hypothesis, neurodegeneratlon in AD could be
linked to an abnormal amyloid precursor protein (APP) processing
through the activity of the beta-site APP cleaving enzyme 1 (BACE1)
and presenilin 1, leading to the production of toxic A.beta.
oligomers that accumulate in fibrillar A.beta. peptides before
forming A.beta. plaques. A.beta. accumulations can lead to synaptic
dysfunction, altered kinase activities resulting in NFTs formation,
neuronal loss and dementia (Hardy and Higgins, 1992, Science 256:
184-5). AD pathogenesis is thus believed to be triggered by the
accumulation of A.beta., whereby A.beta. self-aggregates into
oligomers, which can be of various sizes, and forms diffuse and
neuritic plaques in the parenchyma and blood vessels. A.beta.
oligomers and plaques are potent synaptotoxins, block proteasome
function, inhibit mitochondrial activity, alter intracellular
Ca.sup.2+ levels and stimulate inflammatory processes. Loss of the
normal physiological functions of A.beta. is also thought to
contribute to neuronal dysfunction. A.beta. interacts with the
signalling pathways that regulate the phosphorylation of the
microtubule-associated protein tau. Hyperphosphorylation of tau
disrupts its normal function in regulating axonal transport and
leads to the accumulation of neurofibrillary tangles (NFTs) and
toxic species of soluble tau. Furthermore, degradation of
hyperphosphorylated tau by the proteasome is inhibited by the
actions of A.beta.. These two proteins and their associated
signalling pathways therefore represent important therapeutic
targets for AD.
[0049] C-Jun N-terminal kinases UNKs) are serine-threonine protein
kinases, coded by three genes JNK1, JNK2, and JNK3, expressed as
ten different isoforms by mRNA alternative splicing, each isoforms
being expressed as a short form (46 kDa) and a long form (54 kDa)
(Davis, 2000, Cell 103: 239-52). While JNK1 and JNK2 are
ubiquitous, JNK3 is mainly expressed in the brain (Kyriakis and
Avruch, 2001, Physiol Rev 81: 807-69). JNKs are activated by
phosphorylation (pJNK) through MAPKinase activation by
extracellular stimuli, such as ultraviolet stress, cytokines and
A.beta. peptides and they have multiple functions including gene
expression regulation, cell proliferation and apoptosis
(Dhanasekaran and Reddy, 2008, Oncogene 27: 6245-51).
[0050] According to the present invention, it is assumed that the
JNK inhibitors according to the present invention reduce tau
hyperphosphorylation and, thus, neuronal loss. Therefore, the JNK
inhibitors according to the present invention can be useful for
treating and/or preventing tauopathies. Tauopathies are a class of
neurodegenerative diseases associated with the pathological
aggregation of tau protein in the human brain. The best-known
tauopathy is Alzheimer's disease (AD), wherein tau protein is
deposited within neurons in the form of neurofibrillary tangles
(NFTs), which are formed by hyperphosphorylation of tau protein.
The degree of NFT involvement in AD is defined by Braak stages.
Braak stages I and II are used when NFT involvement is confined
mainly to the transentorhinal region of the brain, stages III and
IV when there is also involvement of limbic regions such as the
hippocampus and V and VI when there is extensive neocortical
involvement. This should not be confused with the degree of senile
plaque involvement, which progresses differently. Thus, the JNK
inhibitors can be used according to the present invention for
treating and/or preventing tauopathies, in particular Alzheimer's
disease with NFT involvement, for example AD with Braak stage I, AD
with Braak stage II, AD with Braak stage III, AD with Braak stage
IV and/or AD with Braak stage V.
[0051] Further tauopathies, i.e. conditions in which
neurofibrillary tangles (NFTs) are commonly observed, and which can
thus be treated and/or prevented by the JNK inhibitors according to
the present invention, include progressive supranuclear palsy
although with straight filament rather than PHF (paired helical
filaments) tau; dementia pugilistica (chronic traumatic
encephalopathy); frontotemporal dementia and parkinsonism linked to
chromosome 17. however without detectable .beta.-amyloid plaques;
Lytico-Bodig disease (Parkinson-dementia complex of Guam);
tangle-predominant dementia, with NFTs similar to AD, but without
plaques; ganglioglioma and gangliocvtoma meningioangiomatosis;
subacute sclerosing panencephalitis and/or lead encephalopathy,
tuberous sclerosis. Hallervorden-Spatz disease. and lipofuscinosis.
Further tauopathies, which can be treated and/or prevented by the
JNK inhibitors according to the present invention, include Pick's
disease: corticobasal degeneration; Argyrophilic grain disease
(AGD); frontotemporal dementia and frontotemporal lobar
degeneration. In Pick's disease and corticobasal degeneration tau
proteins are deposited in the form of inclusion bodies within
swollen or "ballooned" neurons. Argyrophilic grain disease (AGD),
another type of dementia. which is sometimes considered as a type
of Alzheimer disease and which may co-exist with other tauopathies
such as progressive supranuclear palsy, corticobasal degeneration,
and also Pick's disease, is marked by the presence of abundant
argyrophilic grains and coiled bodies on microscopic examination of
brain tissue. The non-Alzheimer's tauopathies are sometimes grouped
together as "Pick's complex".
[0052] It is also preferred according to the present invention,
that the disorder/disease to be prevented and/or treated by the JNK
inhibitor according to the present invention is Mild Cognitive
Impairment (MCI), in particular MCI due to Alzheimer's Disease.
Typically, Mild Cognitive Impairment (MCI) is different from
Alzheimer's Disease, i.e. Mild Cognitive Impairment (MCI) is
typically not Alzheimer's Disease, but is a disease on its own
classified by ICD-10 in F06.7. In ICD-10 (F06.7), MCI is described
as a disorder characterized by impairment of memory, learning
difficulties, and reduced ability to concentrate on a task for more
than brief periods. There is often a marked feeling of mental
fatigue when mental tasks are attempted, and new learning is found
to be subjectively difficult even when objectively successful. None
of these symptoms is so severe that a diagnosis of either dementia
(F00-F03) or delirium (F05.-) can be made. The disorder may
precede, accompany, or follow a wide variety of infections and
physical disorders, both cerebral and systemic, but direct evidence
of cerebral involvement is not necessarily present. It can be
differentiated from postencephalitic syndrome (F07.1) and
postconcussional syndrome (F07.2) by its different etiology, more
restricted range of generally milder symptoms, and usually shorter
duration. Mild cognitive impairment (MCI), in particular MCI due to
Alzheimer's Disease, causes a slight but noticeable and measurable
decline in cognitive abilities, including memory and thinking
skills. MCI involves the onset and evolution of cognitive
impairments whatever type beyond those expected based on the age
and education of the individual, but which are not significant
enough to interfere with their daily activities. The diagnosis of
MCI is described for example by Albert M S, DeKosky S T, Dickson D,
Dubois B, Feldman H H, Fox N C, Gamst A, Holtzman D M, Jagust W J,
Petersen R C, Snyder P J, Carrillo M C, Thies B, Phelps C H (2011)
The diagnosis of mild cognitive impairment due to Alzheimer's
disease: recommendations from the National Institute on
Aging-Alzheimer's Association workgroups on diagnostic guidelines
for Alzheimer's disease; Alzheimers Dement.; 7(3):270-9. MCI may be
at the onset of whatever type of dementia or represents an
ephemeric form of cognitive impairment which may disappear over
time without resulting in a clinical manifestation of dementia. A
person with MCI is at an increased risk of developing Alzheimer's
or another dementia, in particular at an increased risk of
developing Alzheimer's Disease, without however necessarily
developing dementia, in particular Alzheimer's Disease. No
medications are currently approved by the U.S. Food and Drug
Administration (FDA) to treat Mild Cognitive Impairment. Drugs
approved to treat symptoms of Alzheimer's Disease have not shown
any lasting benefit in delaying or preventing progression of MCI to
dementia.
[0053] According to another preferred embodiment, the
disorder/disease to be prevented and/or treated is an inflammatory
disease of the mouth or the jaw bone, in particular pulpitis,
periimplantitis, periodontitis, gingivitis, stomatitis, mucositis,
desquamative disorders, and/or temporomandibular joint disorder,
preferably periodontitis.
[0054] According to another preferred embodiment, the
disorder/disease to be prevented and/or treated is a graft
rejection or transplant rejection reaction, in particular a liver,
lung, kidney, pancreas, skin or heart transplant graft rejection,
e.g. graft versus host or host versus graft.
[0055] According to still another preferred embodiment, the
disorder/disease to be prevented and/or treated is a nephrological
disease (kidney disease), in particular selected from
glomerulonephritis, for example nonproliferative
glomerulonephritis, in particular minimal change disease, focal
segmental glomerulosclerosis, focal segmental glomerular hyalinosis
and/or sclerosis, focal glomerulonephritis, membranous
glomerulonephritis, and/or thin basement membrane disease, and
proliferative glomerulonephritis, in particular
membrano-proliferative glomerulonephritis, mesangio-proliferative
glomerulonephritis, endocapillary proliferative glomerulonephritis,
mesangiocapillary proliferative glomerulonephritis, dense deposit
disease (membranoproliferative glomerulonephritis type II),
extracapillary glomerulonephritis (crescentic glomerulonephritis),
rapidly progressive glomerulonephritis (RPGN), in particular Type I
RPGN, Type II RPGN, Type III RPGN, and Type IV RPGN, acute
proliferate glomerulonephritis, post-infectious glomerulonephritis,
and/or IgA nephropathy (Berger's disease); acute nephritic
syndrome; rapidly progressive nephritic syndrome; recurrent and
persistent hematuria; chronic nephritic syndrome; nephrotic
syndrome; proteinuria with specified morphological lesion;
glomerulitis; glomerulopathy; glomerulosclerosis; acute kidney
injury ("AKI", also called "acute renal failure" or "acute kidney
failure") in general, in particular prerenal AKI, intrinsic AKI,
postrenal AKI, AKI with tubular necrosis for example acute tubular
necrosis, renal tubular necrosis, AKI with cortical necrosis for
example acute cortical necrosis and renal cortical necrosis, AKI
with medullary necrosis, for example medullary (papillary)
necrosis, acute medullary (papillary) necrosis and chronic
medullary (papillary) necrosis, or other AKI; chronic kidney
disease; preferably the disorder/disease to be prevented and/or
treated is glomerulonephritis. It is also preferred that the kidney
disorder/disease to be prevented and/or treated is a nephropathy,
in particular selected from membranous nephropathy, diabetic
nephropathy, IgA nephropathy, hereditary nephropathy, analgesic
nephropathy, CFHR5 nephropathy, contrast-induced nephropathy,
amyloid nephropathy, reflux nephropathy and/or Mesoamerican
nephropathydiabetic nephropathy, preferably the disorder/disease to
be prevented and/or treated is diabetic nephropathy.
[0056] According to still another preferred embodiment, the
disorder/disease to be prevented and/or treated is a disease and/or
disorder of the urinary system, in particular selected from
ureteritis; urinary tract infection (bladder infection, acute
cystitis); cystitis in general, in particular interstitial
cystitis, Hunner's ulcer, trigonitis and/or hemorrhagic cystitis;
urethritis, in particular nongonococcal urethritis or gonococcal
urethritis; painful bladder syndrome; IC/PBS; urethral syndrome;
and/or retroperitoneal fibrosis, preferably cystitis in general, in
particular interstitial cystitis. In this context it is noted that
interstitial cystitis (IC) varies very much in symptoms and
severity and, thus, most researchers believe it is not one, but
several diseases. In recent years, scientists have started to use
the terms "bladder pain syndrome" (BPS) or "painful bladder
syndrome" (PBS) to describe cases with painful urinary symptoms
that may not meet the strictest definition of IC. The term "IC/PBS"
includes all cases of urinary pain that can't be attributed to
other causes, such as infection or urinary stones. The term
interstitial cystitis, or IC, is typically used alone when
describing cases that meet all of the IC criteria, for example as
established by the National Institute of Diabetes and Digestive and
Kidney Diseases (NIDDK).
[0057] According to still another preferred embodiment, the
disorder/disease to be prevented and/or treated is a cancer and/or
tumor disease, in particular selected from solid tumors in general;
hematologic tumors in general, in particular leukemia, for example
acute lymphocytic leukemia (L1, L2, L3), acute lymphoid leukaemia
(ALL), acute myelogenous leukemia (AML), chronic lymphocytic
leukaemia (CLL), chronic myeloid leukaemia (CML), promyelocytic
leukemia (M3), monocytic leukemia (MS), myeloblastic leukemia (M1),
myeloblastic leukemia (M2), megakaryoblastic leukemia (M7) and
myelomonocytic leukemia (M4); myeloma, for example multiple
myeloma; lymphomas, for example non-Hodgkin's lymphomas, mycosis
fungoides, Burkitt's lymphoma, and Hodgkin's syndrome; pancreatic
cancer, in particular pancreatic carcinoma; ovarian cancer, in
particular ovarian carcinoma; liver cancer and liver carcinoma in
general, in particular liver metastases, liver cell carcinoma,
hepatocellular carcinoma, hepatoma, intrahepatic bile duct
carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma (of
liver), and other specified or unspecified sarcomas and carcinomas
of the liver; skin cancer; melanoma, in particular malignant
melanoma; squamous cell carcinoma; glioblastoma; colon cancer and
colon carcinoma in general, in particular cecum carcinoma, appendix
carcinoma, ascending colon carcinoma, hepatic flexure carcinoma,
transverse colon carcinoma, splenic flexure carcinoma, descending
colon carcinoma, sigmoid colon carcinoma, carcinoma of overlapping
sites of colon and/or malignant carcinoid tumors of the colon;
prostate cancer and prostate tumours, in particular prostate
carcinoma.
[0058] Moreover, in the following further diseases to be treated
are disclosed:
[0059] The JNK inhibitors of the present invention may be used for
example for the treatment of inflammatory diseases including for
example acute inflammation as well as chronic inflammation. The JNK
inhibitors of the present invention may be used to treat any type
of tissue inflammation, e.g. inflammation in the eye, inflammation
in the mouth, inflammation of the respiratory system including in
particular the lung, inflammation of the skin, inflammation within
the cardiovascular system, inflammation of the brain, inflammation
in the ear, etc. Some non-limiting examples for such inflammatory
disease states are mucositis, stomatitis, peri-implantitis,
retinitis, chorioiditis, keratoconjunctivitis sicca, inflammatory
bowel diseases (IBD), uveitis (e.g. anterior uveitis, intermediate
uveitis, posterior uveitis), periodontitis, COPD, asthma, pulpitis,
rheumatoid arthritis, osteoarthritis, Crohn's disease, psoriatic
arthritis, vasculitis, interstitial cystitis; acute inflammation at
a site of infection or wound, meningitis, encephalitis, pneumonia,
pharyngitis, tonsillitis, otitis (including otitis media),
vasculitis, synovitis, enteritis, Crohn's disease, ulcerative
colitis, graft rejection; post-surgery or post-trauma inflammation,
in particular intraocular inflammation following ocular anterior
and/or posterior segment surgery, etc.
[0060] The JNK inhibitors as disclosed herein may for example be
used in methods of treatment of ear diseases (in particular
diseases of the inner ear), hearing loss (in particular acute
hearing loss), damaged hair cell stereocilia, hair cell apoptosis,
noise trauma, otitis, otitis media etc. Hearing loss and associated
hair cell apoptosis are non-limiting examples for disorders
resulting from stress situations for cells in which JNK inhibition
can modulate the stress response and for example block
apoptosis.
[0061] The JNK inhibitors of the present invention may also be used
for the treatment of metabolic disorders, for example for the
treatment of diabetes in general, in particular type 1 diabetes
mellitus, type 2 diabetes mellitus, diabetes mellitus due to
underlying condition, for example due to congenital rubella,
Cushing's syndrome, cystic fibrosis, malignant neoplasm,
malnutrition, or pancreatitis and other diseases of the pancreas,
drug or chemical induced diabetes mellitus, and/or other diabetes
mellitus, Fabry disease, Gaucher disease, hypothermia, hyperthermia
hypoxia, lipid histiocytosis, lipidoses, metachromatic
leukodystrophy, mucopolysaccharidosis, Niemann Pick disease,
obesity, and Wolman's disease. Hypothermia, hyperthermia and
hypoxia are again non-limiting examples for stress situations for
cells in which JNK inhibition can modulate the stress response and
for example block apoptosis.
[0062] Likewise, the JNK inhibitors of the present invention may be
used for the treatment of neural, neuronal and/or neurodegenerative
diseases, respectively. Examples for such diseases are for example
Alexander disease; tauopathies, in particular Alzheimer's disease,
for example Alzheimer's disease with early onset, Alzheimer's
disease with late onset, Alzheimer's dementia senile and presenile
forms; Mild Cognitive Impairment, in particular Mild Cognitive
Impairment due to Alzheimer's Disease; amyotrophic lateral
sclerosis (ALS), apoplexy, Ataxia Telangiectasia, cut or otherwise
disrupted axons, axotomy, brain lesions, CMT (Charcot-Marie-Tooth),
corticobasal degeneration, dementia, diseases or disorders of the
nervous system, dystonia, epilepsy, Farber's disease, Friedreich
ataxia (SCA), gangliosidoses, Guillain-Barre syndrome, hereditary
spastic paraplegia, Hirschsprung's disease, human immunodeficiency
virus dementia, Huntington's disease, infarct of the brain,
ischemic stroke, Krabbe disease, Lennox Gastaut Syndrome,
lissencephaly, multiple sclerosis, myelodysplastic syndromes,
myelopathy, AIDS-related neurodegenerative diseases,
neurofibromatosis type 2 (NF-2), neurolatyerism, neuronal
apoptosis, neuronal death, neuropathic pain, neuropathy,
chemotherapy induced neuropathy, diabetes induced neuropathy,
NMDA-induced neurotoxicity, pain, Parkinson's disease,
parkinsonism, Pick's Disease, polyneuropathy, progressive
supranuclear palsy, Sandhoff disease, spina bifida, stroke, Tay
Sachs, TBI (diffuse axonal injury), treatment of dark neurone
induced for example by an inflammatory pain, West Syndrome, spinal
muscular atrophy etc.
[0063] With respect to autoimmune disorders, the JNK inhibitor
peptides of the present invention may for example be used in a
method of treatment of autoimmune diseases of the CNS,
auto-inflammatory diseases, Celiac disease; Sjogren's syndrome,
systemic lupus erythematosus etc.
[0064] Examples for bone diseases which may be treated with the JNK
inhibitors of the present invention are for example arthritis, disc
herniation, fibrodysplasia ossificans progressiva (FOP),
osteoarthritis, osteopetrosis, osteoporosis, in particular diabetes
induced osteoporosis, Paget's Disease, rheumatoid arthritis,
etc.
[0065] Examples for preferred skin diseases which can be treated
with the JNK inhibitors of the present invention are psoriasis and
lupus erythematosus. In more general terms, skin diseases and
diseases of the subcutaneous tissue, which can preferably be
treated and/or prevented with the JNK inhibitors as disclosed
herein are papulosquamous disorders. These include psoriasis,
parapsoriasis, pityriasis rosea, lichen planus and other
papulosquamous disorders for example pityriasis rubra pilaris,
lichen nitidus, lichen striatus, lichen ruber moniliformis, and
infantile popular acrodermatitis. Preferably the disease to be
treated and/or prevented by the JNK inhibitor according to the
invention is selected from the group of psoriasis and
parapsoriasis, whereby psoriasis is particularly preferred.
Examples for psoriasis include psoriasis vulgaris, nummular
psoriasis, plaque psoriasis, generalized pustular psoriasis,
impetigo herpetiformis, Von Zumbusch's disease, acrodermatitis
continua, guttate psoriasis, arthropathis psoriasis, distal
interphalangeal psoriatic arthropathy, psoriatic arthritis
mutilans, psoriatic spondylitis, psoriatic juvenile arthropathy,
psoriatic arthropathy in general, and/or flexural psoriasis.
Examples for parapsoriasis include large-plaque parapsoriasis,
small-plaque parapsoriasis, retiform parapsoriasis, pityriasis
lichenoides and lymphomatoid papulosis.
[0066] Further examples for preferred skin diseases which can be
treated with the JNK inhibitors of the present invention are
eczema; dermatitis in general, in particular atopic dermatitis for
example Besnier's prurigo, atopic or diffuse neurodermatitis,
flexural eczema, infantile eczema, intrinsic eczema, allergic
eczema, other atopic dermatitis, seborrheic dermatitis for example
seborrhea capitis, seborrheic infantile dermatitis, other
seborrheic dermatitis, diaper dermatitis for example diaper
erythema, diaper rash and psoriasiform diaper rash, allergic
contact dermatitis, in particular due to metals, due to adhesives,
due to cosmetics, due to drugs in contact with skin, due to dyes,
due to other chemical products, due to food in contact with skin,
due to plants except food, due to animal dander, and/or due to
other agents, irritant contact dermatitis, in particular due to
detergents, due to oils and greases, due to solvents, due to
cosmetics, due to drugs in contact with skin, due to other chemical
products, due to food in contact with skin, due to plants except
food, due to metal, and/or due to other agents, unspecified contact
dermatitis, exfoliative dermatitis, dermatitis for example general
and localized skin eruption due to substances taken internally, in
particular due to drugs and medicaments, due to ingested food, due
to other substances, nummular dermatitis, dermatitis gangrenosa,
dermatitis herpetiformis, dry skin dermatitis, factitial
dermatitis, perioral dermatitis, radiation-related disorders of the
skin and subcutaneous tissue, stasis dermatitis, Lichen simplex
chronicus and prurigo, pruritus, dyshidrosis, cutaneous
autosensitization, infective dermatitis, erythema intertrigo and/or
pityriasis alba; cellulitis (bacterial infection involving the
skin); lymphangitis, in particular acute or chronic lymphangitis;
panniculitis in general, in particular lobular panniculitis without
vasculitis, for example acute panniculitis, previously termed
Weber-Christian disease and systemic nodular panniculitis, lobular
panniculitis with vasculitis, septal panniculitis without
vasculitis and/or septal panniculitis with vasculitis;
lymphadenitis, in particular acute lymphadenitis; pilonidal cyst
and sinus; pyoderma in general, in particular pyoderma gangrenosum,
pyoderma vegetans, dermatitis gangrenosa, purulent dermatitis,
septic dermatitis and suppurative dermatitis; erythrasma;
omphalitis; pemphigus, in particular pemphigus vulgaris, pemphigus
vegetans, pemphigus foliaceous, Brazilian pemphigus, pemphigus
erythematosus, drug-induced pemphigus, IgA pemphigus, for example
subcorneal pustular dermatosis and intraepidermal neutrophilic IgA
dermatosis, and/or paraneoplastic pemphigus; acne in general, in
particular acne vulgaris, acne conglobata, acne varioliformis, acne
necrotica miliaris, acne tropica, infantile acne acne excoride des
jeunes filles, Picker's acne, and/or acne keloid; mouth and other
skin ulcers; urticaria in general, in particular allergic
urticaria, idiopathic urticarial, urticarial due to cold and heat,
dermatographic urticarial, vibratory urticarial, cholinergic
urticarial, and/or contact urticarial; erythema in general, in
particular erythema multiforme for example nonbullous erythema
multiforme, Stevens-Johnson syndrome, toxic epidermal necrolysis
(Lyell), and Stevens-Johnson syndrome-toxic epidermal necrolysis
overlap syndrome, erythema nodosum, toxic erythema, erythema
annulare centrifugum, erythema marginatum and/or other chronic
figurate erythema; sunburn and other acute skin changes due to
ultraviolet radiation; skin changes due to chronic exposure to
nonionizing radiation; radiodermatitis; folliculitis;
perifolliculitis; pseudofolliculitis barbae; hidradenititis
suppurativa; sarcoidose; vascularitis; adult linear IgA disease;
rosacea, in particular perioral dermatitis, rhinophyma, and other
rosacea; and/or follicular cysts of skin and subcutaneous tissue,
in particular epidermal cyst, pilar cyst, trichodermal cyst,
steatocystoma multiplex, sebaceous cyst and/or other follicular
cysts.
[0067] Diseases of the eye, which may be treated with the JNK
inhibitors of the present invention involve for example age-related
macular degeneration (AMD), in particular in the wet and dry form;
angioid streaks; anterior ischemic optic neuropathy; anterior
uveitis; cataract, in particular age related cataract; central
exudative chorioretinopathy; central serous chorioretinopathy;
chalazion; chorioderemia; chorioiditis; choroidal sclerosis;
conjunctivitis; cyclitis; diabetic retinopathy; dry eye syndrome;
endophthalmitis; episcleritis; eye infection; fundus albipunctatus;
gyrate atrophy of choroid and retina; hordeolum; inflammatory
diseases of the blephara; inflammatory diseases of the choroid;
inflammatory diseases of the ciliary body; inflammatory diseases of
the conjunctiva; inflammatory diseases of the cornea; inflammatory
diseases of the iris; inflammatory diseases of the lacrimal gland;
inflammatory diseases of the orbital bone; inflammatory diseases of
the sclera; inflammatory diseases of the vitreous body;
inflammatory diseases of the uvea; inflammatory diseases of the
retina; intermediate uveitis; irititis; keratitis; Leber's disease;
multifocal choroiditis; myositis of the eye muscle; neovascular
maculopathy (e.g. caused by high myopia, tilted disc syndrome,
choroidal osteoma or the like); NMDA induced retinotoxicity;
non-chronic or chronic inflammatory eye diseases; Oguchi's disease;
optic nerve disease; orbital phlegmon; panophtalmitis; panuveitis;
post caspule opacification; posterior capsule opacification (PCO)
(a cataract after-surgery complication); posterior uveitis;
proliferative vitreoretinopathy; retinal artery occlusion; retinal
detachment, retinal diseases; retinal injuries; retinal
macroaneurysm; retinal pigment epithelium detachment; retinal vein
occlusion; retinitis; retinitis pigmentosa; retinitis punctata
albescens; retinopathy, in particular retinopathy of prematurity
and diabetic retinopathy; scleritis; Stargardt's disease; treatment
of inflamed ocular wounds and/or ocular wound edges; treatment of
intraocular inflammation after eye surgery or trauma, preferably
intraocular inflammation following anterior and/or posterior
segment surgery; uveitis; vitelliform macular dystrophy; etc.
[0068] In particular the JNK inhibitors of the present invention
can be used to treat and/or prevent inflammatory diseases of the
eye, whereby such diseases can relate to the eye as a whole or to
different parts of the eye. For example, the JNK inhibitors of the
present invention can be used to treat and/or prevent
panophthalmitis, which is the inflammation of all coats of the eye
including intraocular structures. Further inflammatory diseases of
the eye, which can be treated and/or prevented with the JNK
inhibitors of the present invention include for example
endophthalmitis, for example purulent and parasitic
endophthalmitis; blebitis; hordeolum; chalazion; blepharitis;
dermatitis and other inflammations of the eyelid; dacryoadenititis;
canaliculitis, in particular acute and chronic lacrimal
canaliculitis; dacryocystitis; inflammation of the orbit, in
particular cellulitis of orbit, periostitis of orbit, tenonitis of
orbit, orbital granuloma (granulomatous inflammation) and orbital
myositis.
[0069] Furthermore, the JNK inhibitors of the present invention can
be used to treat and/or prevent inflammatory diseases of the
conjunctiva, in particular conjunctivitis, for example acute
conjunctivitis, mucopurulent conjunctivitis, atopic conjunctivitis,
toxic conjunctivitis, pseudomembraneous conjunctivitis, serous
conjunctivitis, chronic conjunctivitis, giant pupillary
conjunctivitis, follicular conjunctivitis vernal conjunctivitis,
blepharoconjunctivitis, and/or pingueculitis. Conjunctivitis is an
inflammation of the conjunctiva, which is commonly due to an
infection or an allergic reaction.
[0070] In particular, the JNK inhibitors of the present invention
can be used to treat and/or prevent inflammatory diseases of the
sclera, the cornea, the iris, the ciliary body, the retina and/or
the choroid of the eye. Preferably, the JNK inhibitors of the
present invention can be used to treat and/or prevent uveitis, i.e.
an inflammation of the uvea. The uvea consists of the middle,
pigmented vascular structures of the eye and includes the iris, the
ciliary body, and the choroid. Typically, uveitis is classified as
anterior uveitis, intermediate uveitis, posterior uveitis, and/or
panuveitis, whereby the latter is the inflammation of all the
layers of the uvea. Furthermore, uveitis includes sympathetic
ophthalmia (sympathetic uveitis), which is a bilateral diffuse
granulomatous uveitis of both eyes following trauma to one eye.
Anterior uveitis, which is particularly preferred to be treated
with the JNK inhibitors of the present invention, includes
iridocyclitis and iritis. Iritis is the inflammation of the
anterior chamber and iris. Iridocyclitis presents the same symptoms
as iritis, but also includes inflammation in the vitreous cavity.
Examples of iridocyclitis to be prevented and/or treated with the
JNK inhibitors of the present invention include--but are not
limited to--acute iridocyclitis, subacute iridocyclitis and chronic
iridocyclitis, primary iridocyclitis, recurrent iridocyclitis and
secondary iridocyclitis, lens-induced iridocyclitis, Fuchs'
heterochromic cyclitis, and Vogt-Koyanagi syndrome. Intermediate
uveitis, also known as pars planitis, in particular includes
vitritis, which is inflammation of cells in the vitreous cavity,
sometimes with "snowbanking" or deposition of inflammatory material
on the pars plana. Posterior uveitis includes in particular
chorioretinitis, which is the inflammation of the retina and
choroid, and chorioditis (choroid only). In more general terms, the
JNK inhibitors as disclosed herein can be used to treat and/or
prevent chorioretinal inflammation in general, for example focal
and/or disseminated chorioretinal inflammation, chorioretinitis,
chorioditis, retinochoroiditis, posterior cyclitis, Harada's
disease, chorioretinal inflammation in infectious and parasitic
diseases and/or retinitis, i.e. an inflammation of the retina.
Inflammatory diseases damaging the retina of the eye in general are
included, in addition to retinitis in particular retinal
vasculitis, for example Eales disease and retinal perivasculitis.
Further inflammatory diseases of the sclera, the cornea, the iris,
the ciliary body, the retina and/or the choroid of the eye to be
treated and/or prevented with the JNK inhibitors as disclosed
herein include scleritis, i.e. an inflammation of the sclera, for
example anterior scleritis, brawny scleritis, posterior scleritis,
scleritis with corneal involvement and scleromalacia perforans;
episcleritis, in particular episcleritis periodica fugax and
nodular episcleritis; and keratitis, which is an inflammation of
the cornea, in particular corneal ulcer, superficial keratitis,
macular keratitis, filamentary keratitis, photokeratitis, punctate
keratitis, keratoconjunctivitis, for example exposure
keratoconjunctivitis, keratoconjunctivitis sicca (dry eyes),
neurotrophic keratoconjunctivitis, ophthalmia nodosa, phlyctenular
keratoconjunctivitis, vernal keratoconjunctivitis and other
keratoconjunctivitis, interstitial and deep keratitis, sclerosing
keratitis, corneal neovascularization and other keratitis.
[0071] In addition, the JNK inhibitors as disclosed herein are
particularly useful to treat and/or prevent post-surgery (or
"post-procedural") or post-trauma (intraocular) inflammation of the
eye. "Post-surgery" refers in particular to a surgery performed on
and/or in the eye, preferably anterior and/or posterior segment
surgery, for example cataract surgery, laser eye surgery, glaucoma
surgery, refractive surgery, corneal surgery, vitreo-retinal
surgery, eye muscle surgery, oculoplastic surgery, ocular oncology
surgery, conjunctival surgery including pterygium, and/or surgery
involving the lacrimal apparatus. Preferably, the surgery referred
to in "post-surgery" is a complex eye surgery and/or an
uncomplicated eye surgery. Particularly preferred is the use of JNK
inhibitors as disclosed herein to treat and/or prevent post-surgery
or post-trauma intraocular inflammation, in particular intraocular
inflammation following anterior and/or posterior segment
surgery.
[0072] Another particularly preferred eye disease to be treated
and/or prevented with the JNK inhibitors according to the invention
is retinopathy. Non-limiting examples of retinopathy include
diabetic retinopathy, hypertensive retinopathy (e.g., arterial
hypertension induced), exudative retinopathy, radiation induced
retinopathy, sun-induced solar retinopathy, trauma-induced
retinopathy, e.g. Purtscher's retinopathy, retinopathy of
prematurity (ROP) and/or hyperviscosity-related retinopathy,
non-diabetic proliferative retinopathy, and/or proliferative
vitreo-retinopathy. The JNK inhibitors as disclosed herein are
particularly preferred for the treatment and/or prevention of
diabetic retinopathy and retinopathy of prematurity,
respectively.
[0073] Furthermore, the JNK inhibitors as disclosed herein are
preferably used in the treatment of diseases and/or disorders
relating to degeneration of the macula and/or posterior pole in
general. In particular, the treatment and/or prevention of
age-related macular degeneration (AMD) is preferred, in particular
the wet and/or the dry form of age-related macular degeneration,
exudative and/or non-exudative age-related macular
degeneration.
[0074] Exemplary diseases of the mouth which may be treated with
the JNK inhibitors as disclosed herein are periodontitis, in
particular chronic periodontitis; mucositis, oral desquamative
disorders, oral liquen planus, pemphigus vulgaris, pulpitis;
stomatitis; temporomandibular joint disorder, peri-implantitis etc.
Preferred diseases of the mouth or the jaw bone to be prevented
and/or treated with the JNK inhibitors according to the present
invention can be selected from the group consisting of pulpitis in
general, in particular acute pulpitis, chronic pulpitis,
hyperplastic pulpitis, ulcerative pulpitis, irreversible pulpitis
and/or reversible pulpitis; periimplantitis; periodontitis in
general, in particular chronic periodontitis, complex
periodontitis, simplex periodontitis, aggressive periodontitis,
and/or apical periodontitis, e.g. of pulpal origin; periodontosis,
in particular juvenile periodontosis; gingivitis in general, in
particular acute gingivitis, chronic gingivitis, plaque-induced
gingivitis, and/or non-plaque-induced gingivitis; pericoronitis, in
particular acute and chronic pericoronitis; sialadenitis
(sialoadenitis); parotitis, in particular infectious parotitis and
autoimmune parotitis; stomatitis in general, in particular aphthous
stomatitis (e.g., minor or major), Bednar's aphthae, periadenitis
mucosa necrotica recurrens, recurrent aphthous ulcer, stomatitis
herpetiformis, gangrenous stomatitis, denture stomatitis,
ulcerative stomatitis, vesicular stomatitis and/or
gingivostomatitis; mucositis, in particular mucositis due to
antineoplastic therapy, due to (other) drugs, or due to radiation,
ulcerative mucositis and/or oral mucositis; cheilitis in general,
in particular chapped lips, actinic cheilitis, angular cheilitis,
eczematous cheilitis, infectious cheilitis, granulomatous
cheilitis, drug-related cheilitis, exfoliative cheilitis, cheilitis
glandularis, and/or plasma cell cheilitis; cellulitis (bacterial
infection), in particular of mouth and/or lips; desquamative
disorders, in particular desquamative gingivitis; and/or
temporomandibular joint disorder.
[0075] The present invention is also suitable for use in the
treatment of diseases resulting in loss of bladder function (e.g.,
urinary incontinence, overactive bladder, interstitial cystitis, or
bladder cancer). In particular, diseases and/or disorders of the
urinary system can be treated and/or prevented with the JNK
inhibitors as disclosed herein. Such diseases are in particular
selected from ureteritis; urinary tract infection (bladder
infection, acute cystitis); cystitis in general, in particular
interstitial cystitis, Hunner's ulcer, trigonitis and/or
hemorrhagic cystitis; urethritis, in particular nongonococcal
urethritis or gonococcal urethritis; urethral syndrome; and/or
retroperitoneal fibrosis.
[0076] In addition, kidney diseases and/or disorders can be treated
and/or prevented with the JNK inhibitor according to the present
invention. Particularly preferred kidney diseases to be treated
and/or prevented with the JNK inhibitor according to the present
invention include glomerulopathies, in particular
glomerulonephritis, acute kidney injury and nephropathies.
[0077] Glomerulonephritis refers to several renal diseases, whereby
many of the diseases are characterised by inflammation either of
the glomeruli or small blood vessels in the kidneys, but not all
diseases necessarily have an inflammatory component. Non-limiting
examples of glomerulonephritis diseases to be treated and/or
prevented with the JNK inhibitor according to the present invention
include nonproliferative glomerulonephritis, in particular minimal
change disease, focal segmental glomerulosclerosis, focal segmental
glomerular hyalinosis and/or sclerosis, focal glomerulonephritis,
membranous glomerulonephritis, and/or thin basement membrane
disease, and proliferative glomerulonephritis, in particular
membrano-proliferative glomerulonephritis, mesangio-proliferative
glomerulonephritis, endocapillary proliferative glomerulonephritis,
mesangiocapillary proliferative glomerulonephritis, dense deposit
disease (membranoproliferative glomerulonephritis type II),
extracapillary glomerulonephritis (crescentic glomerulonephritis),
rapidly progressive glomerulonephritis (RPGN), in particular Type I
RPGN, Type II RPGN, Type III RPGN, and Type IV RPGN, acute
proliferate glomerulonephritis, post-infectious glomerulonephritis,
and/or IgA nephropathy (Berger's disease). Furthermore, diseases to
be treated and/or prevented with the JNK inhibitor according to the
present invention include acute nephritic syndrome; rapidly
progressive nephritic syndrome; recurrent and persistent hematuria;
chronic nephritic syndrome; nephrotic syndrome; proteinuria with
specified morphological lesion; glomerulitis; glomerulopathy; and
glomerulosclerosis. Acute kidney injury ("AKI", also called "acute
renal failure" or "acute kidney failure") is an abrupt loss of
kidney function, which is often investigated in a renal
ischemia/reperfusion injury model, and which includes for example
prerenal AKI, intrinsic AKI, postrenal AKI, AKI with tubular
necrosis for example acute tubular necrosis, renal tubular
necrosis, AKI with cortical necrosis for example acute cortical
necrosis and renal cortical necrosis, AKI with medullary necrosis,
for example medullary (papillary) necrosis, acute medullary
(papillary) necrosis and chronic medullary (papillary) necrosis, or
other AKI. Nephropathies, i.e. damage to or disease of a kidney,
includes also nephrosis, which is non-inflammatory nephropathy, and
nephritis, which is inflammatory kidney disease. The JNK inhibitor
according to the present invention are preferably used to treat
and/or prevent nephropathies, in particular membranous nephropathy,
diabetic nephropathy, IgA nephropathy, hereditary nephropathy,
analgesic nephropathy, CFHR5 nephropathy, contrast-induced
nephropathy, amyloid nephropathy, reflux nephropathy and/or
Mesoamerican nephropathy; nephritis in general, in particular lupus
nephritis, pyelonephritis, interstitial nephritis,
tubulointerstitial nephritis, chronic nephritis or acute nephritis,
diffuse proliferative nephritis, and/or focal proloferative
nephritis, tubulo-interstitial nephritis, infectious interstitial
nephritis, pyelitis, pyelonephritits, interstitial nephritis;
tubulopathy, tubulitis, in particular RTA (RTA1 and RTA2), Fanconi
syndrome, Bartter syndrome, Gitelman syndrome, Liddle's syndrome,
nephrogenic diabetes insipidus, renal papillary necrosis,
hydronephrosis, pyonephrosis and/or acute tubular necrosis chronic
kidney disease (CKD); Goodpasture syndrome (anti-glomerular
basement antibody disease); granulomatosis with polyangiitis;
microscopic polyangiitis; and/or Churg-Strauss syndrome. A
particularly preferred nephropathy to be treated and/or prevented
is diabetic nephropathy.
[0078] Another field of use is the treatment of pain, in particular
neuropathic, incident, breakthrough, psychogenic, or phantom pain,
all of these types of pain either in the acute or chronic form.
[0079] Likewise the JNK inhibitors of the present invention may--as
already previously proposed for other JNK inhibitors--be used for
the treatment of proliferative diseases like cancer and tumor
diseases, such as acusticus neurinoma; lung carcinomas; acute
lymphocytic leukemia (L1, L2, L3); acute lymphoid leukaemia (ALL);
acute myelogenous leukemia (AML); adenocarcinomas; anal carcinoma;
bronchial carcinoma; cervix carcinoma; cervical cancer;
astrocytoma; basalioma; cancer with Bcr-Abl transformation; bladder
cancer; blastomas; bone cancer; brain metastases; brain tumours;
breast cancer; Burkitt's lymphoma; carcinoids; cervical cancer;
chronic lymphocytic leukaemia (CLL); chronic myeloid leukaemia
(CML); colon cancer and colon carcinoma in general, in particular
cecum carcinoma, appendix carcinoma, ascending colon carcinoma,
hepatic flexure carcinoma, transverse colon carcinoma, splenic
flexure carcinoma, descending colon carcinoma, sigmoid colon
carcinoma, carcinoma of overlapping sites of colon and/or malignant
carcinoid tumors of the colon; corpus carcinoma;
craniopharyngeomas; CUP syndrome; virus-induced tumours;
EBV-induced B cell lymphoma; endometrium carcinoma; erytholeukemia
(M6); esophagus cancer; gallbladder cancer; gastrointestinal
cancer; gastrointestinal stromal tumors; gastrointestinal tumours;
genitourinary cancer; glaucoma; glioblastoma; gliomas; head/neck
tumours; hepatitis B-induced tumours; hepatocell or hepatocellular
carcinomas; hepatomas; herpes virus-induced tumours; Hodgkin's
syndrome; HTLV-1-induced lymphomas; HTLV-2-induced lymphomas;
insulinomas; intestinal cancer; Kaposi's sarcoma; kidney cancer;
kidney carcinomas; laryngeal cancer; leukemia; lid tumour; liver
cancer and liver carcinoma in general, in particular liver
metastases, liver cell carcinoma, hepatocellular carcinoma,
hepatoma; lung cancer; lymphoid cancer; lymphomas; malignant
melanomas; mammary carcinomas; mantle cell lymphoma;
medulloblastoma; megakaryoblastic leukemia (M7); melanoma, in
particular malignant melanoma; meningioma; mesothelioma; monocytic
leukemia (MS); multiple myeloma; mycosis fungoides; myeloblastic
leukemia (M1); myeloblastic leukemia (M2); myelomonocytic leukemia
(M4); neurinoma; non-Hodgkin's lymphomas; non-small cell carcinoma;
non-small cell carcinoma of the lung; oesophageal cancer;
oesophageal carcinoma; oligodendroglioma; ovarian cancer; ovarian
carcinoma; pancreatic cancer; pancreatic carcinoma; papilloma
virus-induced carcinomas; penis cancer; pituitary tumour;
plasmocytoma; promyelocytic leukemia (M3); prostate cancer;
prostate tumours; rectal tumours; rectum carcinoma; renal-cell
carcinoma; retinoblastoma; sarcomas; Schneeberger's disease; small
cell lung carcinomas; small intestine cancer; small intestine
tumours; soft tissue tumours; spinalioma; squamous cell carcinoma;
stomach cancer; testicular cancer; throat cancer; thymoma; thyroid
cancer; thyroid carcinoma; tongue cancer; undifferentiated AML
(MO); urethral cancer; uterine cancer; vaginal cancer; Von Hippel
Lindau disease; vulval cancer; Wilms' Tumor; Xeroderma pigmentosum;
etc.
[0080] Since JNK signalling is also involved in many cardiovascular
diseases and disorders, the use of JNK inhibitors in the treatment
of such diseases has already been suggested in the past. The
inhibitors of the present invention may be used accordingly, e.g.
for the treatment of cardiovascular diseases such as arterial
hypertension; arteriosclerosis; arteriosclerotic lesions; Behcet's
syndrome; bifurcations of blood vessels; cardiac hypertrophy;
cardiavascular hypertrophy; cardiomyopathies, in particular
chemotherapy induced cardiomyopathies; cerebral ischemia; coronary
heart diseases; dilatation of the abdominal aorta; focal cerebral
ischemia; global cerebral ischemia; heart hypertrophy; infrarenal
aneurism hypertension; ischemia; myocardial infarct, in particular
acute myocardial infarction; myocarditis; reperfusion; restenosis;
vasculitis; Wegener's granulomatosis; etc.
[0081] The JNK inhibitors of the present invention may in the
context of cardiovascular diseases also be used complementary to
coronary artery bypass graft surgery (CABG surgery); percutaneous
transluminal coronary angioplasty (PTCA); and/or stent treatment,
for example to prevent or treat intimal hyperplasia resulting from
said (surgical) treatment.
[0082] Diseases of the respiratory system and in particular lung
diseases which may be treated effectively with the JNK inhibitors
of the present invention are for example acute respiratory distress
syndrome (ARDS); asthma; chronic illnesses involving the
respiratory system; chronic obstructive pulmonary disease (COPD);
cystic fibrosis; inflammatory lung diseases; pneumonia; pulmonary
fibrosis; etc.
[0083] Like the inhibitors in the prior art the inhibitors of the
present invention may also be used to treat disease of the
intestinal tract, e.g. colitis (e.g. atypical colitis, chemical
colitis; collagenous colitis, distal colitis, diversion colitis;
fulminant colitis, indeterminate colitis, infectious colitis,
ischemic colitis, lymphocytic colitis, or microscopic colitis),
Crohn's disease, gastroenteritis, Hirschsprung's disease,
inflammatory digestive diseases; inflammatory bowel disease (IBD),
Morbus Crohn, non-chronic or chronic digestive diseases,
non-chronic or chronic inflammatory digestive diseases; regional
enteritis; ulcerative colitis etc.
[0084] The JNK inhibitors of the present invention may also serve
as therapeutic agent for the treatment of infectious diseases
resulting from e.g. bacterial or viral infection. The JNK
inhibitors as disclosed herein may for example prevent or
ameliorate inflammatory reactions caused by said infections.
Examples for such diseases states, which are not considered to be
limiting, are viral encephalitis; viral induced cancers (e.g. as
mentioned above), human immunodeficiency virus dementia,
meningitis, meningoencephalitis, encephalomyelitis, tonsillitis,
varicella zoster virus infections, etc.
[0085] There are many other diseases, disease states and disorders
for which the JNK inhibitors of the present invention can be used
as treatment, for example Aarskog syndrome, acetaminophen
hepatotoxicity; Alder-Reilly anomaly; alopecia areata;
alpha-1-antitrypsin deficiency; anaphylaxis; apoptosis; apoptotic
cell death; atypical hemolytic uremic syndrome; basopenia;
basophilia; bipolar disorders; burns; cellular shear stress;
Chedial-Higashi syndrome; DNA damage due to chemotherapeutic drugs;
cholestasis; chromosome 11, Partial Monosomy 11q; chromosome 22,
Trisomy Mosaic; chronic granulomatous disease; hepatitis, such as
chronic or fulminant hepatitis; clinical depression; common
variable hypogammaglobulinemia; congenital C3 deficiency; CTL
protection from activation-induced cell death (AICD); deafness;
depression and depressive disorders (in particular prevention of
depressive disorders develop on a background of cytokine-induced
sickness behaviour), DiGeorge's syndrome; diseases caused by
defective apoptosis; diseases of the liver; diseases of the spine;
diseases of the uterus; diseases states and symptoms due to
exposure to DNA damaging agents and/or ionizing radiation and
resulting cellular stress; Down Syndrome; Duchenne muscular
dystrophy; ectodermal dysplasias; endometriosis; eosinopenia;
eosinophilia; exocitoxic cell death; fetal alcohol syndrome;
fibrosis; fibrotic disease; formation of fibrous tissue; free
radicals (leading to cellular stress); graft rejection; Graft
versus host Disease, in particular skin graft versus host; hair
loss; hemolytic uremic syndrome; hepatotoxicity; hyperalgesia, such
as diabetes induced hyperalgesia; hyperthermia; hypoglycemia;
hypothyroidism; idiopathic hypereosinophilic syndrome; IgA
nephropathy; infantile sex-linked agammaglobulinemia; inflammatory
pain; infrarenal aneyrism; islet regeneration; islet
transplantation; Job's syndrome (hyper-IgE); lazy leukocyte
syndrome; leukocyte glucose-6-phosphate dehydrogenase deficiency;
leukodystrophy; leukopenia; lymphocytic leukocytosis;
lymphocytopenia; lymphocytosis; major depression; mania; maniac
depression; Marfan syndrome; mastocytosis; May Hegglin Anomaly;
membranoproliferative glomerulonephritis Type II; monocytopenia;
monocytosis; myeloperoxidase deficiency-benign; myopathies;
neutropenia; neutrophilia; Nezelof's syndrome; organ
transplantation; oxidative stress injuries; Pelger-Huet anomaly;
polycystic kidney diseases; post-dialysis syndrome; radiation
syndromes; radiotherapy; renal diseases; renal failure; rescuing
CTL from activation induced cell death; severe combined
immunodeficiency disease; transplant rejection; transplantation;
trisomy; unipolar depression; UV-induced injuries; Wiskott Aldrich
syndrome; wound healing; etc.
[0086] The inventors of the present invention consider
temporomandibular joint disorder, mucositis, stomatitis, oral
liquen planus (desquamative disorder), Pemphigus vulgaris
(desquamative disorder), periodontitis, chronic periodontitis,
pulpitis, peri-implantitis, uveitis (anterior uveitis, intermediate
uveitis, posterior uveitis), keratoconjunctivitis sicca (dry eye
syndrome), age-related macular degeneration (AMD), in particular in
the wet and dry form, retinopathy, in particular diabetic
retinopathy, post-surgery or post-trauma intraocular inflammation,
preferably intraocular inflammation following anterior and/or
posterior segment surgery, glomerulonephritis, nephropathy, in
particular diabetic nephropathy, interstitial cystitis, coronary
artery bypass graft surgery (CABG surgery), acute myocardial
infarction, prevention of intimal hyperplasia following
percutaneous transluminal coronary angioplasty (PTCA), prevention
of intimal hyperplasia following stent placement, atherosclerosis,
COPD, asthma, rheumatoid arthritis, osteoarthritis, Crohn's
disease, inflammatory bowel disease (IBD), psoriasis, diabetes,
stroke, Parkinson's disease, Alzheimer's disease, systemic lupus
erythematosus, and vasculitis, in particular Wegener's
granulomatosis, to be particularly useful for treatment with the
JNK inhibitors of the present invention.
[0087] According to another aspect the present invention provides a
JNK inhibitor sequence comprising less than 150 amino acids in
length for the (in vitro) treatment of a tissue or organ transplant
prior to or after its transplantation. The term "prior to its
transplantation" comprises the time of isolation and the time of
perfusion/transport. Thus, the treatment of a tissue or organ
transplant "prior to its transplantation" refers for example to
treatment during the isolation and/or during perfusion and/or
during transport. In particular, a solution used for isolation of a
tissue or organ transplant as well as a solution used for
perfusion, transport and/or otherwise treatment of a tissue or
organ transplant can preferably contain the JNK inhibitor according
to the invention.
[0088] In transplantation the tolerable cold ischemia time (CIT)
and the tolerable warm ischemia time (WIT) play critical roles. CIT
is the length of time that elapses between an organ being removed
from the donor, in particular the time of perfusion/treatment of an
organ by cold solutions, to its transplantation into the recipient.
WIT is in general a term used to describe ischemia of cells and
tissues under normothermic conditions. In particular WIT refers to
the length of time that elapses between a donor's death, in
particular from the time of cross-clamping or of asystole in
non-heart-beating donors, until cold perfusion is commenced.
Additionally, WIT may also refer to ischemia during implantation,
from removal of the organ from ice until reperfusion. In
allotransplantation usually, a transplant originating from a
brain-dead donor is typically not subjected to WIT, but has 8-12
hrs of CIT (time needed for transportation from the procurement
hospital to the isolation lab), whereas a transplant from a
non-heart beating donor is typically exposed to a longer WIT and
also 8-12 hrs of CIT. However, such transplantation is currently
not used routinely because of concerns about damage due to the WIT.
In autotransplantation, WIT may occur, however, CIT is usually
limited (typically 1-2 hrs, for example in islet
autotransplantation in patients with chronic pancreatitis).
[0089] Depending on the donor, the organ and/or tissue is not
perfused with blood for a variable amount of time prior to its
transplantation, leading to ischemia. Ischemia is an inevitable
event accompanying transplantation, for example kidney
transplantation. Ischemic changes start with brain death, which is
associated with severe hemodynamic disturbances: increasing
intracranial pressure results in bradycardia and decreased cardiac
output; the Cushing reflex causes tachycardia and increased blood
pressure; and after a short period of stabilization, systemic
vascular resistance declines with hypotension leading to cardiac
arrest. Free radical-mediated injury releases proinflammatory
cytokines and activates innate immunity. It has been suggested that
all of these changes--the early innate response and the ischemic
tissue damage play roles in the development of adaptive responses,
which in turn may lead to transplant rejection. Hypothermic storage
of the organ and/or tissue of various durations before
transplantation add to ischemic tissue damage. The final stage of
ischemic injury occurs during reperfusion. Reperfusion injury, the
effector phase of ischemic injury, develops hours or days after the
initial insult. Repair and regeneration processes occur together
with cellular apoptosis, autophagy, and necrosis; the fate of the
organ depends on whether cell death or regeneration prevails. The
whole process has been described as the ischemia-reperfusion (I-R)
injury. It has a profound influence on not only the early but also
the late function of a transplanted organ or tissue. Prevention of
I-R injury can thus already be started before organ recovery by
donor pretreatment.
[0090] It was found that such transplants may be (pre-)treated by
the JNK inhibitors according to the present invention in order to
improve their viability and functionality until transplanted to the
host. For that aspect of the invention, the transplant is a kidney,
heart, lung, pancreas, in particular pancreatic islets (also called
islets of Langerhans), liver, blood cell, bone marrow, cornea,
accidental severed limb, in particular fingers, hand, foot, face,
nose, bone, cardiac valve, blood vessel or intestine transplant,
preferably a kidney, heart, pancreas, in particular pancreatic
islets (also called islets of Langerhans), or skin transplant.
[0091] Moreover, in a further aspect, the present invention
provides a JNK inhibitor as defined herein for the treatment of a
tissue or organ transplant, or an animal or human who received a
tissue or organ transplant during or after transplantation. The
term "after transplantation" refers in particular to reperfusion of
the organ or tissue, for example a kidney, whereby reperfusion
begins for example by unclamping the respective blood flow. The
treatment with a JNK inhibitor according to the present invention
after transplantation refers in particular to the time interval of
up to four hours after reperfusion, preferably up to two hours
after reperfusion, more preferably up to one hour after reperfusion
and/or at the day(s) subsequent to transplantation. For the
treatment after transplantation, for example after kidney
transplantation, the JNK inhibitor according to the present
invention may be administered for example to an animal or human who
received a tissue or organ transplant as pharmaceutical composition
as described herein, for example systemically, in particular
intravenously, in a dose in the range of 0.01-10 mg/kg, preferably
in the range of 0.1-5 mg/kg, more preferably in the range of 0.5-2
mg/kg at a single dose or repeated doses.
[0092] For that aspect of the invention, the transplant is in
particular a kidney, heart, lung, pancreas, in particular
pancreatic islets (also called islets of Langerhans), liver, blood
cell, bone marrow, cornea, accidental severed limb, in particular
fingers, hand, foot, face, nose, bone, cardiac valve, blood vessel
or intestine transplant, preferably a kidney, heart, pancreas, in
particular pancreatic islets (also called islets of Langerhans), or
skin transplant.
[0093] Since JNK inhibitor sequences as known in the art only
proved usability for a limited number of diseases, it was a
surprising finding that JNK inhibitor sequences as defined herein
may be used and are suitable for the treatment of diseases or
disorders strongly related to JNK signaling as mentioned above.
This was neither obvious nor suggested by the prior art, even
though JNK inhibitor sequences in general have been known from the
art.
[0094] Typically, a JNK inhibitor sequence as defined above may be
derived from a human or rat IB1 sequence, preferably from an amino
acid sequence as defined or encoded by any of sequences according
to SEQ ID NO: 102 (depicts the IB1 cDNA sequence from rat and its
predicted amino acid sequence), SEQ ID NO: 103 (depicts the IB1
protein sequence from rat encoded by the exon-intron boundary of
the rIB1 gene--splice donor), SEQ ID NO: 104 (depicts the IB1
protein sequence from Homo sapiens), or SEQ ID NO: 105 (depicts the
IB1 cDNA sequence from Homo sapiens), more preferably from an amino
acid sequence as defined or encoded by any of sequences according
to SEQ ID NO: 104 (depicts the IB1 protein sequence from Homo
sapiens), or SEQ ID NO: 105 (depicts the IB1 cDNA sequence from
Homo sapiens), or from any fragments or variants thereof. In other
words, the JNK inhibitor sequence comprises a fragment, variant, or
variant of such fragment of a human or rat IB1 sequence. Human or
rat IB sequences are defined or encoded, respectively, by the
sequences according to SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:
104 or SEQ ID NO: 105.
[0095] Preferably, such a JNK inhibitor sequence as used herein
comprises a total length of less than 150 amino acid residues,
preferably a range of 5 to 150 amino acid residues, more preferably
10 to 100 amino acid residues, even more preferably 10 to 75 amino
acid residues and most preferably a range of 10 to 50 amino acid
residues, e.g. 10 to 30, 10 to 20, or 10 to 15 amino acid
residues.
[0096] More preferably, such a JNK inhibitor sequence and the above
ranges may be selected from any of the above mentioned sequences,
even more preferably from an amino acid sequence as defined
according to SEQ ID NO: 104 or as encoded by SEQ ID NO: 105, even
more preferably in the region between nucleotides 420 and 980 of
SEQ ID NO: 105 or amino acids 105 and 291 of SEQ ID NO: 104, and
most preferably in the region between nucleotides 561 and 647 of
SEQ ID NO: 105 or amino acids 152 and 180 of SEQ ID NO: 104.
[0097] According to a particular embodiment, a JNK inhibitor
sequence as used herein typically binds JNK and/or inhibits the
activation of at least one JNK activated transcription factor, e.g.
c-Jun or ATF2 (see e.g. SEQ ID NOs: 15 and 16, respectively) or
Elk1.
[0098] Likewise, the JNK inhibitor sequence as used herein
preferably comprises or consists of at least one amino acid
sequence according to any one of SEQ ID NOs: 1 to 4, 13 to 20 and
33 to 100, or a fragment, derivative or variant thereof. More
preferably, the JNK inhibitor sequence as used herein may contain
1, 2, 3, 4 or even more copies of an amino acid sequence according
to SEQ ID NOs: 1 to 4, 13 to 20 and 33 to 100, or a variant,
fragment or derivative thereof. If present in more than one copy,
these amino acid sequences according to SEQ ID NOs: 1 to 4, 13 to
20 and 33 to 100, or variants, fragments, or derivatives thereof as
used herein may be directly linked with each other without any
linker sequence or via a linker sequence comprising 1 to 10,
preferably 1 to 5 amino acids. Amino acids forming the linker
sequence are preferably selected from glycine or proline as amino
acid residues. More preferably, these amino acid sequences
according to SEQ ID NOs: 1 to 4, 13 to 20 and 33 to 100, or
fragments, variants or derivatives thereof, as used herein, may be
separated by each other by a hinge of two, three or more proline
residues.
[0099] The JNK inhibitor sequences as used herein may be composed
of L-amino acids, D-amino acids, or a combination of both.
Preferably, the JNK inhibitor sequences as used herein comprise at
least 1 or even 2, preferably at least 3, 4 or 5, more preferably
at least 6, 7, 8 or 9 and even more preferably at least 10 or more
D- and/or L-amino acids, wherein the D- and/or L-amino acids may be
arranged in the JNK inhibitor sequences as used herein in a
blockwise, a non-blockwise or in an alternate manner.
[0100] According to one preferred embodiment the JNK inhibitor
sequences as used herein may be exclusively composed of L-amino
acids. The JNK inhibitor sequences as used herein may then comprise
or consist of at least one "native JNK inhibitor sequence"
according to SEQ ID NO: 1 or 3. In this context, the term "native"
or "native JNK inhibitor sequence(s)" is referred to non-altered
JNK inhibitor sequences according to any of SEQ ID NOs: 1 or 3, as
used herein, entirely composed of L-amino acids.
[0101] Accordingly, the JNK inhibitor sequence as used herein may
comprise or consist of at least one (native) amino acid sequence
NH.sub.2--X.sub.n.sup.b--X.sub.n.sup.a-RPTTLXLXXXXXXXQD-X.sub.n.sup.b--CO-
OH (L-IB generic (s)) [SEQ ID NO: 3] and/or the JNK binding domain
(JBDs) of IB1 XRPTTLXLXXXXXXXQDS/TX (L-IB (generic)) [SEQ ID NO:
19]. In this context, each X typically represents an amino acid
residue, preferably selected from any (native) amino acid residue.
X.sub.n.sup.a typically represents one amino acid residue,
preferably selected from any amino acid residue except serine or
threonine, wherein n (the number of repetitions of X) is 0 or 1.
Furthermore, each X.sub.n.sup.b may be selected from any amino acid
residue, wherein n (the number of repetitions of X) is 0-5, 5-10,
10-15, 15-20, 20-30 or more, provided that if n (the number of
repetitions of X) is 0 for X.sub.n.sup.a, X.sub.n.sup.b does
preferably not comprise a serine or threonine at its C-terminus, in
order to avoid a serine or threonine at this position. Preferably,
X.sub.n.sup.b represents a contiguous stretch of peptide residues
derived from SEQ ID NO: 1 or 3. X.sub.n.sup.a and X.sub.n.sup.b may
represent either D or L amino acids. Additionally, the JNK
inhibitor sequence as used herein may comprise or consist of at
least one (native) amino acid sequence selected from the group
comprising the JNK binding domain of IB1 DTYRPKRPTTLNLFPQVPRSQDT
(L-IB1) [SEQ ID NO: 17]. More preferably, the JNK inhibitor
sequence as used herein further may comprise or consist of at least
one (native) amino acid sequence NH.sub.2-RPKRPTTLNLFPQVPRSQD-COOH
(L-IB1(s)) [SEQ ID NO: 1]. Furthermore, the JNK inhibitor sequence
as used herein may comprise or consist of at least one (native)
amino acid sequence selected from the group comprising the JNK
binding domain of IB1 L-IB1(s1) (NH.sub.2-TLNLFPQVPRSQD-COOH, SEQ
ID NO: 33); L-IB1 (s2) (NH.sub.2-TTLNLFPQVPRSQ-COOH, SEQ ID NO:
34); L-IB1(s3) (NH.sub.2-PTTLNLFPQVPRS-COOH, SEQ ID NO: 35);
L-IB1(s4) (NH.sub.2--RPTTLNLFPQVPR-COOH, SEQ ID NO: 36); L-IB1(s5)
(NH.sub.2-KRPTTLNLFPQVP-COOH, SEQ ID NO: 37); L-IB1(s6)
(NH.sub.2-PKRPTTLNLFPQV-COOH, SEQ ID NO: 38); L-IB1(s7)
(NH.sub.2--RPKRPTTLNLFPQ-COOH, SEQ ID NO: 39); L-IB1(s8)
(NH.sub.2-LNLFPQVPRSQD-COOH, SEQ ID NO: 40); L-IB1(s9)
(NH.sub.2-TLNLFPQVPRSQ-COOH, SEQ ID NO: 41); L-IB1(s10)
(NH.sub.2-TTLNLFPQVPRS-COOH, SEQ ID NO: 42); L-IB1(s11)
(NH.sub.2-PTTLNLFPQVPR-COOH, SEQ ID NO: 43); L-IB1(s12)
(NH.sub.2-RPTTLNLFPQVP-COOH, SEQ ID NO: 44); L-IB1(s13)
(NH.sub.2--KRPTTLNLFPQV-COOH, SEQ ID NO: 45); L-IB1(s14)
(NH.sub.2-PKRPTTLNLFPQ-COOH, SEQ ID NO: 46); L-IB1(s15)
(NH.sub.2-RPKRPTTLNLFP-COOH, SEQ ID NO: 47); L-IB1(s16)
(NH.sub.2--NLFPQVPRSQD-COOH, SEQ ID NO: 48); L-IB1(s17)
(NH.sub.2-LNLFPQVPRSQ-COOH, SEQ ID NO: 49); L-IB1(s18)
(NH.sub.2-TLNLFPQVPRS-COOH, SEQ ID NO: 50); L-IB1(s19)
(NH.sub.2-TTLNLFPQVPR-COOH, SEQ ID NO: 51); L-IB1(s20)
(NH.sub.2-PTTLNLFPQVP-COOH, SEQ ID NO: 52); L-IB1(s21)
(NH.sub.2-RPTTLNLFPQV-COOH, SEQ ID NO: 53); L-IB1(s22)
(NH.sub.2--KRPTTLNLFPQ-COOH, SEQ ID NO: 54); L-IB1(s23)
(NH.sub.2-PKRPTTLNLFP-COOH, SEQ ID NO: 55); L-IB1(s24)
(NH.sub.2-RPKRPTTLNLF-COOH, SEQ ID NO: 56); L-IB1(s25)
(NH.sub.2-LFPQVPRSQD-COOH, SEQ ID NO: 57); L-IB1(s26)
(NH.sub.2-NLFPQVPRSQ-COOH, SEQ ID NO: 58); L-IB1(s27)
(NH.sub.2-LNLFPQVPRS-COOH, SEQ ID NO: 59); L-IB1(s28)
(NH.sub.2-TLNLFPQVPR-COOH, SEQ ID NO: 60); L-IB1(s29)
(NH.sub.2-TTLNLFPQVP-COOH, SEQ ID NO: 61); L-IB1(s30)
(NH.sub.2-PTTLNLFPQV-COOH, SEQ ID NO: 62); L-IB1(s31)
(NH.sub.2-RPTTLNLFPQ-COOH, SEQ ID NO: 63); L-IB1(s32)
(NH.sub.2-KRPTTLNLFP-COOH, SEQ ID NO: 64); L-IB1(s33)
(NH.sub.2-PKRPTTLNLF-COOH, SEQ ID NO: 65); and L-IB1(s34)
(NH.sub.2-RPKRPTTLNL-COOH, SEQ ID NO: 66).
[0102] Additionally, the JNK inhibitor sequence as used herein may
comprise or consist of at least one (native) amino acid sequence
selected from the group comprising the (long) JNK binding domain
(JBDs) of IB1 PGTGCGDTYRPKRPTTLNLFPQVPRSQDT (IB1-long) [SEQ ID NO:
13], the (long) JNK binding domain of IB2
IPSPSVEEPHKHRPTTLRLTTLGAQDS (IB2-long) [SEQ ID NO: 14], the JNK
binding domain of c-Jun GAYGYSNPKILKQSMTLNLADPVGNLKPH (c-Jun) [SEQ
ID NO: 15], the JNK binding domain of ATF2
TNEDHLAVHKHKHEMTLKFGPARNDSVIV (ATF2) [SEQ ID NO: 16] (see e.g. FIG.
1A-1). In this context, an alignment revealed a partially conserved
8 amino acid sequence (see e.g. FIG. 1 A) and a further comparison
of the JBDs of IB1 and IB2 revealed two blocks of seven and three
amino acids that are highly conserved between the two
sequences.
[0103] According to another preferred embodiment the JNK inhibitor
sequences as used herein may be composed in part or exclusively of
D-amino acids as defined above. More preferably, these JNK
inhibitor sequences composed of D-amino acids are non-native D
retro-inverso sequences of the above (native) JNK inhibitor
sequences. The term "retro-inverso sequences" refers to an isomer
of a linear peptide sequence in which the direction of the sequence
is reversed and the chirality of each amino acid residue is
inverted (see e.g. Jameson et al., Nature, 368,744-746 (1994);
Brady et al., Nature, 368, 692-693 (1994)). The advantage of
combining D-enantiomers and reverse synthesis is that the positions
of carbonyl and amino groups in each amide bond are exchanged,
while the position of the side-chain groups at each alpha carbon is
preserved. Unless specifically stated otherwise, it is presumed
that any given L-amino acid sequence or peptide as used according
to the present invention may be converted into an D retro-inverso
sequence or peptide by synthesizing a reverse of the sequence or
peptide for the corresponding native L-amino acid sequence or
peptide.
[0104] The D retro-inverso sequences as used herein and as defined
above have a variety of useful properties. For example, D
retro-inverso sequences as used herein enter cells as efficiently
as L-amino acid sequences as used herein, whereas the D
retro-inverso sequences as used herein are more stable than the
corresponding L-amino acid sequences.
[0105] Accordingly, the JNK inhibitor sequences as used herein may
comprise or consist of at least one D retro-inverso sequence
according to the amino acid sequence
NH.sub.2--X.sub.n.sup.b-DQXXXXXXXLXLTTPR-X.sub.n.sup.a--X.sub.n.sup.b--CO-
OH (D-IB1 generic (s)) [SEQ ID NO: 4] and/or XS/TDQXXXXXXXLXLTTPRX
(D-IB (generic)) [SEQ ID NO: 20]. As used in this context,
X.sub.n.sup.b X.sub.n.sup.a and X.sub.n.sup.b are as defined above
(preferably, representing D amino acids), wherein X.sub.n.sup.b
preferably represents a contiguous stretch of residues derived from
SEQ ID NO: 2 or 4. Additionally, the JNK inhibitor sequences as
used herein may comprise or consist of at least one D retro-inverso
sequence according to the amino acid sequence comprising the JNK
binding domain (BDs) of IB1 TDQSRPVQPFLNLTTPRKPRYTD (D-IB1) [SEQ ID
NO: 18]. More preferably, the JNK inhibitor sequences as used
herein may comprise or consist of at least one D retro-inverso
sequence according to the amino acid sequence
NH.sub.2-DQSRPVQPFLNLTTPRKPR-COOH (D-IB1 (s)) [SEQ ID NO: 2].
Furthermore, the JNK inhibitor sequences as used herein may
comprise or consist of at least one D retro-inverso sequence
according to the amino acid sequence comprising the JNK binding
domain (JBDs) of IB1 D-IB1(s1) (NH.sub.2-QPFLNLTTPRKPR-COOH, SEQ ID
NO: 67); D-IB1 (s2) (NH.sub.2-VQPFLNLTTPRKP-COOH, SEQ ID NO: 68);
D-IB1(s3) (NH.sub.2-PVQPFLNLTTPRK-COOH, SEQ ID NO: 69); D-IB1(s4)
(NH.sub.2-RPVQPFLNLTTPR-COOH, SEQ ID NO: 70); D-IB1(s5)
(NH.sub.2-SRPVQPFLNLTTP-COOH, SEQ ID NO: 71); D-IB1(s6)
(NH.sub.2-QSRPVQPFLNLTT-COOH, SEQ ID NO: 72); D-IB1(s7)
(NH.sub.2-DQSRPVQPFLNLT-COOH, SEQ ID NO: 73); D-IB1(s8)
(NH.sub.2-PFLNLTTPRKPR-COOH, SEQ ID NO: 74); D-IB1(s9)
(NH.sub.2-QPFLNLTTPRKP-COOH, SEQ ID NO: 75); D-IB1(s10)
(NH.sub.2-VQPFLNLTTPRK-COOH, SEQ ID NO: 76); D-IB1(s11)
(NH.sub.2-PVQPFLNLTTPR-COOH, SEQ ID NO: 77); D-IB1(s12)
(NH.sub.2-RPVQPFLNLTTP-COOH, SEQ ID NO: 78); D-IB1(s13)
(NH.sub.2-SRPVQPFLNLTT-COOH, SEQ ID NO: 79); D-IB1(s14)
(NH.sub.2-QSRPVQPFLNLT-COOH, SEQ ID NO: 80); D-IB1(s15)
(NH.sub.2-DQSRPVQPFLNL-COOH, SEQ ID NO: 81); D-IB1(s16)
(NH.sub.2-FLNLTTPRKPR-COOH, SEQ ID NO: 82); D-IB1(s17)
(NH.sub.2-PFLNLTTPRKP-COOH, SEQ ID NO: 83); D-IB1(s18)
(NH.sub.2-QPFLNLTTPRK-COOH, SEQ ID NO: 84); D-IB1(s19)
(NH.sub.2-VQPFLNLTTPR-COOH, SEQ ID NO: 85); D-IB1(s20)
(NH.sub.2-PVQPFLNLTTP-COOH, SEQ ID NO: 86); D-IB1(s21)
(NH.sub.2-RPVQPFLNLTT-COOH, SEQ ID NO: 87); D-IB1(s22)
(NH.sub.2-SRPVQPFLNLT-COOH, SEQ ID NO: 88); D-IB1(s23)
(NH.sub.2-QSRPVQPFLNL-COOH, SEQ ID NO: 89); D-IB1(s24)
(NH.sub.2-DQSRPVQPFLN-COOH, SEQ ID NO: 90); D-IB1(s25)
(NH.sub.2-DQSRPVQPFL-COOH, SEQ ID NO: 91); D-IB1(s26)
(NH.sub.2-QSRPVQPFLN-COOH, SEQ ID NO: 92); D-IB1(s27)
(NH.sub.2-SRPVQPFLNL-COOH, SEQ ID NO: 93); D-IB1(s28)
(NH.sub.2-RPVQPFLNLT-COOH, SEQ ID NO: 94); D-IB1(s29)
(NH.sub.2-PVQPFLNLTT-COOH, SEQ ID NO: 95); D-IB1(s30)
(NH.sub.2-VQPFLNLTP-COOH, SEQ ID NO: 96); D-IB1(s31)
(NH.sub.2-QPFLNLTTPR-COOH, SEQ ID NO: 97); D-IB1(s32)
(NH.sub.2-PFLNLTTPRK-COOH, SEQ ID NO: 98); D-IB1(s33)
(NH.sub.2-FLNLTTPRKP-COOH, SEQ ID NO: 99); and D-IB1(s34)
(NH.sub.2-LNLTTPRKPR-COOH, SEQ ID NO: 100).
[0106] The JNK inhibitor sequences as used herein and as disclosed
above are presented in Table 1 (SEQ ID NO:s 1-4, 13-20 and 33-100).
The table presents the name of the JNK inhibitor sequences as used
herein, as well as their sequence identifier number, their length,
and amino acid sequence. Furthermore, Table 1 shows sequences as
well as their generic formulas, e.g. for SEQ ID NO's: 1, 2, 5, 6, 9
and 11 and SEQ ID NO's: 3, 4, 7, 8, 10 and 12, respectively. Table
1 furthermore discloses the chimeric sequences SEQ ID NOs: 9-12 and
23-32 (see below), L-IB1 sequences SEQ ID NOs: 33 to 66 and D-IB1
sequences SEQ ID NOs: 67 to 100.
TABLE-US-00001 TABLE 1 SEQ SEQUENCE/PEPTIDE NAME ID NO AA SEQUENCE
L-IB1(s) 1 19 RPKRPTTLNLFPQVPRSQD
(NH.sub.2-RPKRPTTLNLFPQVPRSQD-COOH) D-IB1(s) 2 19
DQSRPVQPFLNLTTPRKPR (NH.sub.2-DQSRPVQPFLNLTTPRKPR-COOH) L-IB
(generic) (s) 3 19
NH.sub.2-X.sub.n.sup.b-X.sub.n.sup.a-RPTTLXLXXXXXXXQD-X.sub.n.sup.b-COOH
D-IB (generic) (s) 4 19
NH.sub.2-X.sub.n.sup.b-DQXXXXXXXLXLTTPR-X.sub.n.sup.a-X.sub.n.sup.b-COOH
L-TAT 5 10 GRKKRRQRRR (NH.sub.2-GRKKRRQRRR-COOH) D-TAT 6 10
RRRQRRKKRG (NH.sub.2-RRRQRRKKRG-COOH) L-generic-TAT (s) 7 11
NH.sub.2-X.sub.n.sup.b-RKKRRQRRR-X.sub.n.sup.b-COOH D-generic-TAT
(s) 8 11 NH.sub.2-X.sub.n.sup.b-RRRQRRKKR-X.sub.n.sup.b-COOH
L-TAT-IB1(s) 9 31 GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQD
(NH.sub.2-GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQD-COOH) L-TAT-IB (generic)
(s) 10 29
NH.sub.2-X.sub.n.sup.b-RKKRRQRRR-X.sub.n.sup.b-X.sub.n.sup.a-RPTTLXLXXXXX-
XXQD-X.sub.n.sup.b-COOH D-TAT-IB(s) 11 31
DQSRPVQPFLNLTTPRKPRPPRRRQRRKKRG
(NH.sub.2-DQSRPVQPFLNLTTPRKPRPPRRRQRRKKRG-COOH) D-TAT-IB (generic)
(s) 12 29
NH.sub.2-X-DQXXXXXXXLXLTTPR-X.sub.n.sup.a-X.sub.n.sup.b-RRRQRRKKR-X.sub.n-
.sup.b-COOH IB1-long 13 29 PGTGCGDTYRPKRPTTLNLFPQVPRSQDT
(NH.sub.2-PGTGCGDTYRPKRPTTLNLFPQVPRSQDT-COOH) IB2-long 14 27
IPSPSVEEPHKHRPTTLRLTTLGAQDS
(NH.sub.2-IPSPSVEEPHKHRPTTLRLTTLGAQDS-COOH) c-Jun 15 29
GAYGYSNPKILKQSMTLNLADPVGNLKPH
(NH.sub.2-GAYGYSNPKILKQSMTLNLADPVGNLKPH-COOH) ATF2 16 29
TNEDHLAVHKHKHEMTLKFGPARNDSVIV
(NH.sub.2-TNEDHLAVHKHKHEMTLKFGPARNDSVIV-COOH) L-IB1 17 23
DTYRPKRPTTLNLFPQVPRSQDT (NH.sub.2-DTYRPKRPTTLNLFPQVPRSQDT-COOH)
D-IB1 18 23 TDQSRPVQPFLNLTTPRKPRYTD
(NH.sub.2-TDQSRPVQPFLNLTTPRKPRYTD-COOH) L-IB (generic) 19 19
XRPTTLXLXXXXXXXQDS/TX (NH.sub.2-XRPTTLXLXXXXXXXQDS/TX-COOH) D-IB
(generic) 20 19 XS/TDQXXXXXXXLXLTTPRX
(NH.sub.2-XS/TDQXXXXXXXLXLTTPRX-COOH) L-generic-TAT 21 17
XXXXRKKRRQRRRXXXX (NH.sub.2-XXXXRKKRRQRRRXXXX-COOH) D-generic-TAT
22 17 XXXXRRRQRRKKRXXXX (NH.sub.2-XXXXRRRQRRKKRXXXX-COOH) L-TAT-IB1
23 35 GRKKRRQRRRPPDTYRPKRPTTLNLFPQVPRSQDT
(NH.sub.2-GRKKRRQRRRPPDTYRPKRPTTLNLFPQVPRSQDT-COOH) L-TAT-IB
(generic) 24 42 XXXXXXXRKKRRQRRRXXXXXXXXRPTTLXLXXXXXXXQDS/TX
(NH.sub.2-XXXXXXXRKKRRQRRRXXXXXXXXRPTTLXLXXXXXXXQDS/TX-COOH)
D-TAT-IB1 25 35 TDQSRPVQPFLNLTTPRKPRYTDPPRRRQRRKKRG
(NH.sub.2-TDQSRPVQPFLNLTTPRKPRYTDPPRRRQRRKKRG-COOH) D-TAT-IB
(generic) 26 42 XT/SDQXXXXXXXLXLTTPRXXXXXXXXRRRQRRKKRXXXXXXX
(NH.sub.2-XT/SDQXXXXXXXLXLTTPRXXXXXXXXRRRQRRKKRXXXXXXX-COOH)
L-TAT-IB1(s1) 27 30 RKKRRQRRRPPRPKRPTTLNLFPQVPRSQD
(NH.sub.2-RKKRRQRRRPPRPKRPTTLNLFPQVRPSQD-COOH) L-TAT-IB1(s2) 28 30
GRKKRRQRRRX.sub.n.sup.cRPKRPTTLNLFPQVPRSQD
(NH.sub.2-GRKKRRQRRRX.sub.n.sup.cRPKRPTTLNLFPQVPRSQD-COOH)
L-TAT-IB1(s3) 29 29 RKKRRQRRRX.sub.n.sup.cRPKRPTTLNLFPQVPRSQD
(NH.sub.2-RKKRRQRRRX.sub.n.sup.cRPKRPTTLNLFPQVPRSQD-COOH)
D-TAT-IB1(s1) 30 30 DQSRPVQPFLNLTTPRKPRPPRRRQRRKKR
(NH.sub.2-DQSRPVQPFLNLTTPRKPRPPRRRQRRKKR-COOH) D-TAT-IB1(s2) 31 30
DQSRPVQPFLNLTTPRKPRX.sub.n.sup.cRRRQRRKKRG
(NH.sub.2-DQSRPVQPFLNLTTPRKPRX.sub.n.sup.cRRRQRRKKRG-COOH)
D-TAT-IB1(s3) 32 29 DQSRPVQPFLNLTTPRKPRX.sub.n.sup.cRRRQRRKKR
(NH.sub.2-DQSRPVQPFLNLTTPRKPRX.sub.n.sup.cRRRQRRKKR-COOH) L-IB1(s1)
33 13 TLNLFPQVPRSQD (NH.sub.2-TLNLFPQVPRSQD-COOH) L-IB1(s2) 34 13
TTLNLFPQVPRSQ (NH.sub.2-TTLNLFPQVPRSQ-COOH) L-IB1(s3) 35 13
PTTLNLFPQVPRS (NH.sub.2-PTTLNLFPQVPRS-COOH) L-IB1(s4) 36 13
RPTTLNLFPQVPR (NH.sub.2-RPTTLNLFPQVPR-COOH) L-IB1(s5) 37 13
KRPTTLNLFPQVP (NH.sub.2-KRPTTLNLFPQVP-COOH) L-IB1(s6) 38 13
PKRPTTLNLFPQV (NH.sub.2-PKRPTTLNLFPQV-COOH) L-IB1(s7) 39 13
RPKRPTTLNLFPQ (NH.sub.2-RPKRPTTLNLFPQ-COOH) L-IB1(s8) 40 12
LNLFPQVPRSQD (NH.sub.2-LNLFPQVPRSQD-COOH) L-IB1(s9) 41 12
TLNLFPQVPRSQ (NH.sub.2-TLNLFPQVPRSQ-COOH) L-IB1(s10) 42 12
TTLNLFPQVPRS (NH.sub.2-TTLNLFPQVPRS-COOH) L-IB1(s11) 43 12
PTTLNLFPQVPR (NH.sub.2-PTTLNLFPQVPR-COOH) L-IB1(s12) 44 12
RPTTLNLFPQVP (NH.sub.2-RPTTLNLFPQVP-COOH) L-IB1(s13) 45 12
KRPTTLNLFPQV (NH.sub.2-KRPTTLNLFPQV-COOH) L-IB1(s14) 46 12
PKRPTTLNLFPQ (NH.sub.2-PKRPTTLNLFPQ-COOH) L-IB1(s15) 47 12
RPKRPTTLNLFP (NH.sub.2-RPKRPTTLNLFP-COOH) L-IB1(s16) 48 11
NLFPQVPRSQD (NH.sub.2-NLFPQVPRSQD-COOH) L-IB1(s17) 49 11
LNLFPQVPRSQ (NH.sub.2-LNLFPQVPRSQ-COOH) L-IB1(s18) 50 11
TLNLFPQVPRS (NH.sub.2-TLNLFPQVPRS-COOH) L-IB1(s19) 51 11
TTLNLFPQVPR (NH.sub.2-TTLNLFPQVPR-COOH) L-IB1(s20) 52 11
PTTLNLFPQVP (NH.sub.2-PTTLNLFPQVP-COOH) L-IB1(s21) 53 11
RPTTLNLFPQV (NH.sub.2-RPTTLNLFPQV-COOH) L-IB1(s22) 54 11
KRPTTLNLFPQ (NH.sub.2-KRPTTLNLFPQ-COOH) L-IB1(s23) 55 11
PKRPTTLNLFP (NH.sub.2-PKRPTTLNLFP-COOH) L-IB1(s24) 56 11
RPKRPTTLNLF (NH.sub.2-RPKRPTTLNLF-COOH) L-IB1(s25) 57 10 LFPQVPRSQD
(NH.sub.2-LFPQVPRSQD-COOH) L-IB1(s26) 58 10 NLFPQVPRSQ
(NH.sub.2-NLFPQVPRSQ-COOH) L-IB1(s27) 59 10 LNLFPQVPRS
(NH.sub.2-LNLFPQVPRS-COOH) L-IB1(s28) 60 10 TLNLFPQVPR
(NH.sub.2-TLNLFPQVPR-COOH) L-IB1(s29) 61 10 TTLNLFPQVP
(NH.sub.2-TTLNLFPQVP-COOH) L-IB1(s30) 62 10 PTTLNLFPQV
(NH.sub.2-PTTLNLFPQV-COOH) L-IB1(s31) 63 10 RPTTLNLFPQ
(NH.sub.2-RPTTLNLFPQ-COOH) L-IB1(s32) 64 10 KRPTTLNLFP
(NH.sub.2-KRPTTLNLFP-COOH) L-IB1(s33) 65 10 PKRPTTLNLF
(NH.sub.2-PKRPTTLNLF-COOH) L-IB1(s34) 66 10 RPKRPTTLNL
(NH.sub.2-RPKRPTTLNL-COOH) D-IB1(s1) 67 13 QPFLNLTTPRKPR
(NH.sub.2-QPFLNLTTPRKPR-COOH) D-IB1(s2) 68 13 VQPFLNLTTPRKP
(NH.sub.2-VQPFLNLTTPRKP-COOH) D-IB1(s3) 69 13 PVQPFLNLTTPRK
(NH.sub.2-PVQPFLNLTTPRK-COOH) D-IB1(s4) 70 13 RPVQPFLNLTTPR
(NH.sub.2-RPVQPFLNLTTPR-COOH) D-IB1(s5) 71 13 SRPVQPFLNLTTP
(NH.sub.2-SRPVQPFLNLTTP-COOH) D-IB1(s6) 72 13 QSRPVQPFLNLTT
(NH.sub.2-QSRPVQPFLNLTT-COOH) D-IB1(s7) 73 13 DQSRPVQPFLNLT
(NH.sub.2-DQSRPVQPFLNLT-COOH) D-IB1(s8) 74 12 PFLNLTTPRKPR
(NH.sub.2-PFLNLTTPRKPR-COOH) D-IB1(s9) 75 12 QPFLNLTTPRKP
(NH.sub.2-QPFLNLTTPRKP-COOH) D-IB1(s10) 76 12 VQPFLNLTTPRK
(NH.sub.2-VQPFLNLTTPRK-COOH) D-IB1(s11) 77 12 PVQPFLNLTTPR
(NH.sub.2-PVQPFLNLTTPR-COOH) D-IB1(s12) 78 12 RPVQPFLNLTTP
(NH.sub.2-RPVQPFLNLTTP-COOH) D-IB1(s13) 79 12 SRPVQPFLNLTT
(NH.sub.2-SRPVQPFLNLTT-COOH) D-IB1(s14) 80 12 QSRPVQPFLNLT
(NH.sub.2-QSRPVQPFLNLT-COOH) D-IB1(s15) 81 12 DQSRPVQPFLNL
(NH.sub.2-DQSRPVQPFLNL-COOH)
D-IB1(s16) 82 11 FLNLTTPRKPR (NH.sub.2-FLNLTTPRKPR-COOH) D-IB1(s17)
83 11 PFLNLTTPRKP (NH.sub.2-PFLNLTTPRKP-COOH) D-IB1(s18) 84 11
QPFLNLTTPRK (NH.sub.2-QPFLNLTTPRK-COOH) D-IB1(s19) 85 11
VQPFLNLTTPR (NH.sub.2-VQPFLNLTTPR-COOH) D-IB1(s20) 86 11
PVQPFLNLTTP (NH.sub.2-PVQPFLNLTTP-COOH) D-IB1(s21) 87 11
RPVQPFLNLTT (NH.sub.2-RPVQPFLNLTT-COOH) D-IB1(s22) 88 11
SRPVQPFLNLT (NH.sub.2-SRPVQPFLNLT-COOH) D-IB1(s23) 89 11
QSRPVQPFLNL (NH.sub.2-QSRPVQPFLNL-COOH) D-IB1(s24) 90 11
DQSRPVQPFLN (NH.sub.2-DQSRPVQPFLN-COOH) D-IB1(s25) 91 10 DQSRPVQPFL
(NH.sub.2-DQSRPVQPFL-COOH) D-IB1(s26) 92 10 QSRPVQPFLN
(NH.sub.2-QSRPVQPFLN-COOH) D-IB1(s27) 93 10 SRPVQPFLNL
(NH.sub.2-SRPVQPFLNL-COOH) D-IB1(s28) 94 10 RPVQPFLNLT
(NH.sub.2-RPVQPFLNLT-COOH) D-IB1(s29) 95 10 PVQPFLNLTT
(NH.sub.2-PVQPFLNLTT-COOH) D-IB1(s30) 96 10 VQPFLNLTTP
(NH.sub.2-VQPFLNLTTP-COOH) D-IB1(s31) 97 10 QPFLNLTTPR
(NH.sub.2-QPFLNLTTPR-COOH) D-IB1(s32) 98 10 PFLNLTTPRK
(NH.sub.2-PFLNLTTPRK-COOH) D-IB1(s33) 99 10 FLNLTTPRKP
(NH.sub.2-FLNLTTPRKP-COOH) D-IB1(s34) 100 10 LNLTTPRKPR
(NH.sub.2-LNLTTPRKPR-COOH)
[0107] According to another preferred embodiment, the JNK inhibitor
sequence as used herein comprises or consists of at least one
variant, fragment and/or derivative of the above defined native or
non-native amino acid sequences according to SEQ ID NOs: 1-4, 13-20
and 33-100. Preferably, these variants, fragments and/or
derivatives retain biological activity of the above disclosed
native or non-native JNK inhibitor sequences as used herein,
particularly of native or non-native amino acid sequences according
to SEQ ID NOs: 1-4, 13-20 and 33-100, i.e. binding JNK and/or
inhibiting the activation of at least one JNK activated
transcription factor, e.g. c-Jun, ATF2 or Elk1. Functionality may
be tested by various tests, e.g. binding tests of the peptide to
its target molecule or by biophysical methods, e.g. spectroscopy,
computer modeling, structural analysis, etc. Particularly, an JNK
inhibitor sequence or variants, fragments and/or derivatives
thereof as defined above may be analyzed by hydrophilicity analysis
(see e.g. Hopp and Woods, 1981. Proc Natl Acad Sci USA 78:
3824-3828) that can be utilized to identify the hydrophobic and
hydrophilic regions of the peptides, thus aiding in the design of
substrates for experimental manipulation, such as in binding
experiments, or for antibody synthesis. Secondary structural
analysis may also be performed to identify regions of an JNK
inhibitor sequence or of variants, fragments and/or derivatives
thereof as used herein that assume specific structural motifs (see
e.g. Chou and Fasman, 1974, Biochem 13: 222-223). Manipulation,
translation, secondary structure prediction, hydrophilicity and
hydrophobicity profiles, open reading frame prediction and
plotting, and determination of sequence homologies can be
accomplished using computer software programs available in the art.
Other methods of structural analysis include, e.g. X-ray
crystallography (see e.g. Engstrom, 1974. Biochem Exp Biol 11:
7-13), mass spectroscopy and gas chromatography (see e.g. METHODS
IN PROTEIN SCIENCE, 1997, J. Wiley and Sons, New York, N.Y.) and
computer modeling (see e.g. Fletterick and Zoller, eds., 1986.
Computer Graphics and Molecular Modeling, In: CURRENT
COMMUNICATIONS IN MOLECULAR BIOLOGY, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.) may also be employed.
[0108] Accordingly, the JNK inhibitor sequence as used herein may
comprise or consist of at least one variant of (native or
non-native) amino acid sequences according to SEQ ID NOs: 1-4,
13-20 and 33-100. In the context of the present invention, a
"variant of a (native or non-native) amino acid sequence according
to SEQ ID NOs: 1-4, 13-20 and 33-100" is preferably a sequence
derived from any of the sequences according to SEQ ID NOs: 1-4,
13-20 and 33-100, wherein the variant comprises amino acid
alterations of the amino acid sequences according to SEQ ID NOs:
1-4, 13-20 and 33-100. Such alterations typically comprise 1 to 20,
preferably 1 to 10 and more preferably 1 to 5 substitutions,
additions and/or deletions of amino acids according to SEQ ID NOs:
1-4, 13-20 and 33-100, wherein the variant exhibits a sequence
identity with any of the sequences according to SEQ ID NOs: 1-4,
13-20 and 33-100 of at least about 30%, 50%, 70%, 80%, 90%, 95%,
98% or even 99%.
[0109] If variants of (native or non-native) amino acid sequences
according to SEQ ID NOs: 1-4, 13-20 and 33-100 as defined above and
used herein are obtained by substitution of specific amino acids,
such substitutions preferably comprise conservative amino acid
substitutions. Conservative amino acid substitutions may include
synonymous amino acid residues within a group which have
sufficiently similar physicochemical properties, so that a
substitution between members of the group will preserve the
biological activity of the molecule (see e.g. Grantham, R. (1974),
Science 185, 862-864). It is evident to the skilled person that
amino acids may also be inserted and/or deleted in the
above-defined sequences without altering their function,
particularly if the insertions and/or deletions only involve a few
amino acids, e.g. less than twenty, and preferably less than ten,
and do not remove or displace amino acids which are critical to
functional activity. Moreover, substitutions shall be avoided in
variants as used herein, which lead to additional threonines at
amino acid positions which are accessible for a phosphorylase,
preferably a kinase, in order to avoid inactivation of the
JNK-inhibitor sequence as used herein or of the chimeric peptide as
used herein in vivo or in vitro.
[0110] Preferably, synonymous amino acid residues, which are
classified into the same groups and are typically exchangeable by
conservative amino acid substitutions, are defined in Table 2.
TABLE-US-00002 TABLE 2 Preferred Groups of Synonymous Amino Acid
Residues Amino Acid Synonymous Residue Ser Ser, Thr, Gly, Asn Arg
Arg, Gln, Lys, Glu, His Leu Ile, Phe, Tyr, Met, Val, Leu Pro Gly,
Ala, (Thr), Pro Thr Pro, Ser, Ala, Gly, His, Gln, Thr Ala Gly, Thr,
Pro, Ala Val Met, Tyr, Phe, Ile, Leu, Val Gly Ala, (Thr), Pro, Ser,
Gly Ile Met, Tyr, Phe, Val, Leu, Ile Phe Trp, Met, Tyr, Ile, Val,
Leu, Phe Tyr Trp, Met, Phe, Ile, Val, Leu, Tyr Cys Ser, Thr, Cys
His Glu, Lys, Gln, Thr, Arg, His Gln Glu, Lys, Asn, His, (Thr),
Arg, Gln Asn Gln, Asp, Ser, Asn Lys Glu, Gln, His, Arg, Lys Asp
Glu, Asn, Asp Glu Asp, Lys, Asn, Gln, His, Arg, Glu Met Phe, Ile,
Val, Leu, Met Trp Trp
[0111] A specific form of a variant of SEQ ID NOs: 1-4, 13-20 and
33-100 as used herein is a fragment of the (native or non-native)
amino acid sequences according to SEQ ID NOs: 1, 1-4, 13-20 and
33-100" as used herein, which is typically altered by at least one
deletion as compared to SEQ ID NOs 1-4, 13-20 and 33-100.
Preferably, a fragment comprises at least 4 contiguous amino acids
of any of SEQ ID NOs: 1-4, 13-20 and 33-100, a length typically
sufficient to allow for specific recognition of an epitope from any
of these sequences. Even more preferably, the fragment comprises 4
to 18, 4 to 15, or most preferably 4 to 10 contiguous amino acids
of any of SEQ ID NOs: 1-4, 13-20 and 33-100, wherein the lower
limit of the range may be 4, or 5, 6, 7, 8, 9, or 10. Deleted amino
acids may occur at any position of SEQ ID NOs: 1-4, 13-20 and
33-100, preferably N- or C-terminally.
[0112] Furthermore, a fragment of the (native or non-native) amino
acid sequences according to SEQ ID NOs: 1-4, 13-20 and 33-100, as
described above, may be defined as a sequence sharing a sequence
identity with any of the sequences according to SEQ ID NOs: 1-4,
13-20 and 33-100 as used herein of at least about 30%, 50%, 70%,
80%, 90%, 95%, 98%, or even 99%.
[0113] The JNK inhibitor sequences as used herein may further
comprise or consist of at least one derivative of (native or
non-native) amino acid sequences according to SEQ ID NOs: 1-4,
13-20 and 33-100 as defined above. In this context, a "derivative
of an (native or non-native) amino acid sequence according to SEQ
ID NOs: 1-4, 13-20 and 33-100" is preferably an amino acid sequence
derived from any of the sequences according to SEQ ID NOs: 1-4,
13-20 and 33-100, wherein the derivative comprises at least one
modified L- or D-amino acid (forming non-natural amino acid(s)),
preferably 1 to 20, more preferably 1 to 10, and even more
preferably 1 to 5 modified L- or D-amino acids. Derivatives of
variants or fragments also fall under the scope of the present
invention.
[0114] "A modified amino acid" in this respect may be any amino
acid which is altered e.g. by different glycosylation in various
organisms, by phosphorylation or by labeling specific amino acids.
Such a label is then typically selected from the group of labels
comprising: [0115] (i) radioactive labels, i.e. radioactive
phosphorylation or a radioactive label with sulphur, hydrogen,
carbon, nitrogen, etc.; [0116] (ii) colored dyes (e.g. digoxygenin,
etc.); [0117] (iii) fluorescent groups (e.g. fluorescein, etc.);
[0118] (iv) chemoluminescent groups; [0119] (v) groups for
immobilization on a solid phase (e.g. His-tag, biotin, strep-tag,
flag-tag, antibodies, antigen, etc.); and [0120] (vi) a combination
of labels of two or more of the labels mentioned under (i) to
(v).
[0121] In the above context, an amino acid sequence having a
sequence "sharing a sequence identity" of at least, for example,
95% to a query amino acid sequence of the present invention, is
intended to mean that the sequence of the subject amino acid
sequence is identical to the query sequence except that the subject
amino acid sequence may include up to five amino acid alterations
per each 100 amino acids of the query amino acid sequence. In other
words, to obtain an amino acid sequence having a sequence of at
least 95% identity to a query amino acid sequence, up to 5% (5 of
100) of the amino acid residues in the subject sequence may be
inserted or substituted with another amino acid or deleted.
[0122] For sequences without exact correspondence, a "% identity"
of a first sequence may be determined with respect to a second
sequence. In general, these two sequences to be compared are
aligned to give a maximum correlation between the sequences. This
may include inserting "gaps" in either one or both sequences, to
enhance the degree of alignment. A % identity may then be
determined over the whole length of each of the sequences being
compared (so-called global alignment), that is particularly
suitable for sequences of the same or similar length, or over
shorter, defined lengths (so-called local alignment), that is more
suitable for sequences of unequal length.
[0123] Methods for comparing the identity and homology of two or
more sequences, particularly as used herein, are well known in the
art. Thus for instance, programs available in the Wisconsin
Sequence Analysis Package, version 9.1 (Devereux et al., 1984,
Nucleic Acids Res. 12, 387-395.), for example the programs BESTFIT
and GAP, may be used to determine the % identity between two
polynucleotides and the % identity and the % homology between two
polypeptide sequences. BESTFIT uses the "local homology" algorithm
of (Smith and Waterman (1981), J. Mol. Biol. 147, 195-197.) and
finds the best single region of similarity between two sequences.
Other programs for determining identity and/or similarity between
sequences are also known in the art, for instance the BLAST family
of programs (Altschul et al., 1990, J. Mol. Biol. 215, 403-410),
accessible through the home page of the NCBI at world wide web site
ncbi.nlm.nih.gov) and FASTA (Pearson (1990), Methods Enzymol. 183,
63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci. U. S. A
85, 2444-2448.).
[0124] JNK-inhibitor sequences as used according to the present
invention and as defined above may be obtained or produced by
methods well-known in the art, e.g. by chemical synthesis or by
genetic engineering methods as discussed below. For example, a
peptide corresponding to a portion of an JNK inhibitor sequence as
used herein including a desired region of said JNK inhibitor
sequence, or that mediates the desired activity in vitro or in
vivo, may be synthesized by use of a peptide synthesizer.
[0125] JNK inhibitor sequence as used herein and as defined above,
may be furthermore be modified by a trafficking sequence, allowing
the JNK inhibitor sequence as used herein and as defined above to
be transported effectively into the cells. Such modified JNK
inhibitor sequence are preferably provided and used as chimeric
sequences.
[0126] According to a second aspect the present invention therefore
provides the use of a chimeric peptide including at least one first
domain and at least one second domain, for the preparation of a
pharmaceutical composition for treating diseases or disorders
strongly related to JNK signaling as defined above in a subject,
wherein the first domain of the chimeric peptide comprises a
trafficking sequence, while the second domain of the chimeric
peptide comprises an JNK inhibitor sequence as defined above,
preferably of any of sequences according to SEQ ID NO: 1-4, 13-20
and 33-100 or a derivative or a fragment thereof.
[0127] Typically, chimeric peptides as used according to the
present invention have a length of at least 25 amino acid residues,
e.g. 25 to 250 amino acid residues, more preferably 25 to 200 amino
acid residues, even more preferably 25 to 150 amino acid residues,
25 to 100 and most preferably amino acid 25 to 50 amino acid
residues.
[0128] As a first domain the chimeric peptide as used herein
preferably comprises a trafficking sequence, which is typically
selected from any sequence of amino acids that directs a peptide
(in which it is present) to a desired cellular destination. Thus,
the trafficking sequence, as used herein, typically directs the
peptide across the plasma membrane, e.g. from outside the cell,
through the plasma membrane, and into the cytoplasm. Alternatively,
or in addition, the trafficking sequence may direct the peptide to
a desired location within the cell, e.g. the nucleus, the ribosome,
the endoplasmic reticulum (ER), a lysosome, or peroxisome, by e.g.
combining two components (e.g. a component for cell permeability
and a component for nuclear location) or by one single component
having e.g. properties of cell membrane transport and targeted e.g.
intranuclear transport. The trafficking sequence may additionally
comprise another component, which is capable of binding a
cytoplasmic component or any other component or compartment of the
cell (e.g. endoplasmic reticulum, mitochondria, gloom apparatus,
lysosomal vesicles). Accordingly, e.g. the trafficking sequence of
the first domain and the JNK inhibitor sequence of the second
domain may be localized in the cytoplasm or any other compartment
of the cell. This allows to determine localization of the chimeric
peptide in the cell upon uptake.
[0129] Preferably, the trafficking sequence (being included in the
first domain of the chimeric peptide as used herein) has a length
of 5 to 150 amino acid sequences, more preferably a length of 5 to
100 and most preferably a length of from 5 to 50, 5 to 30 or even 5
to 15 amino acids.
[0130] More preferably, the trafficking sequence (contained in the
first domain of the chimeric peptide as used herein) may occur as a
continuous amino acid sequence stretch in the first domain.
Alternatively, the trafficking sequence in the first domain may be
splitted into two or more fragments, wherein all of these fragments
resemble the entire trafficking sequence and may be separated from
each other by 1 to 10, preferably 1 to 5 amino acids, provided that
the trafficking sequence as such retains its carrier properties as
disclosed above. These amino acids separating the fragments of the
trafficking sequence may e.g. be selected from amino acid sequences
differing from the trafficking sequence. Alternatively, the first
domain may contain a trafficking sequence composed of more than one
component, each component with its own function for the transport
of the cargo JNK inhibitor sequence of the second domain to e.g. a
specific cell compartment.
[0131] The trafficking sequence as defined above may be composed of
L-amino acids, D-amino acids, or a combination of both. Preferably,
the trafficking sequence (being included in the first domain of the
chimeric peptide as used herein) may comprise at least 1 or even 2,
preferably at least 3, 4 or 5, more preferably at least 6, 7, 8 or
9 and even more preferably at least 10 or more D- and/or L-amino
acids, wherein the D- and/or L-amino acids may be arranged in the
JNK trafficking sequences in a blockwise, a non-blockwise or in an
alternate manner.
[0132] According to one alternative embodiment, the trafficking
sequence of the chimeric peptide as used herein may be exclusively
composed of L-amino acids. More preferably, the trafficking
sequence of the chimeric peptide as used herein comprises or
consists of at least one "native" trafficking sequence as defined
above. In this context, the term "native" is referred to
non-altered trafficking sequences, entirely composed of L-amino
acids.
[0133] According to another alternative embodiment the trafficking
sequence of the chimeric peptide as used herein may be exclusively
composed of D-amino acids. More preferably, the trafficking
sequence of the chimeric peptide as used herein may comprise a D
retro-inverso peptide of the sequences as presented above.
[0134] The trafficking sequence of the first domain of the chimeric
peptide as used herein may be obtained from naturally occurring
sources or can be produced by using genetic engineering techniques
or chemical synthesis (see e.g. Sambrook, J., Fritsch, E. F.,
Maniatis, T. (1989) Molecular cloning: A laboratory manual. 2nd
edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.).
[0135] Sources for the trafficking sequence of the first domain may
be employed including, e.g. native proteins such as e.g. the TAT
protein (e.g. as described in U.S. Pat. Nos. 5,804,604 and
5,674,980, each of these references being incorporated herein by
reference), VP22 (described in e.g. WO 97/05265; Elliott and
O'Hare, Cell 88: 223-233 (1997)), non-viral proteins (Jackson et
al, Proc. Natl. Acad. Sci. USA 89: 10691-10695 (1992)), trafficking
sequences derived from Antennapedia (e.g. the antennapedia carrier
sequence) or from basic peptides, e.g. peptides having a length of
5 to 15 amino acids, preferably 10 to 12 amino acids and comprising
at least 80%, more preferably 85% or even 90% basic amino acids,
such as e.g. arginine, lysine and/or histidine. Furthermore,
variants, fragments and derivatives of one of the native proteins
used as trafficking sequences are disclosed herewith. With regard
to variants, fragments and derivatives it is referred to the
definition given above for JNK inhibitor sequences as used herein.
Variants, fragments as well as derivatives are correspondingly
defined as set forth above for JNK inhibitor sequences as used
herein. Particularly, in the context of the trafficking sequence, a
variant or fragment or derivative may be defined as a sequence
sharing a sequence identity with one of the native proteins used as
trafficking sequences as defined above of at least about 30%, 50%,
70%, 80%, 90%, 95%, 98%, or even 99%.
[0136] In a preferred embodiment of the chimeric peptide as used
herein, the trafficking sequence of the first domain comprises or
consists of a sequence derived from the human immunodeficiency
virus (HIV)1 TAT protein, particularly some or all of the 86 amino
acids that make up the TAT protein.
[0137] For a trafficking sequence (being included in the first
domain of the chimeric peptide as used herein), partial sequences
of the full-length TAT protein may be used forming a functionally
effective fragment of a TAT protein, i.e. a TAT peptide that
includes the region that mediates entry and uptake into cells. As
to whether such a sequence is a functionally effective fragment of
the TAT protein can be determined using known techniques (see e.g.
Franked et al., Proc. Natl. Acad. Sci, USA 86: 7397-7401 (1989)).
Thus, the trafficking sequence in the first domain of the chimeric
peptide as used herein may be derived from a functionally effective
fragment or portion of a TAT protein sequence that comprises less
than 86 amino acids, and which exhibits uptake into cells, and
optionally the uptake into the cell nucleus. More preferably,
partial sequences (fragments) of TAT to be used as carrier to
mediate permeation of the chimeric peptide across the cell
membrane, are intended to comprise the basic region (amino acids 48
to 57 or 49 to 57) of full-length TAT.
[0138] According to a more preferred embodiment, the trafficking
sequence (being included in the first domain of the chimeric
peptide as used herein) may comprise or consist of an amino acid
sequence containing TAT residues 48-57 or 49 to 57, and most
preferably a generic TAT sequence
NH.sub.2--X.sub.n.sup.b-RKKRRQRRR-X.sub.n.sup.b--COOH
(L-generic-TAT (s)) [SEQ ID NO: 7] and/or XXXXRKKRRQ RRRXXXX
(L-generic-TAT) [SEQ ID NO: 21], wherein X or X.sub.n.sup.b is as
defined above. Furthermore, the number of "X.sub.n.sup.b" residues
in SEQ ID NOs :8 is not limited to the one depicted, and may vary
as described above. Alternatively, the trafficking sequence being
included in the first domain of the chimeric peptide as used herein
may comprise or consist of a peptide containing e.g. the amino acid
sequence NH.sub.2-GRKKRRQRRR-COOH (L-TAT) [SEQ ID NO: 5].
[0139] According to another more preferred embodiment the
trafficking sequence (being included in the first domain of the
chimeric peptide as used herein) may comprise a D retro-inverso
peptide of the sequences as presented above, i.e. the D
retro-inverso sequence of the generic TAT sequence having the
sequence NH.sub.2--X.sub.n.sup.b-RRRQRRKKR-X.sub.n.sup.b--COOH
(D-generic-TAT (s)) [SEQ ID NO : 8] and/or XXXXRRRQRRKKRXXXX
(D-generic-TAT) [SEQ ID NO: 22]. Also here, X.sub.n.sup.b is as
defined above (preferably representing D amino acids). Furthermore,
the number of "X.sub.n.sup.b" residues in SEQ ID NOs :8 is not
limited to the one depicted, and may vary as described above. Most
preferably, the trafficking sequence as used herein may comprise
the D retro-inverso sequence NH.sub.2-RRRQRRKKRG-COOH (D-TAT) [SEQ
ID NO: 6].
[0140] According to another embodiment the trafficking sequence
being included in the first domain of the chimeric peptide as used
herein may comprise or consist of variants of the trafficking
sequences as defined above. A "variant of a trafficking sequence"
is preferably a sequence derived from a trafficking sequence as
defined above, wherein the variant comprises a modification, for
example, addition, (internal) deletion (leading to fragments)
and/or substitution of at least one amino acid present in the
trafficking sequence as defined above. Such (a) modification(s)
typically comprise(s) 1 to 20, preferably 1 to 10 and more
preferably 1 to 5 substitutions, additions and/or deletions of
amino acids. Furthermore, the variant preferably exhibits a
sequence identity with the trafficking sequence as defined above,
more preferably with any of SEQ ID NOs: 5 to 8 or 21-22, of at
least about 30%, 50%/o, 70%, 80%, 90%, 95%, 98% or even 99%.
[0141] Preferably, such a modification of the trafficking sequence
being included in the first domain of the chimeric peptide as used
herein leads to a trafficking sequence with increased or decreased
stability. Alternatively, variants of the trafficking sequence can
be designed to modulate intracellular localization of the chimeric
peptide as used herein. When added exogenously, such variants as
defined above are typically designed such that the ability of the
trafficking sequence to enter cells is retained (i.e. the uptake of
the variant of the trafficking sequence into the cell is
substantially similar to that of the native protein used a
trafficking sequence). For example, alteration of the basic region
thought to be important for nuclear localization (see e.g. Dang and
Lee, J. Biol. Chem. 264: 18019-18023 (1989); Hauber et al., J.
Virol. 63: 1181-1187 (1989); et al., J. Virol. 63: 1-8 (1989)) can
result in a cytoplasmic location or partially cytoplasmic location
of the trafficking sequence, and therefore, of the JNK inhibitor
sequence as component of the chimeric peptide as used herein.
Additional to the above, further modifications may be introduced
into the variant, e.g. by linking e.g. cholesterol or other lipid
moieties to the trafficking sequence to produce a trafficking
sequence having increased membrane solubility. Any of the above
disclosed variants of the trafficking sequences being included in
the first domain of the chimeric peptide as used herein can be
produced using techniques typically known to a skilled person (see
e.g. Sambrook, J., Fritsch, E. F., Maniatis, T. (1989) Molecular
cloning: A laboratory manual. 2nd edition. Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.)
[0142] As a second domain the chimeric peptide as used herein
typically comprises an JNK inhibitor sequence, selected from any of
the JNK inhibitor sequences as defined above, including variants,
fragments and/or derivatives of these JNK inhibitor sequences.
[0143] Both domains, i.e. the first and the second domain(s), of
the chimeric peptide as used herein, may be linked such as to form
a functional unit. Any method for linking the first and second
domain(s) as generally known in the art may be applied.
[0144] According to one embodiment, the first and the second
domain(s) of the chimeric peptide as used herein are preferably
linked by a covalent bond. A covalent bond, as defined herein, may
be e.g. a peptide bond, which may be obtained by expressing the
chimeric peptide as defined above as a fusion protein. Fusion
proteins, as described herein, can be formed and used in ways
analogous to or readily adaptable from standard recombinant DNA
techniques, as described below. However, both domains may also be
linked via side chains or may be linked by a chemical linker
moiety.
[0145] The first and/or second domains of the chimeric peptide as
used herein may occur in one or more copies in said chimeric
peptide. If both domains are present in a single copy, the first
domain may be linked either to the N-terminal or the C-terminal end
of the second domain. If present in multiple copies, the first and
second domain(s) may be arranged in any possible order. E.g. the
first domain can be present in the chimeric peptide as used herein
in a multiple copy number, e.g. in two, three or more copies, which
are preferably arranged in consecutive order. Then, the second
domain may be present in a single copy occurring at the N- or
C-terminus of the sequence comprising the first domain.
Alternatively, the second domain may be present in a multiple copy
number, e.g. in two, three or more copies, and the first domain may
be present in a single copy. According to both alternatives, first
and second domain(s) can take any place in a consecutive
arrangement. Exemplary arrangements are shown in the following:
e.g. first domain-first domain-first domain-second domain; first
domain-first domain-second domain-first domain; first domain-second
domain-first domain-first domain; or e.g. second domain-first
domain-first domain-first domain. It is well understood for a
skilled person that these examples are for illustration purposes
only and shall not limit the scope of the invention thereto. Thus,
the number of copies and the arrangement may be varied as defined
initially.
[0146] Preferably, the first and second domain(s) may be directly
linked with each other without any linker. Alternatively, they may
be linked with each other via a linker sequence comprising 1 to 10,
preferably 1 to 5 amino acids. Amino acids forming the linker
sequence are preferably selected from glycine or proline as amino
acid residues. More preferably, the first and second domain(s) may
be separated by each other by a hinge of two, three or more proline
residues between the first and second domain(s).
[0147] The chimeric peptide as defined above and as used herein,
comprising at least one first and at least one second domain, may
be composed of L-amino acids, D-amino acids, or a combination of
both. Therein, each domain (as well as the linkers used) may be
composed of L-amino acids, D-amino acids, or a combination of both
(e.g. D-TAT and L-IB1(s) or L-TAT and D-IB1(s), etc.). Preferably,
the chimeric peptide as used herein may comprise at least 1 or even
2, preferably at least 3, 4 or 5, more preferably at least 6, 7, 8
or 9 and even more preferably at least 10 or more D- and/or L-amino
acids, wherein the D- and/or L-amino acids may be arranged in the
chimeric peptide as used herein in a blockwise, a non-blockwise or
in an alternate manner.
[0148] According to a specific embodiment the chimeric peptide as
used herein comprises or consists of the L-amino acid chimeric
peptides according to the generic L-TAT-IB peptide
NH.sub.2--X.sub.n.sup.b-RKKRRQRRR-X.sub.n.sup.b--X.sub.n.sup.a-RPTTLXLXXX-
XXXXQD-X.sub.n.sup.b--COOH (L-TAT-IB (generic) (s)) [SEQ ID NO:
10], wherein X, X.sub.n.sup.a and X.sub.n.sup.b are preferably as
defined above. More preferably, the chimeric peptide as used herein
comprises or consists of the L-amino acid chimeric peptide
NH.sub.2-GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQD-COOH (L-TAT-IB1 (s)) [SEQ
ID NO: 9]. Alternatively or additionally, the chimeric peptide as
used herein comprises or consists of the L-amino acid chimeric
peptide sequence GRKKRRQRRR PPDTYRPKRP TTLNLFPQVP RSQDT (L-TAT-IB1)
[SEQ ID NO: 23], or XXXXXXXRKK RRQRRRXXXX XXXXRPTTLX LXXXXXXXQD
S/TX (L-TAT-IB generic) [SEQ ID NO: 24], wherein X is preferably
also as defined above, or the chimeric peptide as used herein
comprises or consists of the L-amino acid chimeric peptide sequence
RKKRRQRRRPPRPKRPTTLNLFPQVPRSQD (L-TAT-IB1(s1)) [SEQ ID NO: 27],
GRKKRRQRRRX.sub.n.sup.cRPKRPTTLNLFPQVPRSQD (L-TAT-IB1(s2)) [SEQ ID
NO: 28], or RKKRRQRRRX.sub.n.sup.cRPKRPTTLNLFPQVPRSQD
(L-TAT-IB1(s3)) [SEQ ID NO: 29]. In this context, each X typically
represents an amino acid residue as defined above, more preferably
X.sub.n.sup.c represents a contiguous stretch of peptide residues,
each X independently selected from each other from glycine or
proline, e.g. a monotonic glycine stretch or a monotonic proline
stretch, wherein n (the number of repetitions of X.sub.n.sup.c) is
typically 0-5, 5-10, 10-15, 15-20, 20-30 or even more, preferably
0-5 or 5-10. X.sub.n.sup.c may represent either D or L amino
acids.
[0149] According to an alternative specific embodiment the chimeric
peptide as used herein comprises or consists of D-amino acid
chimeric peptides of the above disclosed L-amino acid chimeric
peptides. Exemplary D retro-inverso chimeric peptides according to
the present invention are e.g. the generic D-TAT-IB peptide
NH.sub.2--X.sub.n.sup.b-DQXXXXXXXLXLTTPR-X.sub.n.sup.a--X.sub.n.sup.b-RRR-
QRRKKR-X.sub.n.sup.b--COOH (D-TAT-IB (generic) (s)) [SEQ ID NO:
12]. Herein, X, X.sub.n.sup.a and X.sub.n.sup.b are preferably as
defined above (preferably representing D amino acids). More
preferably, the chimeric peptide as used herein comprises or
consists of D-amino acid chimeric peptides according to the TAT-IB1
peptide NH.sub.2-DQSRPVQPFLNLTTPRKPRPPRRRQRRKKRG-COOH (D-TAT-IB1
(s)) [SEQ ID NO: 11]. Alternatively or additionally, the chimeric
peptide as used herein comprises or consists of the D-amino acid
chimeric peptide sequence TDQSRPVQPFLNLTTPRKPRYTDPPRRRQRRKKRG
(D-TAT-IB1) [SEQ ID NO: 25], or
XT/SDQXXXXXXXLXLTTPRXXXXXXXXRRRQRRKKRXXXXXXX (D-TAT-IB generic)
[SEQ ID NO: 26], wherein X is preferably also as defined above, or
the chimeric peptide as used herein comprises or consists of the
D-amino acid chimeric peptide sequence
DQSRPVQPFLNLTTPRKPRPPRRRQRRKKR (D-TAT-IB1(s1)) [SEQ ID NO: 30],
DQSRPVQPFLNLTTPRKPRX.sub.n.sup.cRRRQRRKKRG (D-TAT-IB1(s2)) [SEQ ID
NO: 31], or DQSRPVQPFLNLTTPRKPRX.sub.n.sup.cRRRQRRKKR
(D-TAT-IB1(s3)) [SEQ ID NO: 32]. X.sub.n.sup.c may be as defined
above.
[0150] The first and second domain(s) of the chimeric peptide as
defined above may be linked to each other by chemical or
biochemical coupling carried out in any suitable manner known in
the art, e.g. by establishing a peptide bond between the first and
the second domain(s) e.g. by expressing the first and second
domain(s) as a fusion protein, or e.g. by crosslinking the first
and second domain(s) of the chimeric peptide as defined above.
[0151] Many known methods suitable for chemical crosslinking of the
first and second domain(s) of the chimeric peptide as defined above
are non-specific, i.e. they do not direct the point of coupling to
any particular site on the transport polypeptide or cargo
macromolecule. As a result, use of non-specific crosslinking agents
may attack functional sites or sterically block active sites,
rendering the conjugated proteins biologically inactive. Thus,
preferably such crosslinking methods are used, which allow a more
specific coupling of the first and second domain(s).
[0152] In this context, one way to increasing coupling specificity
is a direct chemical coupling to a functional group present only
once or a few times in one or both of the first and second
domain(s) to be crosslinked. For example, cysteine, which is the
only protein amino acid containing a thiol group, occurs in many
proteins only a few times. Also, for example, if a polypeptide
contains no lysine residues, a crosslinking reagent specific for
primary amines will be selective for the amino terminus of that
polypeptide. Successful utilization of this approach to increase
coupling specificity requires that the polypeptide have the
suitably rare and reactive residues in areas of the molecule that
may be altered without loss of the molecule's biological activity.
Cysteine residues may be replaced when they occur in parts of a
polypeptide sequence where their participation in a crosslinking
reaction would otherwise likely interfere with biological activity.
When a cysteine residue is replaced, it is typically desirable to
minimize resulting changes in polypeptide folding. Changes in
polypeptide folding are minimized when the replacement is
chemically and sterically similar to cysteine. For these reasons,
serine is preferred as a replacement for cysteine. As demonstrated
in the examples below, a cysteine residue may be introduced into a
polypeptide's amino acid sequence for crosslinking purposes. When a
cysteine residue is introduced, introduction at or near the amino
or carboxy terminus is preferred. Conventional methods are
available for such amino acid sequence modifications, wherein the
polypeptide of interest is produced by chemical synthesis or via
expression of recombinant DNA.
[0153] Coupling of the first and second domain(s) of the chimeric
peptide as defined above and used herein can also be accomplished
via a coupling or conjugating agent. There are several
intermolecular crosslinking reagents which can be utilized (see for
example, Means and Feeney, CHEMICAL MODIFICATION OF PROTEINS,
Holden-Day, 1974, pp. 39-43). Among these reagents are, for
example, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or
N,N'-(1,3-phenylene) bismaleimide (both of which are highly
specific for sulfhydryl groups and form irreversible linkages);
N,N'-ethylene-bis-(iodoacetamide) or other such reagent having 6 to
11 carbon methylene bridges (which are relatively specific for
sulfhydryl groups); and 1,5-difluoro-2,4-dinitrobenzene (which
forms irreversible linkages with amino and tyrosine groups). Other
crosslinking reagents useful for this purpose include:
p,p'-difluoro-m, m'-dinitrodiphenylsulfone which forms irreversible
crosslinkages with amino and phenolic groups); dimethyl adipimidate
(which is specific for amino groups); phenol-1,4 disulfonylchloride
(which reacts principally with amino groups);
hexamethylenediisocyanate or diisothiocyanate, or
azophenyl-p-diisocyanate (which reacts principally with amino
groups); glutaraldehyde (which reacts with several different side
chains) and disdiazobenzidine (which reacts primarily with tyrosine
and histidine).
[0154] Crosslinking reagents used for crosslinking the first and
second domain(s) of the chimeric peptide as defined above may be
homobifunctional, i.e. having two functional groups that undergo
the same reaction. A preferred homobifunctional crosslinking
reagent is bismaleimidohexane ("BMH"). BMH contains two maleimide
functional groups, which react specifically with
sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7).
The two maleimide groups are connected by a hydrocarbon chain.
Therefore, BMH is useful for irreversible crosslinking of
polypeptides that contain cysteine residues.
[0155] Crosslinking reagents used for crosslinking the first and
second domain(s) of the chimeric peptide as defined above may also
be heterobifunctional. Heterobifunctional crosslinking agents have
two different functional groups, for example an amine-reactive
group and a thiol-reactive group, that will crosslink two proteins
having free amines and thiols, respectively. Examples of
heterobifunctional crosslinking agents are succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC"),
m-maleimidobenzoyl-N-hydroxysuccinimide ester ("MBS"), and
succinimide 4-(p-maleimidophenyl)butyrate ("SMPB"), an extended
chain analog of MBS. The succinimidyl group of these crosslinkers
reacts with a primary amine, and the thiol-reactive maleimide forms
a covalent bond with the thiol of a cysteine residue.
[0156] Crosslinking reagents suitable for crosslinking the first
and second domain(s) of the chimeric peptide as defined above often
have low solubility in water. A hydrophilic moiety, such as a
sulfonate group, may thus be added to the crosslinking reagent to
improve its water solubility. In this respect, Sulfo-MBS and
Sulfo-SMCC are examples of crosslinking reagents modified for water
solubility, which may be used according to the present
invention.
[0157] Likewise, many crosslinking reagents yield a conjugate that
is essentially non-cleavable under cellular conditions. However,
some crosslinking reagents particularly suitable for crosslinking
the first and second domain(s) of the chimeric peptide as defined
above contain a covalent bond, such as a disulfide, that is
cleavable under cellular conditions. For example, Traut's reagent,
dithiobis(succinimidylpropionate) ("DSP"), and N-succinimidyl
3-(2-pyridyldithio)propionate ("SPDP") are well-known cleavable
crosslinkers. The use of a cleavable crosslinking reagent permits
the cargo moiety to separate from the transport polypeptide after
delivery into the target cell. Direct disulfide linkage may also be
useful.
[0158] Numerous crosslinking reagents, including the ones discussed
above, are commercially available. Detailed instructions for their
use are readily available from the commercial suppliers. A general
reference on protein crosslinking and conjugate preparation is:
Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSSLINKING, CRC Press
(1991).
[0159] Chemical crosslinking of the first and second domain(s) of
the chimeric peptide as defined above may include the use of spacer
arms. Spacer arms provide intramolecular flexibility or adjust
intramolecular distances between conjugated moieties and thereby
may help preserve biological activity. A spacer arm may be in the
form of a polypeptide moiety that includes spacer amino acids, e.g.
proline. Alternatively, a spacer arm may be part of the
crosslinking reagent, such as in "long-chain SPDP" (Pierce Chem.
Co., Rockford, Ill., cat. No. 21651 H).
[0160] Preferably, any of the peptides disclosed herein, in
particular the JNK inhibitor, the trafficking sequence and the
chimeric peptide as disclosed herein, preferably the JNK inhibitor
according to SEQ ID NO: 11, may have a modification at one or both
of their termini, i.e. either at the C- or at the N-terminus or at
both. The C-Terminus may preferably be modified by an amide
modification, whereas the N-terminus may be modified by any
suitable NH2-protection group, such as e.g. acylation. More
preferably, the JNK inhibitor and the chimeric peptide as disclosed
herein, preferably the JNK inhibitor according to SEQ ID NO: 11, is
modified by an amide modification at the C-terminus.
[0161] It is also preferred that any of the peptides disclosed
herein, in particular the JNK inhibitor, the trafficking sequence
(e.g. of the chimeric peptide) and the chimeric peptide as
disclosed herein, preferably the JNK inhibitor according to SEQ ID
NO: 11, may be deleted at their N- and/or C-terminus by 1, 2 or 3
amino acids. For example, in a chimeric peptide according to the
present invention each domain, i.e. the JNK-inhibitor and the
trafficking sequence domain, may be deleted at their N- and/or
C-terminus by 1, 2 or 3 amino acids and/or the chimeric peptide
according to the present invention may be deleted at its N- and/or
C-terminus by 1, 2 or 3 amino acids. More preferably, the inventive
chimeric peptide comprises or consists of a D-amino acid chimeric
peptide according to the TAT-IB1 peptide
[NH.sub.2-DQSRPVQPFLNLTTPRKPRPPRRRQRRKKRG-COOH, SEQ ID NO: 11] and
the linking portion of the first and second domain (instead of PP)
may be composed of --X.sub.n.sup.a--X.sub.n.sup.b--, which are as
defined above. In particular, the second domain(s) of SEQ ID NO:
11, eventually with --X.sub.n.sup.a--X.sub.n.sup.b-- instead of
(PP), may be deleted at their N- and/or C-terminus by 1, 2 or 3
amino acids. In another preferred embodiment, the first domain of
SEQ ID NO: 11 may be deleted at its N- and or C-terminus by 1, 2 or
3 amino acids. This/these deletion/s may be combined with the
deletion/s disclosed for the amino acid residues of the termini of
the second domain. Again, the shorter the peptides are, the less
their (unspecific) cell toxicity. However, the peptides must retain
their biological function, i.e. their cell membrane permeability
(first domain) and their JNK inhibitory function (second
domain).
[0162] Furthermore, variants, fragments or derivatives of one of
the above disclosed chimeric peptides may be used herein. With
regard to fragments and variants it is generally referred to the
definition given above for JNK inhibitor sequences.
[0163] Particularly, in the context of the present invention, a
"variant of a chimeric peptide" is preferably a sequence derived
from any of the sequences according to SEQ ID NOs: 9 to 12 and 23
to 32, wherein the chimeric variant comprises amino acid
alterations of the chimeric peptides according to SEQ ID NOs: 9 to
12 and 23 to 32 as used herein. Such alterations typically comprise
1 to 20, preferably 1 to 10 and more preferably 1 to 5
substitutions, additions and/or deletions (leading to fragments) of
amino acids according to SEQ ID NOs: 9 to 12 and 23 to 32, wherein
the altered chimeric peptide as used herein exhibits a sequence
identity with any of the sequences according to SEQ ID NOs: 9-12
and 23 to 32 of at least about 30%, 50%, 70%, 80%, or 95%, 98%, or
even 99%. Preferably, these variants retain the biological activity
of the first and the second domain as contained in the chimeric
peptide as used herein, i.e. the trafficking activity of the first
domain as disclosed above and the activity of the second domain for
binding JNK and/or inhibiting the activation of at least one JNK
activated transcription factor.
[0164] Accordingly, the chimeric peptide as used herein also
comprises fragments of the afore disclosed chimeric peptides,
particularly of the chimeric peptide sequences according to any of
SEQ ID NOs: 9 to 12 and 23 to 32. Thus, in the context of the
present invention, a "fragment of the chimeric peptide" is
preferably a sequence derived any of the sequences according to SEQ
ID NOs: 9 to 12 and 23 to 32, wherein the fragment comprises at
least 4 contiguous amino acids of any of SEQ ID NOs: 9 to 12 and 23
to 32. This fragment preferably comprises a length which is
sufficient to allow specific recognition of an epitope from any of
these sequences and to transport the sequence into the cells, the
nucleus or a further preferred location. Even more preferably, the
fragment comprises 4 to 18, 4 to 15, or most preferably 4 to 10
contiguous amino acids of any of SEQ ID NOs: 9 to 12 and 23 to 32.
Fragments of the chimeric peptide as used herein further may be
defined as a sequence sharing a sequence identity with any of the
sequences according to any of SEQ ID NOs: 9 to 12 and 23 to 32 of
at least about 30%, 50%, 70%, 80%, or 95%, 98%, or even 99%.
[0165] Finally, the chimeric peptide as used herein also comprises
derivatives of the afore disclosed chimeric peptides, particularly
of the chimeric peptide sequences according to any of SEQ ID NOs: 9
to 12 and 23 to 32.
[0166] The present invention additionally refers to the use of
nucleic acid sequences encoding JNK inhibitor sequences as defined
above, chimeric peptides or their fragments, variants or
derivatives, all as defined above, for the preparation of a
pharmaceutical composition for treating diseases or disorders
strongly related to JNK signaling as defined above in a subject. A
preferable suitable nucleic acid encoding an JNK inhibitor sequence
as used herein is typically chosen from human IB1 nucleic acid
(GenBank Accession No. (AF074091), rat IB1 nucleic acid (GenBank
Accession No. AF 108959), or human IB2 (GenBank Accession No
AF218778) or from any nucleic acid sequence encoding any of the
sequences as defined above, i.e. any sequence according to SEQ ID
NO: 1-26.
[0167] Nucleic acids encoding the JNK inhibitor sequences as used
herein or chimeric peptides as used herein may be obtained by any
method known in the art (e.g. by PCR amplification using synthetic
primers hybridizable to the 3'- and 5'-termini of the sequence
and/or by cloning from a cDNA or genomic library using an
oligonucleotide sequence specific for the given gene sequence).
[0168] Additionally, nucleic acid sequences are disclosed herein as
well, which hybridize under stringent conditions with the
appropriate strand coding for a (native) JNK inhibitor sequence or
chimeric peptide as defined above. Preferably, such nucleic acid
sequences comprise at least 6 (contiguous) nucleic acids, which
have a length sufficient to allow for specific hybridization. More
preferably, such nucleic acid sequences comprise 6 to 38, even more
preferably 6 to 30, and most preferably 6 to 20 or 6 to 10
(contiguous) nucleic acids.
[0169] "Stringent conditions" are sequence dependent and will be
different under different circumstances. Generally, stringent
conditions can be selected to be about 5.degree. C. lower than the
thermal melting point (TM) for the specific sequence at a defined
ionic strength and pH. The TM is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Typically, stringent
conditions will be those in which the salt concentration is at
least about 0.02 molar at pH 7 and the temperature is at least
about 60.degree. C. As other factors may affect the stringency of
hybridization (including, among others, base composition and size
of the complementary strands), the presence of organic solvents and
the extent of base mismatching, the combination of parameters is
more important than the absolute measure of any one.
[0170] "High stringency conditions" may comprise the following,
e.g. Step 1: Filters containing DNA are pretreated for 8 hours to
overnight at 65.degree. C. in buffer composed of 6*SSC, 50 mM
Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA,
and 500 .mu.g/ml denatured salmon sperm DNA. Step 2: Filters are
hybridized for 48 hours at 65.degree. C. in the above
prehybridization mixture to which is added 100 mg/ml denatured
salmon sperm DNA and 5-20*10.sup.6 cpm of .sup.32P-labeled probe.
Step 3: Filters are washed for 1 hour at 37.degree. C. in a
solution containing 2*SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA.
This is followed by a wash in 0.1*SSC at 50.degree. C. for 45
minutes. Step 4: Filters are autoradiographed. Other conditions of
high stringency that may be used are well known in the art (see
e.g. Ausubel et at, (eds.), 1993, Current Protocols in Molecular
Biology, John Wiley and Sons, NY; and Kriegler, 1990, Gene Transfer
and Expression, a Laboratory Manual, Stockton Press, NY).
[0171] "Moderate stringency conditions" can include the following:
Step 1: Filters containing DNA are pretreated for 6 hours at
55.degree. C. in a solution containing 6*SSC, 5*Denhardt's
solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA. Step
2: Filters are hybridized for 18-20 hours at 55.degree. C. in the
same solution with 5-20*10.sup.6 cpm .sup.32P-labeled probe added.
Step 3: Filters are washed at 37.degree. C. for 1 hour in a
solution containing 2*SSC, 0.1% SDS, then washed twice for 30
minutes at 60.degree. C. in a solution containing 1*SSC and 0.1%
SDS. Step 4: Filters are blotted dry and exposed for
autoradiography. Other conditions of moderate stringency that may
be used are well-known in the art (see e.g. Ausubel et al., (eds.),
1993, Current Protocols in Molecular Biology, John Wiley and Sons,
NY; and Kriegler, 1990, Gene Transfer and Expression, a Laboratory
Manual, Stockton Press, NY).
[0172] Finally, "low stringency conditions" can include: Step 1:
Filters containing DNA are pretreated for 6 hours at 40.degree. C.
in a solution containing 35% formamide, 5.times.SSC, 50 mM Tris-HCl
(pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500
.mu.g/ml denatured salmon sperm DNA. Step 2: Filters are hybridized
for 18-20 hours at 40.degree. C. in the same solution with the
addition of 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 .mu.g/ml salmon
sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20.times.106 cpm
.sup.32P-labeled probe. Step 3: Filters are washed for 1.5 hours at
55 C in a solution containing 2.times.SSC, 25 mM Tris-HCl (pH 7.4),
5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh
solution and incubated an additional 1.5 hours at 60.degree. C.
Step 4: Filters are blotted dry and exposed for autoradiography. If
necessary, filters are washed for a third time at 65-68.degree. C.
and reexposed to film. Other conditions of low stringency that may
be used are well known in the art (e.g. as employed for
cross-species hybridizations). See e.g. Ausubel et al., (eds.),
1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons,
NY; and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY
MANUAL, Stockton Press, NY.
[0173] The nucleic acid sequences as defined above according to the
present invention can be used to express peptides, i.e. an JNK
inhibitor sequence as used herein or an chimeric peptide as used
herein for analysis, characterization or therapeutic use; as
markers for tissues in which the corresponding peptides (as used
herein) are preferentially expressed (either constitutively or at a
particular stage of tissue differentiation or development or in
disease states). Other uses for these nucleic acids include, e.g.
molecular weight markers in gel electrophoresis-based analysis of
nucleic acids.
[0174] According to a further embodiment of the present invention,
expression vectors may be used for the above purposes for
recombinant expression of one or more JNK inhibitor sequences
and/or chimeric peptides as defined above. The term "expression
vector" is used herein to designate either circular or linear DNA
or RNA, which is either double-stranded or single-stranded. It
further comprises at least one nucleic acid as defined above to be
transferred into a host cell or into a unicellular or multicellular
host organism. The expression vector as used herein preferably
comprises a nucleic acid as defined above encoding the JNK
inhibitor sequence as used herein or a fragment or a variant
thereof, or the chimeric peptide as used herein, or a fragment or a
variant thereof. Additionally, an expression vector according to
the present invention preferably comprises appropriate elements for
supporting expression including various regulatory elements, such
as enhancers/promoters from viral, bacterial, plant, mammalian, and
other eukaryotic sources that drive expression of the inserted
polynucleotide in host cells, such as insulators, boundary
elements, LCRs (e.g. described by Blackwood and Kadonaga (1998),
Science 281, 61-63) or matrix/scaffold attachment regions (e.g.
described by Li, Harju and Peterson, (1999), Trends Genet. 15,
403-408). In some embodiments, the regulatory elements are
heterologous (i.e. not the native gene promoter). Alternately, the
necessary transcriptional and translational signals may also be
supplied by the native promoter for the genes and/or their flanking
regions.
[0175] The term "promoter" as used herein refers to a region of DNA
that functions to control the transcription of one or more nucleic
acid sequences as defined above, and that is structurally
identified by the presence of a binding site for DNA-dependent
RNA-polymerase and of other DNA sequences, which interact to
regulate promoter function. A functional expression promoting
fragment of a promoter is a shortened or truncated promoter
sequence retaining the activity as a promoter. Promoter activity
may be measured by any assay known in the art (see e.g. Wood, de
Wet, Dewji, and DeLuca, (1984), Biochem Biophys. Res. Commun. 124,
592-596; Seliger and McElroy, (1960), Arch. Biochem. Biophys. 88,
136-141) or commercially available from Promega.RTM.).
[0176] An "enhancer region" to be used in the expression vector as
defined herein, typically refers to a region of DNA that functions
to increase the transcription of one or more genes. More
specifically, the term "enhancer", as used herein, is a DNA
regulatory element that enhances, augments, improves, or
ameliorates expression of a gene irrespective of its location and
orientation vis-a-vis the gene to be expressed, and may be
enhancing, augmenting, improving, or ameliorating expression of
more than one promoter.
[0177] The promoter/enhancer sequences to be used in the expression
vector as defined herein, may utilize plant, animal, insect, or
fungus regulatory sequences. For example, promoter/enhancer
elements can be used from yeast and other fungi (e.g. the GAL4
promoter, the alcohol dehydrogenase promoter, the phosphoglycerol
kinase promoter, the alkaline phosphatase promoter). Alternatively,
or in addition, they may include animal transcriptional control
regions, e.g. (i) the insulin gene control region active within
pancreatic beta-cells (see e.g. Hanahan, et al., 1985. Nature 315:
115-122); (ii) the immunoglobulin gene control region active within
lymphoid cells (see e.g. Grosschedl, et al., 1984, Cell 38:
647-658); (iii) the albumin gene control region active within liver
(see e.g. Pinckert, et al., 1987. Genes and Dev 1: 268-276; (iv)
the myelin basic protein gene control region active within brain
oligodendrocyte cells (see e.g. Readhead, et al., 1987, Cell 48:
703-712); and (v) the gonadotropin-releasing hormone gene control
region active within the hypothalamus (see e.g. Mason, et al.,
1986, Science 234: 1372-1378), and the like.
[0178] Additionally, the expression vector as defined herein may
comprise an amplification marker. This amplification marker may be
selected from the group consisting of, e.g. adenosine deaminase
(ADA), dihydrofolate reductase (DHFR), multiple drug resistance
gene (MDR), ornithine decarboxylase (ODC) and
N-(phosphonacetyl)-L-aspartate resistance (CAD).
[0179] Exemplary expression vectors or their derivatives suitable
for the present invention particularly include, e.g. human or
animal viruses (e.g. vaccinia virus or adenovirus); insect viruses
(e.g. baculovirus); yeast vectors; bacteriophage vectors (e.g.
lambda phage); plasmid vectors and cosmid vectors.
[0180] The present invention additionally may utilize a variety of
host-vector systems, which are capable of expressing the peptide
coding sequence(s) of nucleic acids as defined above. These
include, but are not limited to: (i) mammalian cell systems that
are infected with vaccinia virus, adenovirus, and the like; (ii)
insect cell systems infected with baculovirus and the like; (iii)
yeast containing yeast vectors or (iv) bacteria transformed with
bacteriophage, DNA, plasmid DNA, or cosmid DNA. Depending upon the
host-vector system utilized, any one of a number of suitable
transcription and translation elements may be used.
[0181] Preferably, a host cell strain, suitable for such a
host-vector system, may be selected that modulates the expression
of inserted sequences of interest, or modifies or processes
expressed peptides encoded by the sequences in the specific manner
desired. In addition, expression from certain promoters may be
enhanced in the presence of certain inducers in a selected host
strain; thus facilitating control of the expression of a
genetically-engineered peptide. Moreover, different host cells
possess characteristic and specific mechanisms for the
translational and post-translational processing and modification
(e.g. glycosylation, phosphorylation, and the like) of expressed
peptides. Appropriate cell lines or host systems may thus be chosen
to ensure the desired modification and processing of the foreign
peptide is achieved. For example, peptide expression within a
bacterial system can be used to produce an non-glycosylated core
peptide; whereas expression within mammalian cells ensures "native"
glycosylation of a heterologous peptide.
[0182] The present invention further provides the use of antibodies
directed against the JNK inhibitor sequences and/or chimeric
peptides as described above, for preparing a pharmaceutical
composition for the treatment of diseases or disorders strongly
related to JNK signaling as defined herein. Furthermore, efficient
means for production of antibodies specific for JNK inhibitor
sequences according to the present invention, or for chimeric
peptides containing such an inhibitor sequence, are described and
may be utilized for this purpose.
[0183] According to the invention, JNK inhibitor sequences and/or
chimeric peptides as defined herein, as well as, fragments,
variants or derivatives thereof, may be utilized as immunogens to
generate antibodies that immunospecifically bind these peptide
components. Such antibodies include, e.g. polyclonal, monoclonal,
chimeric, single chain, Fab fragments and a Fab expression library.
In a specific embodiment the present invention provides antibodies
to chimeric peptides or to JNK inhibitor sequences as defined
above. Various procedures known within the art may be used for the
production of these antibodies.
[0184] By way of example, various host animals may be immunized for
production of polyclonal antibodies by injection with any chimeric
peptide or JNK inhibitor sequence as defined above. Various
adjuvants may be used thereby to increase the immunological
response which include, but are not limited to, Freund's (complete
and incomplete) adjuvant, mineral gels (e.g. aluminum hydroxide),
surface active substances (e.g. lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, dinitrophenol, etc.), CpG,
polymers, Pluronics, and human adjuvants such as Bacille
Calmette-Guerin and Corynebacterium parvum.
[0185] For preparation of monoclonal antibodies directed towards an
chimeric peptide or a JNK inhibitor sequence as defined above, any
technique may be utilized that provides for the production of
antibody molecules by continuous cell line culture. Such techniques
include, but are not limited to, the hybridoma technique (see
Kohler and Milstein, 1975. Nature 256: 495-497); the trioma
technique; the human B-cell hybridoma technique (see Kozbor, et
al., 1983, Immunol Today 4: 72) and the EBV hybridoma technique to
produce human monoclonal antibodies (see Cole, et al., 1985. In:
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96). Human monoclonal antibodies may be utilized in the practice
of the present invention and may be produced by the use of human
hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:
2026-2030) or by transforming human B-cells with Epstein Barr Virus
in vitro (see Cole, et al., 1985. In: Monoclonal Antibodies and
Cancer Therapy (Alan R. Liss, Inc., pp. 77-96).
[0186] According to the invention, techniques can be adapted for
the production of single-chain antibodies specific to the JNK
inhibitor sequences and/or chimeric peptides (see e.g. U.S. Pat.
No. 4,946,778) as defined herein. In addition, methods can be
adapted for the construction of Fab expression libraries (see e.g.
Huse et al., 1989. Science 246: 1275-1281) to allow rapid and
effective identification of monoclonal Fab fragments with the
desired specificity for these JNK inhibitor sequences and/or
chimeric peptides. Non-human antibodies can be "humanized" by
techniques well known in the art (see e.g. U.S. Pat. No.
5,225,539). Antibody fragments that contain the idiotypes to a JNK
inhibitor sequences and/or chimeric peptide as defined herein may
be produced by techniques known in the art including, e.g. (i) a
F(ab').sub.2 fragment produced by pepsin digestion of an antibody
molecule; (ii) a Fab fragment generated by reducing the disulfide
bridges of an F(ab').sub.2 fragment; (iii) a Fab fragment generated
by the treatment of the antibody molecule with papain and a
reducing agent and (iv) Fv fragments.
[0187] In one embodiment of this invention, methods, that may be
utilized for the screening of antibodies and which possess the
desired specificity include, but are not limited to, enzyme-linked
immunosorbent assay (ELISA) and other immunologically-mediated
techniques known within the art. In a specific embodiment,
selection of antibodies that are specific to a particular epitope
of an JNK inhibitor sequence and/or an chimeric peptide as defined
herein (e.g. a fragment thereof typically comprising a length of
from 5 to 20, preferably 8 to 18 and most preferably 8 to 11 amino
acids) is facilitated by generation of hybridomas that bind to the
fragment of an JNK inhibitor sequence and/or an chimeric peptide,
as defined herein, possessing such an epitope. These antibodies
that are specific for an epitope as defined above are also provided
herein.
[0188] The antibodies as defined herein may be used in methods
known within the art referring to the localization and/or
quantification of an JNK inhibitor sequence (and/or correspondingly
to a chimeric peptide as defined above), e.g. for use in measuring
levels of the peptide within appropriate physiological samples, for
use in diagnostic methods, or for use in imaging the peptide, and
the like.
[0189] The JNK inhibitor sequences, chimeric peptides, nucleic
acids, vectors, host cells and/or antibodies as defined according
to the invention can be formulated in a pharmaceutical composition,
which may be applied in the prevention or treatment of any of the
diseases as defined herein, particularly in the prevention or
treatment of diseases or disorders strongly related to JNK
signaling as defined herein. Typically, such a pharmaceutical
composition used according to the present invention includes as an
active component, e.g.: (i) any one or more of the JNK inhibitor
sequences and/or chimeric peptides as defined above, and/or
variants, fragments or derivatives thereof, particularly JNK
inhibitor sequences according to any of sequences of SEQ ID NOs: 1
to 4 and 13 to 20 and 33-100 and/or chimeric peptides according to
any of sequences of SEQ ID NOs: 9 to 12 and 23 to 32, and/or JNK
inhibitor sequences according to any of sequences of SEQ ID NOs: 1
to 4 and 13 to 20 and 33-100 comprising a trafficking sequence
according to any of SEQ ID NOs: 5 to 8 and 21 to 22, or variants or
fragments thereof within the above definitions; and/or (ii) nucleic
acids encoding an JNK inhibitor sequence and/or an chimeric peptide
as defined above and/or variants or fragments thereof, and/or (iii)
cells comprising any one or more of the JNK inhibitor sequences
and/or chimeric peptides, and/or variants, fragments or derivatives
thereof, as defined above and/or (iv) cells transfected with a
vector and/or nucleic acids encoding an JNK inhibitor sequence
and/or an chimeric peptide as defined above and/or variants or
fragments thereof.
[0190] According to a preferred embodiment, such a pharmaceutical
composition as used according to the present invention typically
comprises a safe and effective amount of a component as defined
above, preferably of at least one JNK inhibitor sequence according
to any of sequences of SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100
and/or at least one chimeric peptide according to any of sequences
of SEQ ID NOs: 9 to 12 and 23 to 32, and/or at least one JNK
inhibitor sequence according to any of sequences of SEQ ID NOs: 1
to 4 and 13 to 20 and 33-100 comprising a trafficking sequence
according to any of SEQ ID NOs: 5-8 and 21 to 22, or variants or
fragments thereof within the above definitions, or at least one
nucleic acids encoding same, or at least one vector, host cell or
antibody as defined above. It is particulary preferred that a
pharmaceutical composition as used according to the present
invention comprises as an active component a chimeric peptide
comprising or consisting of the sequence according to SEQ ID NO:
11.
[0191] In addition, the pharmaceutical composition as used
according to the present invention may additionally--i.e. in
addition to any one or more of the JNK inhibitor sequences and/or
chimeric peptides as defined above, and/or variants, fragments or
derivatives thereof--also comprise optionally a further "active
component", which is also useful in the respective disease. In this
context, the pharmaceutical composition according to the present
invention may also combined in the therapy of the diseases
according to the present invention with a further pharmaceutical
composition comprising a further "active component". For example, a
pharmaceutical composition comprising a JNK inhibitor and/or
chimeric peptide according to the present invention may be used in
post-surgery intraocular inflammation as stand-alone therapy or in
combination with corticosteroids, preferably glucocorticoids, e.g.
dexamethasone. Moreover, e.g. a pharmaceutical composition
comprising a JNK inhibitor and/or chimeric peptide according to the
present invention may preferably be used in the prevention and/or
treatment of Alzheimer's Disease and/or Mild Cognitive Impairment,
in particular MCI due to Alzheimer's disease, as stand-alone
therapy or in combination with PKR inhibitors and, optionally, in
addition to the JNK inhibitor according to the present invention
and the PKR inhibitor with a amyloid lowering agent. PKR inhibitors
are in particular peptides, e.g. "SC1481" by Polypeptide Group.
Amyloid lowering agents include .beta.-secretase (BACE1)
inhibitors, .gamma.-secretase inhibitors (GSI) and modulators
(GSM). Examples of such amyloid lowering agents, which are
currently in clinical trials may be retrieved from Vassar R. (2014)
BACE1 inhibitor drugs in clinical trials for Alzheimer's disease.
Alzheimers Res Ther.; 6(9):89 or from Jia Q, Deng Y, Qing H (2014)
Potential therapeutic strategies for Alzheimer's disease targeting
or beyond .beta.-amyloid: insights from clinical trials. Biomed Res
Int. 2014; 2014:837157; for example Pioglitazone, CTS-21166,
MK8931, LY2886721, AZD3293, E2609, NIC5-15, Begacestat, CHF 5074,
EVP-0962, Atorvastatin, Simvastatin, Etazolate,
Epigallocatechin-3-gallate (EGCg), Scyllo-inositol
(ELND005/AZD103), Tramiprosate (3 APS), PBT2, Affitope AD02, and
Affitope AD03. In the case of a combination therapy, separate
pharmaceutical compositions for the active components to be
combined are preferred for better individual dosing, however for
convenience also a single pharmaceutical composition comprising the
active components to be combined is conceivable. In the case of
separate pharmaceutical compositions for the active components to
be combined the administration of the JNK inhibitor according to
the present invention may be before, during (concomitant or
overlapping administration) or after the administration of the
other active component comprised in a separate pharmaceutical
composition, for example the PKR inhibitor, the amyloid lowering
agent or the glucocorticoid. Administration "before" the
administration of the JNK inhibitor preferably means within 24 h,
more preferably within 12 h, even more preferably within 3 h,
particularly preferably within 1 h and most preferably within 30
min before the administration of the JNK inhibitor starts.
Administration "after" the administration of the JNK inhibitor
preferably means within 24 h, more preferably within 12 h, even
more preferably within 3 h, particularly preferably within 1 h and
most preferably within 30 min after the administration of the JNK
inhibitor is finished.
[0192] The inventors of the present invention additionally found,
that the JNK-inhibitor sequence and the chimeric peptide,
respectively, as defined herein, exhibit a particular well uptake
rate into cells involved in the diseases of the present invention.
Therefore, the amount of a JNK-inhibitor sequence and chimeric
peptide, respectively, in the pharmaceutical composition to be
administered to a subject, may--without being limited thereto--have
a very low dose. Thus, the dose may be much lower than for peptide
drugs known in the art, such as DTS-108 (Florence Meyer-Losic et
al., Clin Cancer Res., 2008, 2145-53). This has several positive
aspects, for example a reduction of potential side reactions and a
reduction in costs.
[0193] Preferably, the dose (per kg bodyweight) is in the range of
up to 10 mmol/kg, preferably up to 1 mmol/kg, more preferably up to
100 .mu.mol/kg, even more preferably up to 10 .mu.mol/kg, even more
preferably up to 1 .mu.mol/kg, even more preferably up to 100
nmol/kg, most preferably up to 50 nmol/kg.
[0194] Thus, the dose range may preferably be from about 0.01
pmol/kg to about 1 mmol/kg, from about 0.1 pmol/kg to about 0.1
mmol/kg, from about 1.0 pmol/kg to about 0.01 mmol/kg, from about
10 pmol/kg to about 1 .mu.mol/kg, from about 50 pmol/kg to about
500 nmol/kg, from about 100 pmol/kg to about 300 nmol/kg, from
about 200 pmol/kg to about 100 nmol/kg, from about 300 pmol/kg to
about 50 nmol/kg, from about 500 pmol/kg to about 30 nmol/kg, from
about 250 pmol/kg to about 5 nmol/kg, from about 750 pmol/kg to
about 10 nmol/kg, from about 1 nmol/kg to about 50 nmol/kg, or a
combination of any two of said values.
[0195] In this context, prescription of treatment, e.g. decisions
on dosage etc. when using the above pharmaceutical composition is
typically within the responsibility of general practitioners and
other medical doctors, and typically takes account of the disorder
to be treated, the condition of the individual patient, the site of
delivery, the method of administration and other factors known to
practitioners. Examples of the techniques and protocols mentioned
above can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 16th
edition, Osol, A. (ed), 1980. Accordingly, a "safe and effective
amount" as defined above for components of the pharmaceutical
compositions as used according to the present invention means an
amount of each or all of these components, that is sufficient to
significantly induce a positive modification of diseases or
disorders strongly related to JNK signaling as defined herein. At
the same time, however, a "safe and effective amount" is small
enough to avoid serious side-effects, that is to say to permit a
sensible relationship between advantage and risk. The determination
of these limits typically lies within the scope of sensible medical
judgment. A "safe and effective amount" of such a component will
vary in connection with the particular condition to be treated and
also with the age and physical condition of the patient to be
treated, the severity of the condition, the duration of the
treatment, the nature of the accompanying therapy, of the
particular pharmaceutically acceptable carrier used, and similar
factors, within the knowledge and experience of the accompanying
doctor. The pharmaceutical compositions according to the invention
can be used according to the invention for human and also for
veterinary medical purposes.
[0196] The pharmaceutical composition as used according to the
present invention may furthermore comprise, in addition to one of
these substances, a (compatible) pharmaceutically acceptable
carrier, excipient, buffer, stabilizer or other materials well
known to those skilled in the art.
[0197] In this context, the expression "(compatible)
pharmaceutically acceptable carrier" preferably includes the liquid
or non-liquid basis of the composition. The term "compatible" means
that the constituents of the pharmaceutical composition as used
herein are capable of being mixed with the pharmaceutically active
component as defined above and with one another component in such a
manner that no interaction occurs which would substantially reduce
the pharmaceutical effectiveness of the composition under usual use
conditions. Pharmaceutically acceptable carriers must, of course,
have sufficiently high purity and sufficiently low toxicity to make
them suitable for administration to a person to be treated.
[0198] If the pharmaceutical composition as used herein is provided
in liquid form, the pharmaceutically acceptable carrier will
typically comprise one or more (compatible) pharmaceutically
acceptable liquid carriers. The composition may comprise as
(compatible) pharmaceutically acceptable liquid carriers e.g.
pyrogen-free water; isotonic saline, i.e. a solution of 0.9% NaCl,
or buffered (aqueous) solutions, e.g. phosphate, citrate etc.
buffered solutions, vegetable oils, such as, for example, groundnut
oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from
theobroma; polyols, such as, for example, polypropylene glycol,
glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid,
etc. Particularly for injection and/or infusion of the
pharmaceutical composition as used herein, a buffer, preferably an
aqueous buffer, and/or 0.9% NaCl may be used.
[0199] If the pharmaceutical composition as used herein is provided
in solid form, the pharmaceutically acceptable carrier will
typically comprise one or more (compatible) pharmaceutically
acceptable solid carriers. The composition may comprise as
(compatible) pharmaceutically acceptable solid carriers e.g. one or
more compatible solid or liquid fillers or diluents or
encapsulating compounds may be used as well, which are suitable for
administration to a person. Some examples of such (compatible)
pharmaceutically acceptable solid carriers are e.g. sugars, such
as, for example, lactose, glucose and sucrose; starches, such as,
for example, corn starch or potato starch; cellulose and its
derivatives, such as, for example, sodium carboxymethylcellulose,
ethylcellulose, cellulose acetate; powdered tragacanth; malt;
gelatin; tallow; solid glidants, such as, for example, stearic
acid, magnesium stearate; calcium sulphate, etc.
[0200] The precise nature of the (compatible) pharmaceutically
acceptable carrier or other material may depend on the route of
administration. The choice of a (compatible) pharmaceutically
acceptable carrier may thus be determined in principle by the
manner in which the pharmaceutical composition as used according to
the invention is administered. Various possible routes of
administration are listed in the list "Route of Administration" of
the FDA (cf. FDA: Data Standards Manual-Drug Nomenclature
Monographs-Monograph Number: C-DRG-00301; Version Number 004),
which is incorporated by reference herein. Further guidance for
selecting an appropriate route of administration, in particular for
non-human animals, can be found in Turner P V et al. (2011) Journal
of the American Association for Laboratory Animal Science, Vol. 50,
No 5, p. 600-613, which is also incorporated by reference herein.
Preferred examples for routes for administration include parenteral
routes (e.g. via injection), such as intravenous, intramuscular,
subcutaneous, intradermal, or transdermal routes, etc., enteral
routes, such as oral, or rectal routes, etc., topical routes, such
as nasal, or intranasal routes, etc., or other routes, such as
epidermal routes or patch delivery. Also contemplated (in
particular for eye related diseases) are instillation,
intravitreal, and subconjunctival administration. Likewise,
administration may occur intratympanical, for example, whenever ear
related diseases are treated.
[0201] The pharmaceutical composition as used according to the
invention can be administered, for example, systemically. In
general, routes for systemic administration include, for example,
parenteral routes (e.g. via injection and/or infusion), such as
intravenous, intra-arterial, intraosseous, intramuscular,
subcutaneous, intradermal, -transdermal, or transmucosal routes,
etc., and enteral routes (e.g. as tablets, capsules, suppositories,
via feeding tubes, gastrostomy), such as oral, gastrointestinal or
rectal routes, etc. By systemic administration a system-wide action
can be achieved and systemic administration is often very
convenient, however, depending on the circumstances it may also
trigger unwanted "side-effects" and/or higher concentrations of the
JNK inhibitor according to the invention may be necessary as
compared to local administration. Systemic administration is in
general applicable for the prevention and/or treatment of the
diseases/disorders mentioned above due to its system-wide action.
Preferred routes of systemic administration are intravenous,
intramuscular, subcutaneous, oral and rectal administration,
whereby intravenous and oral administration are particularly
preferred.
[0202] The pharmaceutical composition as used according to the
invention can also be administered, for example, locally, for
example topically. Topical administration typically refers to
application to body surfaces such as the skin or mucous membranes,
whereas the more general term "local administration" additionally
comprises application in and/or into specific parts of the body.
Topical application is particularly preferred for the treatment
and/or prevention of diseases and/or disorders of the skin and/or
subcutaneous tissue as defined herein as well as for certain
diseases of the mouth and/or diseases relating to or are accessible
by mucous membranes.
[0203] Routes for local administration include, for example,
inhalational routes, such as nasal, or intranasal routes,
ophtalamic and otic drugs, e.g. eye drops and ear drops,
administration through the mucous membranes in the body, etc., or
other routes, such as epidermal routes, epicutaneous routes
(application to the skin) or patch delivery and other local
application, e.g. injection and/or infusion, into the organ or
tissue to be treated etc. In local administration side effects are
typically largely avoided. It is of note, that certain routes of
administration may provide both, a local and a systemic effect, for
example inhalation.
[0204] Routes for administration for the pharmaceutical composition
as used according to the invention can be chosen according to the
desired location of the application depending on the
disorder/disease to be prevented or treated.
[0205] For example, an enteral administration refers to the
gastrointestinal tract as application location and includes oral
(p.o.), gastroinstestinal and rectal administration, whereby these
are typically systemic administration routes, which are applicable
to the prevention/treatment of the diseases mentioned above in
general. In addition, enteral administration is preferred to
prevent and/or treat diseases/disorders of the gastrointestinal
tract as mentioned above, for example inflammatory diseases of the
gastrointestinal tract, metabolic diseases, cancer and tumor
diseases, in particular of the gastrointestinal tract etc. For
example, the oral route is usually the most convenient for a
patient and carries the lowest cost. Therefore, oral administration
is preferred for convenient systemic administration, if applicable.
Pharmaceutical compositions for oral administration may be in
tablet, capsule, powder or liquid form. A tablet may include a
solid carrier as defined above, such as gelatin, and optionally an
adjuvant. Liquid pharmaceutical compositions for oral
administration generally may include a liquid carrier as defined
above, such as water, petroleum, animal or vegetable oils, mineral
oil or synthetic oil. Physiological saline solution, dextrose or
other saccharide solution or glycols such as ethylene glycol,
propylene glycol or polyethylene glycol may be included.
[0206] Furthermore, enteral administration also includes
application locations in the proximal gastrointestinal tract
without reaching the intestines, for example sublingual, sublabial,
buccal or intragingival application. Such routes of administration
are preferred for applications in stomatology, i.e.
disease/disorders of the mouth which may be treated and/or
prevented with the JNK inhibitors as disclosed herein, for example
pulpitis in general, in particular acute pulpitis, chronic
pulpitis, hyperplastic pulpitis, ulcerative pulpitis, irreversible
pulpitis and/or reversible pulpitis; periimplantitis; periodontitis
in general, in particular chronic periodontitis, complex
periodontitis, simplex periodontitis, aggressive periodontitis,
and/or apical periodontitis, e.g. of pulpal origin; periodontosis,
in particular juvenile periodontosis; gingivitis in general, in
particular acute gingivitis, chronic gingivitis, plaque-induced
gingivitis, and/or non-plaque-induced gingivitis; pericoronitis, in
particular acute and chronic pericoronitis; sialadenitis
(sialoadenitis); parotitis, in particular infectious parotitis and
autoimmune parotitis; stomatitis in general, in particular aphthous
stomatitis (e.g., minor or major), Bednar's aphthae, periadenitis
mucosa necrotica recurrens, recurrent aphthous ulcer, stomatitis
herpetiformis, gangrenous stomatitis, denture stomatitis,
ulcerative stomatitis, vesicular stomatitis and/or
gingivostomatitis; mucositis, in particular mucositis due to
antineoplastic therapy, due to (other) drugs, or due to radiation,
ulcerative mucositis and/or oral mucositis; cheilitis in general,
in particular chapped lips, actinic cheilitis, angular cheilitis,
eczematous cheilitis, infectious cheilitis, granulomatous
cheilitis, drug-related cheilitis, exfoliative cheilitis, cheilitis
glandularis, and/or plasma cell cheilitis; cellulitis (bacterial
infection), in particular of mouth and/or lips; desquamative
disorders, in particular desquamative gingivitis; and/or
temporomandibular joint disorder. Particularly preferred diseases
to be treated and/or prevented according to the invention by these
routes of administration are selected from periodontitis, in
particular chronic periodontitis, mucositis, oral desquamative
disorders, oral liquen planus, pemphigus vulgaris, pulpitis,
stomatitis, temporomandibular joint disorder, and
peri-implantitis.
[0207] For example, intragingival administration, e.g. by injection
into the gums (gingiva), is preferred in stomatology applications,
for example for preventing and/or treating periodontitis. For
example, disorders/diseases of the mouth, in particular
periodontitis, may be prevented or treated by sublingual,
sublabial, buccal or intragingival application, in particular
intragingival application, of the pharmaceutical composition as
defined above comprising a dose (per kg body weight) of 100 ng/kg
to 100 mg/kg, preferably 10 .mu.g/kg to 10 mg/kg of the JNK
inhibitor according to the present invention, whereby the chimeric
peptide according to a sequence of SEQ ID NO. 11 is particularly
preferred.
[0208] Alternatively, the diseases of the mouth mentioned above may
also be treated and/or prevented by systemic and, preferably,
topical administration of the JNK inhibitor as disclosed herein or
the respective pharmaceutical composition.
[0209] In addition, enteral administration also includes strictly
enteral administration, i.e. directly into the intestines, which
can be used for systemic as well as for local administration.
[0210] Moreover, the JNK inhibitor according to the present
invention, used in the preventention and/or treatment of diseases
and/or disorders according to the present invention may be
administered to the central nervous system (CNS). Such routes of
administration include in particular epidural (peridural),
intra-CSF (intra-cerebrospinal fluid), intracerebroventricular
(intraventricular), intrathecal and intracerebral administration,
for example administration into specific brain regions, whereby
problems relating to the blood-brain-barrier can be avoided. Such
CNS routes of administration are preferred if the disease/disorder
to be treated is a neural, a neurological and/or a
neurodegenerative disease as specified above.
[0211] In addition, the JNK inhibitor according to the present
invention, used in the preventention and/or treatment of diseases
and/or disorders according to the present invention may be
administered at, in or onto the eye. Such routes of administration
include instillation, e.g. eye drops applied topically, for example
onto the conjunctiva, and, in particular, intravitreous (IVT),
subconjunctival, and posterior juxtascleral administration, e.g. by
injection, infusion and/or instillation and/or localized,
sustained-release drug delivery (for example in case of the
subconjunctival route), whereby eyedrops (for topical application),
intravitreous (IVT) and subconjunctival routes of administration
are particularly preferred. The subconjunctival route is safer and
less invasive than the intravitreal route, however, the
intravitreal route involves less systemic exposure than the
subconjunctival route due to the presence of conjunctival and
orbital blood vessels and tissue.
[0212] Local administration onto/in the eye is particularly
preferred for eye-related diseases/disorders to be treated and/or
prevented as disclosed herein, for example age-related macular
degeneration (AMD), in particular in the wet and dry form; angioid
streaks; anterior ischemic optic neuropathy; anterior uveitis;
cataract, in particular age related cataract; central exudative
chorioretinopathy; central serous chorioretinopathy; chalazion;
chorioderemia; chorioiditis; choroidal sclerosis; conjunctivitis;
cyclitis; diabetic retinopathy; dry eye syndrome; endophthalmitis;
episcleritis; eye infection; fundus albipunctatus; gyrate atrophy
of choroid and retina; hordeolum; inflammatory diseases of the
blephara; inflammatory diseases of the choroid; inflammatory
diseases of the ciliary body; inflammatory diseases of the
conjunctiva; inflammatory diseases of the cornea; inflammatory
diseases of the iris; inflammatory diseases of the lacrimal gland;
inflammatory diseases of the orbital bone; inflammatory diseases of
the sclera; inflammatory diseases of the vitreous body;
inflammatory diseases of the uvea; inflammatory diseases of the
retina; intermediate uveitis; irititis; keratitis; Leber's disease;
multifocal choroiditis; myositis of the eye muscle; neovascular
maculopathy (e.g. caused by high myopia, tilted disc syndrome,
choroidal osteoma or the like); NMDA induced retinotoxicity;
non-chronic or chronic inflammatory eye diseases; Oguchi's disease;
optic nerve disease; orbital phlegmon; panophtalmitis; panuveitis;
post caspule opacification; posterior capsule opacification (PCO)
(a cataract after-surgery complication); posterior uveitis;
intraocular inflammation, in particular post-surgery or post-trauma
intraocular inflammation, preferably intraocular inflammation
following anterior and/or posterior segment surgery; proliferative
vitreoretinopathy; retinal artery occlusion; retinal detachment,
retinal diseases; retinal injuries; retinal macroaneurysm; retinal
pigment epithelium detachment; retinal vein occlusion; retinitis;
retinitis pigmentosa; retinitis punctata albescens; retinopathy, in
particular retinopathy of prematurity and diabetic retinopathy;
scleritis; Stargardt's disease; treatment of inflamed ocular wounds
and/or ocular wound edges; treatment of intraocular inflammation
after eye surgery or trauma; uveitis; vitelliform macular
dystrophy; etc.
[0213] In particular, age-related macular degeneration (AMD), in
particular the wet and the dry form of AMD, uveitis, in particular
anterior and/or posterior uveitis, retinopathy, in particular
retinopathy of prematurity and diabetic retinopathy, and
post-surgery or post-trauma eye inflammation, in particular
post-surgery or intraocular inflammation preferably intraocular
inflammation following anterior and/or posterior segment surgery,
are prevented and/or treated by the JNK inhibitor used according to
the present invention by local administration in and/or onto the
eye, preferably by instillation, e.g. eye drops, and/or
intravitreal and/or subconjunctival administration, e.g. by
injection or instillation. Instillation, e.g. eyedrops, and/or
subconjunctival administration, e.g. by injection, are thereby
preferred routes of administration and subconjunctival
administration, e.g. by subconjunctival injection, is particularly
preferred. For these routes of administration, in particular
intravitreal and/or subconjunctival administration, the respective
pharmaceutical composition according to the present invention,
preferably comprises a dose per eye in the range of 100 ng to 10
mg, more preferably in the range of 1 .mu.g to 5 mg, even more
preferably in the range of 50 .mu.g to 1 mg of the JNK inhibitor
according to the present invention, preferably of the chimeric
peptide according to a sequence of SEQ ID NO. 11 (i.e. a dose in
the range of 100 ng to 10 mg, more preferably in the range of 1
.mu.g to 5 mg, even more preferably in the range of 50 .mu.g to 1
mg of the JNK inhibitor administered per eye). One single
administration or more administrations, in particular two, three,
four or five, administrations of such dose(s) may be performed,
whereby a single administration is preferred, however, also
subsequent dose(s) may be administered, for example on different
days of the treatment schedule. For example for intravitreal and/or
subconjunctival administration in humans a single dose (per eye) of
the JNK inhibitor is preferably in the range of 1 .mu.g to 5 mg,
preferably 50 .mu.g to 1.5 mg, more preferably 500 .mu.g to 1 mg,
most preferably 800 .mu.g to 1 mg. The injection volume, in
particular for subconjunctival injection, may be for example 100
.mu.l to 500 .mu.l, e.g. 250 .mu.l. A single subconjuctival
injection of such a dose is for example particularly useful to
treat and/or prevent post-surgery intraocular inflammation in
humans, preferably intraocular inflammation following anterior
and/or posterior segment surgery.
[0214] For topical ocular administration, in particular as
instillation, preferably eyedrops, which may be applied to both
eyes or to one eye only, depending on the need, the pharmaceutical
composition comprising the JNK inhibitor according to the invention
is typically a solution, preferably an ophthalamic solution, e.g.
comprising (sterile) 0.9% NaCl. Such a pharmaceutical composition
comprises in particular 0.001%-10% of the JNK inhibitor as
described herein, preferably 0.01%-5% of the JNK inhibitor as
described herein, more preferably 0.05%-2% of the JNK inhibitor as
described herein, even more preferably 0.1%-1% of the JNK inhibitor
as described herein. The eyedrops may be administered once or
repeatedly, whereby repeated administration is preferred. In
general, the administration depends on the need and may for example
be on demand. In repeated administration, subsequent dose(s) may be
administered on the same and/or different days of the treatment
schedule, whereby on the same day a single dose or more than one
single doses, in particular two, three, four or five, preferably
two to four doses may be administered, whereby such repeated
administration is preferably spaced by intervals of one or more
hour(s), e.g. two, three, four, five, six, seven or eight hours.
For example eye drops may be administered three or four times per
day for several, e.g. two, three, four, five or six weeks.
[0215] In addition, eye diseases as described herein may of course
also be treated and/or prevented by systemic application of the JNK
inhibitor according to the invention (which also applies to the
other diseases/disorders as described herein). The dose for
systemic administration in eye diseases, in particular for
intravenous administration, ranges preferably from 0.001 mg/kg to
10 mg/kg, more preferably from 0.01 mg/kg to 5 mg/kg, even more
preferably from 0.1 mg/kg to 2 mg/kg. Such doses are for example
particularly useful to treat and/or prevent uveitis, whereby the
treatment schedule may comprises a single dose or repeated doses,
whereby subsequent dose(s) may be administered on different days of
the treatment schedule.
[0216] Preferably, for the prevention and/or treatment of uveitis,
preferably anterior uveitis, more preferably acute anterior
uveitis, a single dose or repeated doses of the JNK inhibitor
according to the invention, preferably the JNK inhibitor according
to SEQ ID NO: 11, are administered subconjunctivally. Preferably, a
single dose is administered. However, it is also preferred that
repeated doses are administered, preferably weekly or every second
week. Preferably, the JNK inhibitor according to the invention,
preferably the JNK inhibitor according to SEQ ID NO: 11, is applied
in doses, e.g. for (sub-conjunctival) injection, in the range of
0.01 .mu.g/eye to 10 mg/eye, more preferably 0.1 .mu.g/eye to 5
mg/eye, even more preferably 1 .mu.g/eye to 2 mg/eye, particularly
preferably 100 .mu.g/eye to 1.5 mg/eye, most preferably 500
.mu.g/eye to 1 mg/eye, e.g. 900 .mu.g/eye.
[0217] For example, if more than a single dose is applied, in
particular intravenously, in the treatment and/or prevention of
uveitis, the doses are typically spaced by intervals of at least
one day, preferably by intervals of at least two days, more
preferably by intervals of at least three days, even more
preferably by intervals of at least four days, at least five days,
or at least six days, particularly preferably by intervals of at
least a week, most preferably by intervals of at least ten
days.
[0218] Other routes of administration for the use of the JNK
inhibitor according to the present invention, which are typically
chosen according to the disease to be prevented and/or treated and
the respective pharmacokinetics, include--but are not limited
to-epicutaneous application (onto the skin) and/or intralesional
application (into a skin lesion), for example for skin diseases as
defined herein (mentioned above), in particular selected from
psoriasis, eczema, dermatitis, acne, mouth ulcers, erythema, lichen
plan, sarcoidose, vascularitis, and adult linear IgA disease; nasal
administration, for example for diseases of the respiratory system
and in particular lung diseases, for example acute respiratory
distress syndrome (ARDS), asthma, chronic illnesses involving the
respiratory system, chronic obstructive pulmonary disease (COPD),
cystic fibrosis, inflammatory lung diseases, pneumonia, and
pulmonary fibrosis; intraarticular administration (into a joint
space), for example in arthritis, in particular juvenile idiopathic
arthritis, psoriastic arthritis and rheumatoid arthritis, and
arthrosis, and osteoarthritis; intravesical administration (i.e.
into the urinary bladder), for example for diseases of the urinary
system, in particular the urinary bladder; intracardiac
administration, intracavernous administration, intravaginal
administration, and intradermal administration.
[0219] In general, the method of administration depends on various
factors as mentioned above, for example the selected pharmaceutical
carrier and the nature of the pharmaceutical preparation (e.g. as a
liquid, tablet etc.) as well as the route of administration. For
example, the pharmaceutical composition comprising the JNK
inhibitor according to the invention may be prepared as a liquid,
for example as a solution of the JNK inhibitor according to the
invention, preferably of the chimeric peptide according to a
sequence of SEQ ID NO. 11, in 0.9% NaCl. A liquid pharmaceutical
composition can be administered by various methods, for example as
a spray (e.g., for inhalational, intranasal etc. routes), as a
fluid for topical application, by injection, including bolus
injection, by infusion, for example by using a pump, by
instillation, but also p.o., e.g. as drops or drinking solution, in
a patch delivery system etc.
[0220] Accordingly, for the administration different devices may be
used, in particular for injection and/or infusion, e.g. a syringe
(including a pre-filled syringe); an injection device (e.g. the
INJECT-EASET.TM. and GENJECTT.TM. device); an infusion pump (such
as e.g. Accu-Chek.TM.); an injector pen (such as the GENPENT.TM.);
a needleless device (e.g. MEDDECTOR.TM. and BIOJECTOR.TM.); or an
autoinjector.
[0221] The suitable amount of the pharmaceutical composition to be
used can be determined by routine experiments with animal models.
Such models include, without implying any limitation, for example
rabbit, sheep, mouse, rat, gerbil, dog, pig and non-human primate
models. Preferred unit dose forms for administration, in particular
for injection and/or infusion, include sterile solutions of water,
physiological saline or mixtures thereof. The pH of such solutions
should be adjusted to about 7.4. Suitable carriers for
administration, in particular for injection and/or infusion,
include hydrogels, devices for controlled or delayed release,
polylactic acid and collagen matrices. Suitable pharmaceutically
acceptable carriers for topical application include those, which
are suitable for use in lotions, creams, gels and the like. If the
compound is to be administered perorally, tablets, capsules and the
like are the preferred unit dose form. The pharmaceutically
acceptable carriers for the preparation of unit dose forms, which
can be used for oral administration are well known in the prior
art. The choice thereof will depend on secondary considerations
such as taste, costs and storability, which are not critical for
the purposes of the present invention, and can be made without
difficulty by a person skilled in the art.
[0222] For intravenous, intramuscular, intraperitoneal, cutaneous
or subcutaneous injection and/or infusion, or injection and/or
infusion at the site of affliction, i.e. local injection/infusion,
the active ingredient will be in the form of a parenterally
acceptable aqueous solution which is pyrogen-free and has suitable
pH, isotonicity and stability. Those of relevant skill in the art
are well able to prepare suitable solutions using, for example,
isotonic vehicles such as Sodium Chloride Injection, in particular
0.9% NaCl, Ringer's Injection, Lactated Ringer's Injection.
Preservatives, stabilizers, buffers, antioxidants and/or other
additives may be included, as required. Whether it is a
polypeptide, peptide, or nucleic acid molecule, other
pharmaceutically useful compound according to the present invention
that is to be given to an individual, administration is preferably
in a "prophylactically effective amount or a "therapeutically
effective amount" (as the case may be), this being sufficient to
show benefit to the individual. The actual amount administered, and
rate and time-course of administration, will depend on the nature
and severity of what is being treated. For example, for i.v.
administration in humans, single doses of up to 1 mg per kg body
weight are preferred, more preferably up to 500 .mu.g per kg body
weight, even more preferably up to 100 .mu.g per kg body weight,
for example in the range of 100 ng to 1 mg per kg body weight, more
specifically in the range of 1 .mu.g to 500 .mu.g per kg body
weight, even more specifically in the range of 5 .mu.g to 100 .mu.g
per kg body weight. Such doses may be administered for example as
injection and/or infusion, in particular as infusion, whereby the
duration of the infusion varies for example between 1 to 90 min,
preferably 10 to 70 min, more preferably 30 to 60 min.
[0223] Particularly preferred embodiments of the use of the JNK
inhibitor according to the present invention, for example the
chimeric peptide having a sequence according to SEQ ID NO. 11, in
particular in a pharmaceutical composition as defined herein,
include--but are not limited to--the prevention and/or treatment of
the following diseases/disorders: [0224] (i) respiratory diseases,
in particular lung inflammation and fibrosis, specifically COPD,
wherein the JNK inhibitor is preferably applied in doses (per kg
body weight) in the range of 1 ng/kg to 10 mg/kg, more preferably
10 ng/kg to 1 mg/kg, even more preferably 1 .mu.g/kg to 0.1 mg/kg,
whereby such a single dose may be repeated one, two, three or four
times, and which is preferably applied systemically, e.g. i.v. or
s.c., or intranasally; [0225] (ii) metabolic diseases and
disorders, for example diabetes in general, in particular type 1
diabetes mellitus, type 2 diabetes mellitus, diabetes mellitus due
to underlying condition, for example due to congenital rubella,
Cushing's syndrome, cystic fibrosis, malignant neoplasm,
malnutrition, or pancreatitis and other diseases of the pancreas,
drug or chemical induced diabetes mellitus, and/or other diabetes
mellitus, wherein for the treatment and/or prevention of the
metabolic diseases the JNK inhibitor is preferably applied in doses
(per kg body weight) in the range of 100 .mu.g/kg to 100 mg/kg,
more preferably 1 mg/kg to 50 mg/kg, even more preferably 5 mg/kg
to 15 mg/kg, whereby such a single dose may be repeated daily for
one to several, e.g. four, weeks, and which is preferably applied
systemically, e.g. i.v. or s.c.; [0226] (iii) diseases of the mouth
and/or the jaw bone, in particular inflammatory diseases of the
mouth and/or the jaw bone selected from (i) pulpitis in general, in
particular acute pulpitis, chronic pulpitis, hyperplastic pulpitis,
ulcerative pulpitis, irreversible pulpitis and/or reversible
pulpitis; (ii) periimplantitis; (iii) periodontitis in general, in
particular chronic periodontitis, complex periodontitis, simplex
periodontitis, aggressive periodontitis, and/or apical
periodontitis, e.g. of pulpal origin; periodontosis, in particular
juvenile periodontosis; (iv) gingivitis in general, in particular
acute gingivitis, chronic gingivitis, plaque-induced gingivitis,
and/or non-plaque-induced gingivitis; (v) pericoronitis, in
particular acute and chronic pericoronitis; sialadenitis
(sialoadenitis); parotitis, in particular infectious parotitis and
autoimmune parotitis; (vi) stomatitis in general, in particular
aphthous stomatitis (e.g., minor or major), Bednar's aphthae,
periadenitis mucosa necrotica recurrens, recurrent aphthous ulcer,
stomatitis herpetiformis, gangrenous stomatitis, denture
stomatitis, ulcerative stomatitis, vesicular stomatitis and/or
gingivostomatitis; (vii) mucositis, in particular mucositis due to
antineoplastic therapy, due to (other) drugs, or due to radiation,
ulcerative mucositis and/or oral mucositis; (viii) cheilitis in
general, in particular chapped lips, actinic cheilitis, angular
cheilitis, eczematous cheilitis, infectious cheilitis,
granulomatous cheilitis, drug-related cheilitis, exfoliative
cheilitis, cheilitis glandularis, and/or plasma cell cheilitis; and
(ix) cellulitis (bacterial infection), in particular of mouth
and/or lips; desquamative disorders, in particular desquamative
gingivitis; and/or temporomandibular joint disorder, whereby
periodontitis, periimplantitis, gingivitis, stomatitis and
mucositis are preferred and periodontitis is particularly
preferred; wherein for the treatment and/or prevention of the
diseases of the mouth and/or the jaw bone the JNK inhibitor is
preferably applied in doses (per kg body weight) in the range of
100 .mu.g/kg to 100 mg/kg, more preferably 1 mg/kg to 10 mg/kg,
even more preferably 2 mg/kg to 5 mg/kg, and which is preferably
applied intragingivally or topically, particularly preferably
intragingivally; [0227] (iv) Nephrological Diseases (Kidney
Diseases), in Particular Selected from (i) glomerulonephritis, for
example nonproliferative glomerulonephritis, in particular minimal
change disease, focal segmental glomerulosclerosis, focal segmental
glomerular hyalinosis and/or sclerosis, focal glomerulonephritis,
membranous glomerulonephritis, and/or thin basement membrane
disease, and proliferative glomerulonephritis, in particular
membrano-proliferative glomerulonephritis, mesangio-prol iferative
glomerulonephritis, endocapillary proliferative glomerulonephritis,
mesangiocapillary proliferative glomerulonephritis, dense deposit
disease (membranoproliferative glomerulonephritis type II),
extracapillary glomerulonephritis (crescentic glomerulonephritis),
rapidly progressive glomerulonephritis (RPGN), in particular Type I
RPGN, Type II RPGN, Type III RPGN, and Type IV RPGN, acute
proliferate glomerulonephritis, post-infectious glomerulonephritis,
and/or IgA nephropathy (Berger's disease); acute nephritic
syndrome; rapidly progressive nephritic syndrome; recurrent and
persistent hematuria; chronic nephritic syndrome; nephrotic
syndrome; proteinuria with specified morphological lesion;
glomerulitis; glomerulopathy; glomerulosclerosis; (ii) acute kidney
injury ("AKI", also called "acute renal failure" or "acute kidney
failure") in general, in particular prerenal AKI, intrinsic AKI,
postrenal AKI, AKI with tubular necrosis for example acute tubular
necrosis, renal tubular necrosis, AKI with cortical necrosis for
example acute cortical necrosis and renal cortical necrosis, AKI
with medullary necrosis, for example medullary (papillary)
necrosis, acute medullary (papillary) necrosis and chronic
medullary (papillary) necrosis, or other AKI; chronic kidney
disease; or (iii) nephropathy, in particular selected from
membranous nephropathy, diabetic nephropathy, IgA nephropathy,
hereditary nephropathy, analgesic nephropathy, CFHR5 nephropathy,
contrast-induced nephropathy, amyloid nephropathy, reflux
nephropathy and/or Mesoamerican nephropathydiabetic nephropathy,
diabetic nephropathy, whereby preferably the disorder/disease to be
prevented and/or treated is glomerulonephritis or diabetic
nephropathy, more preferably the disorder/disease to be prevented
and/or treated is glomerulonephritis; wherein for the treatment
and/or prevention of the nephrological diseases (kidney diseases),
preferably of glomerulonephritis, more preferably of
glomerulonephritis with focal segmental glomerulosclerosis and/or
fibrosis, the JNK inhibitor is preferably applied in doses (per kg
body weight) in the range of 10 .mu.g/kg to 100 mg/kg, more
preferably 100 .mu.g/kg to 10 mg/kg, even more preferably 1 mg/kg
to 5 mg/kg, and the JNK inhibitor, preferably the chimeric peptide
having a sequence according to SEQ ID NO. 11, is preferably
administered, if applicable, once or repeatedly, preferably weekly
(once per week) for several, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10, or
more weeks, every second week (once per two weeks) for several,
e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more weeks, monthly (once
per month) for several, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more
months, every sixth week (once per every six weeks) for several,
e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more months, every second
month (once per two months) for several, e.g. 2, 3, 4, 5, 6, 7, 8,
9, or 10, or more months or every third month (once per three
months) for several, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more
weeks, more preferably weekly (once per week) for several, e.g. 2,
3, 4, 5, 6, 7, 8, 9, or 10, or more weeks, every second week (once
per two weeks) for several, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10, or
more weeks, monthly (once per month) for several, e.g. 2, 3, 4, 5,
6, 7, 8, 9, or 10, or more months, even more preferably monthly
(once per month) for several, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10,
or more months, and which is preferably applied systemically, e.g.
i.v. or s.c.; [0228] (v) cancer and tumor diseases, in particular
selected from (i) liver cancer and liver carcinoma in general, in
particular liver metastases, liver cell carcinoma, hepatocellular
carcinoma, hepatoma, intrahepatic bile duct carcinoma,
cholangiocarcinoma, hepatoblastoma, angiosarcoma (of liver), and
other specified or unspecified sarcomas and carcinomas of the
liver; (ii) prostate cancer and/or prostate carcinoma; and/or (iii)
colon cancer and colon carcinoma in general, in particular cecum
carcinoma, appendix carcinoma, ascending colon carcinoma, hepatic
flexure carcinoma, transverse colon carcinoma, splenic flexure
carcinoma, descending colon carcinoma, sigmoid colon carcinoma,
carcinoma of overlapping sites of colon and/or malignant carcinoid
tumors of the colon, wherein for the treatment and/or prevention of
the cancer and tumor diseases the JNK inhibitor is preferably
applied in doses (per kg body weight) in the range of 1 .mu.g/kg to
100 mg/kg, more preferably 10 .mu.g/kg to 50 mg/kg, even more
preferably 0.1 mg/kg to 20 mg/kg, particularly preferably 0.1 mg/kg
to 5 mg/kg, if applicable repeatedly, for example daily, every 2 or
3 days or weekly, for several, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10,
weeks, and which is preferably applied systemically, e.g. p.o.,
i.v. or s.c.; [0229] (vi) diseases of the eye, in particular (i)
age-related macular degeneration (AMD), including exudative and/or
non-exudative age-related macular degeneration, preferably the wet
or the dry form of age-related macular degeneration; (ii)
retinopathy, in particular selected from diabetic retinopathy,
(arterial hypertension induced) hypertensive retinopathy, exudative
retinopathy, radiation induced retinopathy, sun-induced solar
retinopathy, trauma-induced retinopathy, e.g. Purtscher's
retinopathy, retinopathy of prematurity (ROP) and/or
hyperviscosity-related retinopathy, non-diabetic proliferative
retinopathy, and/or proliferative vitreo-retinopathy, whereby
diabetic retinopathy and retinopathy of prematurity (ROP) are
preferred and diabetic retinopathy is particularly preferred; (iii)
post-surgery and/or post-trauma inflammation of the eye, in
particular after a surgery performed on and/or in the eye,
preferably intraocular inflammation following anterior and/or
posterior segment surgery, for example after cataract surgery,
laser eye surgery (e.g. Laser-in-situ-Keratomileusis (LASIK)),
glaucoma surgery, refractive surgery, corneal surgery,
vitreo-retinal surgery, eye muscle surgery, oculoplastic surgery,
ocular oncology surgery, conjunctival surgery including pterygium,
and/or surgery involving the lacrimal apparatus, in particular
after complex eye surgery and/or after uncomplicated eye surgery;
and/or (iv) uveitis, in particular anterior, intermediate and/or
posterior uveitis, sympathetic uveitis and/or panuveitis,
preferably anterior and/or posterior uveitis; wherein for the
treatment and/or prevention of the diseases of the eye, preferably
for the treatment and/or prevention of diabetic retinopathy,
anterior and/or posterior uveitis or post-surgery and/or
post-trauma inflammation of the eye, the JNK inhibitor is
preferably applied in doses, e.g. for injection, in the range of
0.01 .mu.g/eye to 10 mg/eye, more preferably 0.1 .mu.g/eye to 5
mg/eye, even more preferably 1 .mu.g/eye to 2 mg/eye, particularly
preferably 100 .mu.g/eye to 1.5 mg/eye, most preferably 500
.mu.g/eye to 1 mg/eye, e.g. 900 .mu.g/eye, preferably by a single
injection, however, if necessary repeatedly, for example daily,
every 2 or 3 days or weekly, for several, e.g. 2, 3, 4, 5, 6, 7, 8,
9, or 10, weeks, or once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or
more weeks, preferably once every 2, 3, 4, 6, 8, 10, or 12 weeks,
and which is preferably applied i.v. or in or onto the eye, more
preferably intravitreally or subconjunctivally, even more
preferably subconjunctivally. For example, for treating and/or
preventing post-surgery intraocular inflammation, in particular
intraocular inflammation following anterior and/or posterior
segment surgery, for example after cataract surgery, laser eye
surgery (e.g. Laser-in-situ-Keratomileusis (LASIK)), glaucoma
surgery, refractive surgery, corneal surgery, vitreo-retinal
surgery, eye muscle surgery, oculoplastic surgery, ocular oncology
surgery, conjunctival surgery including pterygium, and/or surgery
involving the lacrimal apparatus, in particular after complex eye
surgery and/or after uncomplicated eye surgery, subconjunctival
administration and/or instillation, e.g. eye drops, are
particularly preferred. Thereby, for subconjunctival administration
a single injection after the surgery, preferably within three hours
after surgery, for example just after the end of the surgical
procedure when the patient is still in the operating room, is
particularly preferred. For instillation for example application of
two to four doses, preferably three doses per day for two to four
weeks, preferably three weeks, is preferred, whereby the first dose
may be applied for example just after surgery. Moreover, for
treating and/or preventing post-surgery intraocular inflammation,
in particular intraocular inflammation following anterior and/or
posterior segment surgery, the JNK inhibitors of the present
invention may be administered as stand-alone therapy, however, the
JNK inhibitors of the present invention may also be administered in
combination with other medications, e.g. with corticosteroids,
preferably glucocorticoids, for example dexamethasone, in
particular if the inflammation persists over a predetermined
period. For example, the JNK inhibitors of the present invention
may first be used alone and, if the inflammation persists may be
combined with corticosteroids or, if corticosteroids were used
alone first, they may be combined with the JNK inhibitors of the
present invention if the inflammation persists; [0230] (vii)
diseases and/or disorders of the urinary system, in particular
ureteritis; urinary tract infection (bladder infection, acute
cystitis); cystitis in general, in particular interstitial
cystitis, Hunner's ulcer, trigonitis and/or hemorrhagic cystitis;
urethritis, in particular nongonococcal urethritis or gonococcal
urethritis; painful bladder syndrome; IC/PBS; urethral syndrome;
and/or retroperitoneal fibrosis; preferably IC/PBS; wherein for the
treatment and/or prevention of the diseases and/or disorders of the
urinary system, preferably for the treatment and/or prevention of
IC/PBS, the JNK inhibitor is preferably applied (i) systemically,
more preferably intravenously, e.g. by intravenous injection, in
doses of (per kg body weight) in the range of 100 ng/kg to 10
mg/kg, more preferably 1 .mu.g/kg to 5 mg/kg, even more preferably
10 .mu.g/kg to 2 mg/kg, particularly preferably 0.1 mg/kg to 1
mg/kg, most preferably 0.2 mg/kg to 0.5 mg/kg, preferably
administered in one single dose, however, if applicable also
preferably administered repeatedly, for example daily, every 2 or 3
days or weekly, for several, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10,
weeks; or the JNK inhibitor is also preferably applied (ii)
intravesically, more preferably by intravesical infusion,
preferably at a concentration of 10
.mu.g/ml -1000 mg/ml, more preferably 50 .mu.g/ml-500 mg/ml, even
more preferably 100 .mu.g/ml-100 mg/ml, and particularly preferably
0.5 mg/ml-50 mg/ml, preferably in single doses of 0.1-1000 mg, more
preferably 0.5-500 mg, even more preferably 1-100 mg, and
particularly preferably 2-10 mg, preferably administered in one
single dose, however, if applicable also preferably administered
repeatedly, for example daily, every 2 or 3 days or weekly, for
several, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10, weeks; and [0231]
(viii) neural, neuronal or neurodegenerative disorders, in
particular neurodegenerative disease, preferably Alzheimer's
disease, for example Alzheimer's disease with early onset,
Alzheimer's disease with late onset, Alzheimer's dementia senile
and presenile forms, and/or Mild Cognitive Impairment, in
particular Mild Cognitive Impairment due to Alzheimer's Disease,
wherein for the treatment and/or prevention of the neural, neuronal
or neurodegenerative disorders the JNK inhibitor is preferably
applied in doses (per kg body weight) in the range of 1 .mu.g/kg to
100 mg/kg, more preferably 10 .mu.g/kg to 50 mg/kg, even more
preferably 100 .mu.g/kg to 10 mg/kg, and particularly preferably
500 .mu.g/kg to 1 mg/kg, whereby the JNK inhibitor is preferably
administered, if applicable, once or repeatedly, preferably weekly
(once per week) for several, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10, or
more weeks, every second week (once per two weeks) for several,
e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more weeks, monthly (once
per month) for several, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more
months, every sixth week (once per every six weeks) for several,
e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more months, every second
month (once per two months) for several, e.g. 2, 3, 4, 5, 6, 7, 8,
9, or 10, or more months or every third month (once per three
months) for several, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more
weeks, more preferably weekly (once per week) for several, e.g. 2,
3, 4, 5, 6, 7, 8, 9, or 10, or more weeks, every second week (once
per two weeks) for several, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10, or
more weeks, monthly (once per month) for several, e.g. 2, 3, 4, 5,
6, 7, 8, 9, or 10, or more months, even more preferably monthly
(once per month) for several, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10,
or more months, and which is preferably applied systemically, e.g.
i.v., p.o., i.m., s.c. or intra-CSF (intra-cerebrospinal fluid)
moreover, for treating and/or preventing neural, neuronal or
neurodegenerative disorders, in particular neurodegenerative
disease, preferably Alzheimer's disease, for example Alzheimer's
disease with early onset, Alzheimer's disease with late onset,
Alzheimer's dementia senile and presenile forms, and/or Mild
Cognitive Impairment, in particular Mild Cognitive Impairment due
to Alzheimer's Disease, the JNK inhibitors of the present invention
may be administered as stand-alone therapy, however, the JNK
inhibitors of the present invention may also be administered in
combination with other medications, e.g. with a PKR inhibitor, e.g.
"SC1481" by Polypeptide Group, and, optionally, in addition to the
JNK inhibitor according to the present invention and the PKR
inhibitor with a amyloid lowering agent, whereby amyloid lowering
agents include .beta.-secretase (BACE1) inhibitors,
.gamma.-secretase inhibitors (GSI) and modulators (GSM) and
examples of such inhibitors, which are currently in clinical trials
may be retrieved from Vassar R. (2014) BACE1 inhibitor drugs in
clinical trials for Alzheimer's disease. Alzheimers Res Ther.;
6(9):89 or from Jia Q, Deng Y, Qing H (2014) Potential therapeutic
strategies for Alzheimer's disease targeting or beyond
.beta.-amyloid: insights from clinical trials. Biomed Res Int.
2014; 2014:837157.
[0232] Prevention and/or treatment of a disease as defined herein
typically includes administration of a pharmaceutical composition
as defined above. The term "modulate" includes the suppression of
expression of JNK when it is over-expressed in any of the above
diseases. It also includes suppression of phosphorylation of c-jun,
ATF2 or NFAT4 in any of the above diseases, for example, by using
at least one JNK inhibitor sequence according to any of sequences
of SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100 and/or at least one
chimeric peptide according to any of sequences of SEQ ID NOs: 9 to
12 and 23 to 32, whereby SEQ ID NO: 11 is particularly preferred,
and/or at least one JNK inhibitor sequence according to any of
sequences of SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100 comprising
a trafficking sequence according to any of SEQ ID NOs: 5 to 8 and
21 to 22, or variants or fragments thereof within the above
definitions, as a competitive inhibitor of the natural c-jun, ATF2
and NFAT4 binding site in a cell. The term "modulate" also includes
suppression of hetero- and homomeric complexes of transcription
factors made up of, without being limited thereto, c-jun, ATF2, or
NFAT4 and their related partners, such as for example the AP-1
complex that is made up of c-jun, AFT2 and c-fos. When a disease or
disorder strongly related to JNK signaling as defined above is
associated with JNK overexpression, such suppressive JNK inhibitor
sequences can be introduced to a cell. In some instances,
"modulate" may then include the increase of JNK expression, for
example by use of an IB peptide-specific antibody that blocks the
binding of an IB-peptide to JNK, thus preventing JNK inhibition by
the IB-related peptide.
[0233] Prevention and/or treatment of a subject with the
pharmaceutical composition as disclosed above may be typically
accomplished by administering (in vivo) an ("therapeutically
effective") amount of said pharmaceutical composition to a subject,
wherein the subject may be e.g. any mammal, e.g. a human, a
primate, mouse, rat, dog, cat, cow, horse or pig, whereby a human
is particularly preferred. The term "therapeutically effective"
means that the active component of the pharmaceutical composition
is of sufficient quantity to ameliorate the disease or disorder
strongly related to JNK signaling as defined above.
[0234] Accordingly, any peptide as defined above, e.g. at least one
JNK inhibitor sequence according to any of sequences of SEQ ID NOs:
1 to 4 and 13 to 20 and 33-100 and/or at least one chimeric peptide
according to any of sequences of SEQ ID NOs: 9 to 12 and 23 to 32,
preferably SEQ ID NO: 11, and/or at least one JNK inhibitor
sequence according to any of sequences of SEQ ID NOs: 1 to 4 and 13
to 20 and 33-100 comprising a trafficking sequence according to any
of SEQ ID NOs: 5 to 8 and 21 to 22, or variants or fragments
thereof within the above definitions, may be utilized in a specific
embodiment of the present invention to treat diseases or disorders
strongly related to JNK signaling as defined above, e.g. by
modulating activated JNK signaling pathways.
[0235] However, the above defined peptides may be also encoded by
nucleic acids, which then may form part of the inventive
pharmaceutical compositions, e.g. for use in gene therapy. In this
context, gene therapy refers to therapy that is performed by
administration of a specific nucleic acid as defined above to a
subject, e.g. by way of a pharmaceutical composition as defined
above, wherein the nucleic acid(s) exclusively comprise(s) L-amino
acids. In this embodiment of the present invention, the nucleic
acid produces its encoded peptide(s), which then serve(s) to exert
a therapeutic effect by modulating function of the disease or
disorder. Any of the methods relating to gene therapy available
within the art may be used in the practice of the present invention
(see e.g. Goldspiel, et al., 1993. Clin Pharm 12: 488-505).
[0236] In a preferred embodiment, the nucleic acid as defined above
and as used for gene therapy is part of an expression vector
encoding and expressing any one or more of the IB-related peptides
as defined above within a suitable host, i.e. an JNK inhibitor
sequence according to any of sequences of SEQ ID NOs: 1 to 4 and 13
to 20 and 33-100 and/or a chimeric peptide according to any of
sequences of SEQ ID NOs: 9 to 12 and 23 to 32, and/or an JNK
inhibitor sequence according to any of sequences of SEQ ID NOs: 1
to 4 and 13 to 20 and 33-100 comprising a trafficking sequence
according to any of SEQ ID NOs: 5 to 8 and 21 to 22, or variants or
fragments thereof within the above definitions. In a specific
embodiment, such an expression vector possesses a promoter that is
operably-linked to coding region(s) of a JNK inhibitor sequence.
The promoter may be defined as above, e.g. inducible or
constitutive, and, optionally, tissue-specific.
[0237] In another specific embodiment, a nucleic acid molecule as
defined above is used for gene therapy, in which the coding
sequences of the nucleic acid molecule (and any other desired
sequences thereof) as defined above are flanked by regions that
promote homologous recombination at a desired site within the
genome, thus providing for intra-chromosomal expression of these
nucleic acids (see e.g. Koller and Smithies, 1989. Proc Natl Acad
Sci USA 86: 8932-8935).
[0238] Delivery of the nucleic acid as defined above according to
the invention into a patient for the purpose of gene therapy,
particular in the context of the above mentioned diseases or
disorders strongly related to JNK signaling as defined above may be
either direct (i.e. the patient is directly exposed to the nucleic
acid or nucleic acid-containing vector) or indirect (i.e. cells are
first transformed with the nucleic acid in vitro, then transplanted
into the patient), whereby in general the routes of administration
as mentioned above for the pharmaceutical composition apply as
well, however, a local administration for example by local
injection into the tissue or organ to be treated is preferred.
These two approaches are known, respectively, as in vivo or ex vivo
gene therapy. In a specific embodiment of the present invention, a
nucleic acid is directly administered in vivo, where it is
expressed to produce the encoded product. This may be accomplished
by any of numerous methods known in the art including, e.g.
constructing the nucleic acid as part of an appropriate nucleic
acid expression vector and administering the same in a manner such
that it becomes intracellular (e.g. by infection using a defective
or attenuated retroviral, adeno-associated viral or other viral
vector; see U.S. Pat. No. 4,980,286); directly injecting naked DNA;
using microparticle bombardment (e.g. a "GeneGun"; Biolistic,
DuPont); coating the nucleic acids with lipids; using associated
cell-surface receptors/transfecting agents; encapsulating in
liposomes, microparticles, or microcapsules; administering it in
linkage to a peptide that is known to enter the nucleus; or by
administering it in linkage to a ligand predisposed to
receptor-mediated endocytosis (see e.g. Wu and Wu, 1987. J Biol
Chem 262: 4429-4432), which can be used to "target" cell types that
specifically express the receptors of interest, etc.
[0239] An additional approach to gene therapy in the practice of
the present invention involves transferring a gene (comprising a
nucleic acid as defined above) into cells in in vitro tissue
culture by such methods as electroporation, lipofection, calcium
phosphate-mediated transfection, viral infection, or the like.
Generally, the method of transfer includes the concomitant transfer
of a selectable marker to the cells. The cells are then placed
under selection pressure (e.g. antibiotic resistance) so as to
facilitate the isolation of those cells that have taken up, and are
expressing, the transferred gene. Those cells are then delivered to
a patient. In a specific embodiment, prior to the in vivo
administration of the resulting recombinant cell, the nucleic acid
is introduced into a cell by any method known within the art
including e.g. transfection, electroporation, microinjection,
infection with a viral or bacteriophage vector containing the
nucleic acid sequences of interest, cell fusion,
chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, and similar methods that ensure that
the necessary developmental and physiological functions of the
recipient cells are not disrupted by the transfer. See e.g.
Loeffler and Behr, 1993. Meth Enzymol 217: 599-618. The chosen
technique should provide for the stable transfer of the nucleic
acid to the cell, such that the nucleic acid is expressible by the
cell. Preferably, the transferred nucleic acid is heritable and
expressible by the cell progeny.
[0240] In preferred embodiments of the present invention, the
resulting recombinant cells may be delivered to a patient by
various methods known within the art including, e.g. injection of
epithelial cells (e.g. subcutaneously), application of recombinant
skin cells as a skin graft onto the patient, and intravenous
injection of recombinant blood cells (e.g. hematopoietic stem or
progenitor cells). The total amount of cells that are envisioned
for use depend upon the desired effect, patient state, and the
like, and may be determined by one skilled within the art. Cells
into which a nucleic acid can be introduced for purposes of gene
therapy encompass any desired, available cell type, and may be
xenogeneic, heterogeneic, syngeneic, or autogeneic. Cell types
include, but are not limited to, differentiated cells such as
epithelial cells, endothelial cells, keratinocytes, fibroblasts,
muscle cells, hepatocytes and blood cells, or various stem or
progenitor cells, in particular embryonic heart muscle cells, liver
stem cells (International Patent Publication WO 94/08598), neural
stem cells (Stemple and Anderson, 1992, Cell 71: 973-985),
hematopoietic stem or progenitor cells, e.g. as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver, and
the like. In a preferred embodiment, the cells utilized for gene
therapy are autologous to the patient.
[0241] Alternatively and/or additionally, for treating diseases as
mentioned herein targeting therapies may be used to deliver the JNK
inhibitor sequences, chimeric peptides, and/or nucleic acids as
defined above more specifically to certain types of cell, by the
use of targeting systems such as (a targeting) antibody or cell
specific ligands. Antibodies used for targeting are typically
specific for cell surface proteins of cells associated with any of
the diseases as defined below. By way of example, these antibodies
may be directed to cell surface antibodies such as e.g. B
cell-associated surface proteins such as MHC class II DR protein,
CD18 (LFA-1 beta chain), CD45RO, CD40 or Bgp95, or cell surface
proteins selected from e.g. CD2, CD4, CD5, CD7, CD8, CD9, CD10,
CD13, CD16, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33,
CD34, CD38, CD39, CD4, CD43, CD45, CD52, CD56, CD68, CD71, CD138,
etc. Targeting constructs may be typically prepared by covalently
binding the JNK inhibitor sequences, chimeric peptides, and nucleic
acids as defined herein according to the invention to an antibody
specific for a cell surface protein or by binding to a cell
specific ligand. Proteins may e.g. be bound to such an antibody or
may be attached thereto by a peptide bond or by chemical coupling,
crosslinking, etc. The targeting therapy may then be carried out by
administering the targeting construct in a pharmaceutically
efficient amount to a patient by any of the administration routes
as defined below, e.g. intraperitoneal, nasal, intravenous, oral
and patch delivery routes. Preferably, the JNK inhibitor sequences,
chimeric peptides, or nucleic acids as defined herein according to
the invention, being attached to the targeting antibodies or cell
specific ligands as defined above, may be released in vitro or in
vivo, e.g. by hydrolysis of the covalent bond, by peptidases or by
any other suitable method. Alternatively, if the JNK inhibitor
sequences, chimeric peptides, or nucleic acids as defined herein
according to the invention are attached to a small cell specific
ligand, release of the ligand may not be carried out. If present at
the cell surface, the chimeric peptides may enter the cell upon the
activity of its trafficking sequence. Targeting may be desirable
for a variety of reasons; for example if the JNK inhibitor
sequences, chimeric peptides, and nucleic acids as defined herein
according to the invention are unacceptably toxic or if it would
otherwise require a too high dosage.
[0242] Instead of administering the JNK inhibitor sequences and/or
chimeric peptides as defined herein according to the invention
directly, they could be produced in the target cells by expression
from an encoding gene introduced into the cells, e.g. from a viral
vector to be administered. The viral vector typically encodes the
JNK inhibitor sequences and/or chimeric peptides as defined herein
according to the invention. The vector could be targeted to the
specific cells to be treated. Moreover, the vector could contain
regulatory elements, which are switched on more or less selectively
by the target cells upon defined regulation. This technique
represents a variant of the VDEPT technique (virus-directed enzyme
prodrug therapy), which utilizes mature proteins instead of their
precursor forms.
[0243] Alternatively, the JNK inhibitor sequences and/or chimeric
peptides as defined herein could be administered in a precursor
form by use of an antibody or a virus. These JNK inhibitor
sequences and/or chimeric peptides may then be converted into the
active form by an activating agent produced in, or targeted to, the
cells to be treated. This type of approach is sometimes known as
ADEPT (antibody-directed enzyme prodrug therapy) or VDEPT
(virus-directed enzyme prodrug therapy); the former involving
targeting the activating agent to the cells by conjugation to a
cell-specific antibody, while the latter involves producing the
activating agent, e.g. a JNK inhibitor sequence or the chimeric
peptide, in a vector by expression from encoding DNA in a viral
vector (see for example, EP-A-415731 and WO 90/07936).
[0244] According to another preferred embodiment, the JNK inhibitor
sequences, chimeric peptides, nucleic acid sequences or antibodies
to JNK inhibitor sequences or to chimeric peptides as defined
herein, e.g. an JNK inhibitor sequence according to any of
sequences of SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100 and/or a
chimeric peptide according to any of sequences of SEQ ID NOs: 9 to
12 and 23 to 32, preferably SEQ ID NO: 11, and/or an JNK inhibitor
sequence according to any of sequences of SEQ ID NOs: 1 to 4 and 13
to 20 and 33-100 comprising a trafficking sequence according to any
of SEQ ID NOs: 5 to 8 and 21 to 22, or variants or fragments
thereof within the above definitions, may be utilized for the
treatment of a tissue or organ prior to its transplantation.
Preferably, a solution for the isolation, transport, perfusion,
implantation or the like of an organ and/or tissue to be
transplanted comprises the JNK inhibitor according to the present
invention, preferably in a concentration in the range of 1 to 1000
.mu.M, more preferably in the range of 10 to 500 .mu.M, even more
preferably in the range of 50 to 150 .mu.M. For this aspect of the
invention, the transplant is a kidney, heart, lung, pancreas, in
particular pancreatic islets (also called islets of Langerhans),
liver, blood cell, bone marrow, cornea, accidental severed limb, in
particular fingers, hand, foot, face, nose, bone, cardiac valve,
blood vessel or intestine transplant, preferably a kidney, heart,
pancreas, in particular pancreatic islets (also called islets of
Langerhans), or skin transplant. For example, the JNK inhibitor
according to the invention may be contained in the solution for the
isolation of pancreatic islets. Such a solution may be for example
injected into the pancreatic duct prior to isolation. Moreover, it
is preferred if a solution containing the JNK inhibitor according
to the invention is applied in isolation, transport, perfusion,
transplantation or the like of an organ and/or tissue, in
particular if the time of ischemia exceeds 15 min, more preferably,
if the time of ischemia exceeds 20 min, even more preferably if the
time of ischemia is at least 30 min. These ischemia times may apply
to warm and/or cold ischemia time, however, it is particularly
preferred if they apply exclusively to warm ischemia time (WIT),
whereby WIT refers to the length of time that elapses between a
donor's death, in particular from the time of cross-clamping or of
asystole in non-heart-beating donors, until cold perfusion is
commenced and to ischemia during implantation, from removal of the
organ from ice until reperfusion.
[0245] According to a further embodiment, the JNK inhibitor
sequences, chimeric peptides, nucleic acid sequences or antibodies
to JNK inhibitor sequences or to chimeric peptides as defined
herein, e.g. an JNK inhibitor sequence according to any of
sequences of SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100 and/or a
chimeric peptide according to any of sequences of SEQ ID NOs: 9 to
12 and 23 to 32, and/or an JNK inhibitor sequence according to any
of sequences of SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100
comprising a trafficking sequence according to any of SEQ ID NOs: 5
to 8 and 21 to 22, or variants or fragments thereof within the
above definitions, may be utilized in (in vitro) assays (e.g.
immunoassays) to detect, prognose, diagnose, or monitor various
conditions and disease states selected from diseases or disorders
strongly related to JNK signaling as defined above, or monitor the
treatment thereof. The immunoassay may be performed by a method
comprising contacting a sample derived from a patient with an
antibody to an JNK inhibitor sequence, a chimeric peptide, or a
nucleic acid sequence, as defined above, under conditions such that
immunospecific-binding may occur, and subsequently detecting or
measuring the amount of any immunospecific-binding by the antibody.
In a specific embodiment, an antibody specific for an JNK inhibitor
sequence, a chimeric peptide or a nucleic acid sequence may be used
to analyze a tissue or serum sample from a patient for the presence
of JNK or a JNK inhibitor sequence; wherein an aberrant level of
JNK is indicative of a diseased condition. The immunoassays that
may be utilized include, but are not limited to, competitive and
non-competitive assay systems using techniques such as Western
Blots, radioimmunoassays (RIA), enzyme linked immunosorbent assay
(ELISA), "sandwich" immunoassays, immunoprecipitation assays,
precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, fluorescent
immunoassays, complement-fixation assays, immunoradiometric assays,
and protein-A immunoassays, etc. Alternatively, (in vitro) assays
may be performed by delivering the JNK inhibitor sequences,
chimeric peptides, nucleic acid sequences or antibodies to JNK
inhibitor sequences or to chimeric peptides, as defined above, to
target cells typically selected from e.g. cultured animal cells,
human cells or micro-organisms, and to monitor the cell response by
biophysical methods typically known to a skilled person. The target
cells typically used therein may be cultured cells (in vitro) or in
vivo cells, i.e. cells composing the organs or tissues of living
animals or humans, or microorganisms found in living animals or
humans.
[0246] The present invention additionally provides the use of kits
for diagnostic or therapeutic purposes, particular for the
treatment, prevention or monitoring of diseases or disorders
strongly related to JNK signaling as defined above, wherein the kit
includes one or more containers containing JNK inhibitor sequences,
chimeric peptides, nucleic acid sequences and/or antibodies to
these JNK inhibitor sequences or to chimeric peptides as defined
above, e.g. an anti-JNK inhibitor sequence antibody to an JNK
inhibitor sequence according to any of sequences of SEQ ID NOs: 1
to 4 and 13 to 20 and 33-100, to a chimeric peptide according to
any of sequences of SEQ ID NOs: 9 to 12 and 23 to 32, to an JNK
inhibitor sequence according to any of sequences of SEQ ID NOs: 1
to 4 and 13 to 20 and 33-100 comprising a trafficking sequence
according to any of SEQ ID NOs: 5 to 8 and 21 to 22, or to or
variants or fragments thereof within the above definitions, or such
an anti-JNK inhibitor sequence antibody and, optionally, a labeled
binding partner to the antibody. The label incorporated thereby
into the antibody may include, but is not limited to, a
chemiluminescent, enzymatic, fluorescent, colorimetric or
radioactive moiety. In another specific embodiment, kits for
diagnostic use in the treatment, prevention or monitoring of
diseases or disorders strongly related to JNK signaling as defined
above are provided which comprise one or more containers containing
nucleic acids that encode, or alternatively, that are the
complement to, an JNK inhibitor sequence and/or a chimeric peptide
as defined above, optionally, a labeled binding partner to these
nucleic acids, are also provided. In an alternative specific
embodiment, the kit may be used for the above purposes as a kit,
comprising one or more containers, a pair of oligonucleotide
primers (e.g. each 6-30 nucleotides in length) that are capable of
acting as amplification primers for polymerase chain reaction (PCR;
see e.g. Innis, et al., 1990. PCR PROTOCOLS, Academic Press, Inc.,
San Diego, Calif.), ligase chain reaction, cyclic probe reaction,
and the like, or other methods known within the art used in context
with the nucleic acids as defined above. The kit may, optionally,
further comprise a predetermined amount of a purified JNK inhibitor
sequence as defined above, a chimeric peptide as defined above, or
nucleic acids encoding these, for use as a diagnostic, standard, or
control in the assays for the above purposes.
[0247] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
fall within the scope of the appended claims.
[0248] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entirety.
[0249] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent
from the following detailed description and claims.
DESCRIPTION OF FIGURES
[0250] FIG. 1 are diagrams showing alignments of conserved JBD
domain regions in the indicated transcription factors. JNK
inhibitor sequences used herein were identified by carrying out
sequence alignments. The results of this alignment are exemplarily
shown in FIGS. 1A-1C. FIG. 1A depicts the region of highest
homology between the JBDs of IB1, IB2, c-Jun and ATF2. Panel B
depicts the amino acid sequence of the JBDs of L-IB1(s) and L-IB1
for comparative reasons. Fully conserved residues are indicated by
asterisks, while residues changed to Ala in the GFP-JBD.sub.23Mut
vector are indicated by open circles. FIG. 1C shows the amino acid
sequences of chimeric proteins that include a JNK inhibitor
sequence and a trafficking sequence. In the example shown, the
trafficking sequence is derived from the human immunodeficiency
virus (HIV) TAT polypeptide, and the JNK inhibitor sequence is
derived from an IB1(s) polypeptide. Human, mouse, and rat sequences
are identical in Panels B and C.
[0251] FIG. 2 is a diagram showing sequences of generic TAT-IB
fusion peptides from human, mouse and rat.
[0252] FIG. 3 depicts the results of the inhibition of endogeneous
JNK-activity in HepG2 cells using fusion peptides according to SEQ
ID NOs: 9 and 11 in an one-well approach. As can be seen from FIG.
3, particularly panel d in FIG. 3, D-TAT-IB1(s) according to SEQ ID
NO: 11 (here abbreviated as D-JNKI) effectively inhibits JNK
activity, even better than L-TAT-IB1 (s) according to SEQ ID NO: 9
(here abbreviated as L-JNKI).
[0253] FIG. 4 shows the result of the cytotoxicity assay with a
chimeric JNK inhibitor sequence according to SEQ ID NO: 11, also
termed XG-102 (see Example 12). As can be seen, XG-102 (SEQ ID NO:
11) is not cytotoxic for HFFs. HFFs were seeded in 96-well tissue
culture plates. Medium containing DMSO (same level as the 5 .mu.M
drug), or XG-102 at 1, 2, and 5 .mu.M was added for 24 h. Neutral
Red was briefly added, the cells were fixed, then the dye was
extracted. Absorbance was measured at 540 nm. No difference was
observed between DMSO and 1 .mu.M XG-102.
[0254] FIG. 5 depicts the results of the plaque reduction assay
carried out for testing activity of a chimeric JNK inhibitor
sequence according to SEQ ID NO: 11, also termed XG-102 against
Varizella Zoster Virus (VZV) (see Example 12). As can be seen,
XG-102 (SEQ ID NO: 11) is a potent inhibitor of Varizella Zoster
Virus (VZV), particularly at concentrations of 0.5 .mu.M and 1
.mu.M
[0255] FIG. 6 shows the results of the inhibition of Varizella
Zoster Virus (VZV) in cultured human fibroblasts using a chimeric
JNK inhibitor sequence according to SEQ ID NO: 11, also termed
XG-102 (see Example 12). As can be seen, VZV shows a significant
sensitivity to XG-102 (SEQ ID NO: 11). VZV replication was normal
in the presence of the negative control (XG-100, the Tat peptide
alone). XG-102 (SEQ ID NO: 11) thus prevented VZV replication
already at the lowest concentration tested of 0.25 .mu.M.
[0256] FIG. 7 depicts the activity of XG-102 (SEQ ID NO: 11) on
cell recruitment in lung using MPO in lung homogenization in the
treatment of Chronic Obstructive Pulmonary Disease (COPD) using an
animal model of Bleomycin induced acute lung inflammation. As can
be seen, MPO was not significantly induced after bleomycin
administration. XG-102 (SEQ ID NO: 11) had thus only little effect
on the MPO levels in the lung.
[0257] FIG. 8 depicts the activity of XG-102 (SEQ ID NO: 11) on TNF
levels in the treatment of Chronic Obstructive Pulmonary Disease
(COPD) using an animal model of Bleomycin induced acute lung
fibrosis. When measuring TNF levels, a trend to reduction of the
TNF level in BALF after administration of XG-102 (SEQ ID NO: 11)
was observed in the BLM model. TNF levels are very low after
BLM.
[0258] FIG. 9 depicts the activity of XG-102 (SEQ ID NO: 11) on
cell recruitment in bronchoalveolar lavage space in the treatment
of Chronic Obstructive Pulmonary Disease (COPD) using an animal
model of Bleomycin induced acute lung fibrosis. At 0.1 mg/kg,
XG-102 (SEQ ID NO: 11) reduces significantly the lymphocyte
recruitment and the number of total cells recruited during the
inflammatory stage characterised at this point by the lymphocytes
recruitment. At 0.1 mg/kg, XG-102 (SEQ ID NO: 11) enhances the
lymphocytes recruitment or the number of total cell into the
bronchoalveolar space (n=5 mice per group; *, p<0.05; **,
p<0.001).
[0259] FIG. 10 describes the results of the histology in the
treatment of Chronic Obstructive Pulmonary Disease (COPD) using an
animal model of Bleomycin induced acute lung fibrosis. 3 .mu.m
sections of lungs were stained with haematoxylin and eosin.
Inflammatory cells accumulation, fibrotic areas, loss of lung
architecture were observed 10 days after BLM administration. As can
be seen, a decrease of these parameters is observed after
administration of XG-102 at the low dose (0.001 mg/kg) but not with
the high dose (0.1 mg/kg).
[0260] FIG. 11 shows the effects of a treatment with XG-102 (SEQ ID
NO: 11) on brain A.beta..sub.1-40 and A.beta..sub.1-42 levels
determined by ELISA. The Graphs represent the A.beta..sub.1-40
(left) and A.beta..sub.1-42 (right) levels determined by ELISA in
different brain homogenate fractions with Triton 40 and Triton 42.
Data are represented as scattered dot plot with individual values
(black) and group mean.+-.SEM. Significant differences are marked
with asterisks (* p<0.05; ** p<0.01). Significant group
differences were observed only in Triton X-100 fraction for
A.beta..sub.1-42.
[0261] FIG. 12 depicts the effects of a treatment with XG-102 (SEQ
ID NO: 11) on CSF A.beta..sub.1-40 and A.beta..sub.1-42 levels
determined by ELISA. The Graphs represent the A.beta..sub.1-40
(left) and A.beta..sub.1-42 (right) levels determined by ELISA in
CSF. Data are represented as scattered dot plot with individual
values (black) and group mean.+-.SEM. Significant differences are
marked with asterisks (* p<0.05; ** p<0.01). Treatment with
XG-102 (SEQ ID NO: 11) in both dosages led to a significant
decrease of A.beta..sub.1-40 and A.beta..sub.1-42 in CSF.
[0262] FIG. 13 shows the treatment effects on the ThioflavinS
staining visualized number of plaques in the hAPP Tg mice: The
graphs represent the number of ThioflavinS stained plaques per
mm.sup.2 in the cortex and the hippocampus. XG-102 (SEQ ID NO: 11)
treatment reduced the number of plaques negatively dose-dependent
in the hippocampus. Data are represented as means+SEM. N=8 per
group. * . . . p<0.05; ** . . . p<0.01.
[0263] FIG. 14 depicts the treatment effects on the ThioflavinS
visualized plaque area [%] in the hAPP Tg mice: The Graphs
represent the plaque area [%] of ThioflavinS positive plaques in
the cortex and the hippocampus. XG-102 (SEQ ID NO: 11)
significantly reduced the area obtained by plaques in the
hippocampus. Data are represented as means+SEM. N=8 per group.
[0264] FIG. 15 describes the results of the administration of
XG-102 (SEQ ID NO: 11) on fasting blood glucose levels (absolute
and relative) in the animal model for diabetes type 2. Fasting
blood glucose was measured every third day until day 68 and on a
regular basis until termination at day 111 in groups A and C. We
observed a clear and significant (p<0.001) decrease in the level
of fasting blood glucose of the diabetic db/db mice treated with
XG-102 (SEQ ID NO: 11) (10 mg/kg) as compared to vehicle control.
The fasting blood glucose levels of the mice treated with XG-102
(SEQ ID NO: 11) (10 mg/kg) reached a low plateau of approximately 5
mmol/L. This effect was evident after 14 days of dosing and
persisted throughout the study, thus during the entire wash-out
period from day 21 to day 111. In contrast, we observed no effect
of low dose of XG-102 (SEQ ID NO: 11) (1 mg/kg) during 28 days of
dosing.
[0265] FIG. 16 describes the results of the administration of
XG-102 (SEQ ID NO: 11), 10 mg/kg on body weight in the animal model
for diabetes type 2. We observed a clear and significant
(p<0.001) prevention of body weight increase in mice treated
with XG-102 (SEQ ID NO: 11) (10 mg/kg) as compared to vehicle
control. This effect was evident from day 28 of dosing and remained
until the day of termination day 111. In contrast, we observed no
effect of low dose of XG-102 (SEQ ID NO: 11) (1 mg/kg) on body
weight during 28 days of dosing.
[0266] FIG. 17, 18 describe the effect of vehicle or XG-102 (SEQ ID
NO: 11) (10 mg/kg) in the animal model for diabetes type 2 on 24
hour food and water intake, and urine and faeces production as
measured in metabolic cages on study day 68 in FIGS. 17 (g) and 18
(normalized to g of body weight). We observed no significant
effects of XG-102 (SEQ ID NO: 11) (10 mg/kg) on any of the measured
parameters as compared to vehicle control though a trend towards a
decrease in food intake and urine production was observed.
[0267] FIG. 19, 20 describe the effect of vehicle or XG-102 (SEQ ID
NO: 11) (10 mg/kg) in the animal model for diabetes type 2 as
measured on day 57, 77 and 108 on plasma levels of insulin, MCP-1
and IL-6 in FIG. 19; on plasma levels of tPAI-1, TNF and resistin
in FIG. 20; We observed no significant effects of XG-102 (SEQ ID
NO: 11) (10 mg/kg) on any of the measured parameters as compared to
vehicle control except the levels of plasma resistin, which was
significantly higher in XG-102 (SEQ ID NO: 11) treated animals at
day 77 and 108.
[0268] FIG. 21 shows the effect of vehicle or XG-102 (SEQ ID NO:
11) (10 mg/kg) in the animal model for diabetes type 2 on tissue
weight of epididymal, inguinal subcutaneous, and retroperitoneal
fat pads. We observed a significant decrease of epididymal
(p<0.05) and retroperitoneal (p<0.01) fat mass in the mice
treated with XG-102 as compared to vehicle control.
[0269] FIG. 22 depicts the effect of vehicle or XG-102 (SEQ ID NO:
11) (10 mg/kg) in the animal model for diabetes type 2 on tissue
weight of brain, spleen and heart. We observed no significant
effects of XG-102 (SEQ ID NO: 11) (10 mg/kg) on these parameters as
compared to vehicle control.
[0270] FIG. 23 describes the effect of vehicle or XG-102 (SEQ ID
NO: 11) (10 mg/kg) in the animal model for diabetes type 2 on
tissue weight of kidney and liver. We observed a significant
decrease of kidney (p<0.05) and liver (p<0.01) mass in the
mice treated with XG-102 (SEQ ID NO: 11) as compared to vehicle
control.
[0271] FIG. 24 Primary cultured macrophages were incubated with
XG-102 (SEQ ID NO: 11) and extensively washed. Presence of XG-102
(SEQ ID NO: 11) was revealed using a specific antibody against
XG-102. XG-102 is strongly incorporated into primary
macrophages.
[0272] FIG. 25 Mice were treated via three different routes of
administration (s.c., i.v., i.p.) with radiolabeled peptides with
C.sup.14 (1 mg/kg). Animals were sacrificed 72 hours after
injection and processed for immunoradiography. Sagital sections
were exposed and revealed the accumulation XG-102 peptides in the
liver, spleen, and bone marrow predominantly (XG-102: SEQ ID NO:
11).
[0273] FIG. 26 shows an immunostaining against XG-102 (SEQ ID NO:
11) in the liver of rats injected with 1 mg/kg of XG-102 i.v.
Animals were sacrificed 24 hours after injection. Revelation was
done using DAB substrate. This figure shows again the pronounced
accumulation of XG-102 in the liver, and especially, in the Kupffer
cells (macrophages).
[0274] FIG. 27 shows the inhibition of Cytokine & Chemokine
Release in two cell lines. XG-102 (SEQ ID NO:11) inhibits cytokine
release in both myeloid and lymphoid cell lines, reducing
LPS-induced TNFa, IL-6 and MCP-1 release in THP-1 cells (Panels
A-C) and PMA & ionomycin-induced IFNg, IL-6 and IL-2 production
in Jurkat cells (Panels D-F). The control (XG-101) is less
effective due to its lesser stability.
[0275] FIG. 28 shows the inhibition of cytokine release in primary
cells. XG-102 (SEQ ID NO:11) also inhibits cytokine release in
primary lymphoid and myeloid cells, reducing LPS-induced TNFa, IL-6
and Rantes release in murine macrophages (Panels A-C) and PMA &
ionomycin-induced TNFa and IFNg production in murine T cells
(Panels D-E). Effects occur at non-cytotoxic concentrations of
XG-102 (Panel F)
[0276] FIG. 29 shows the IB1 cDNA sequence from rat and its
predicted amino acid sequence (SEQ ID NO:102)
[0277] FIG. 30 shows the IB1 protein sequence from rat encoded by
the exon-intron boundary of the rIB1 gene-splice donor (SEQ ID
NO:103)
[0278] FIG. 31 shows the IB1 protein sequence from Homo sapiens
(SEQ ID NO:104)
[0279] FIG. 32 shows the IB1 cDNA sequence from Homo sapiens (SEQ
ID NO:105)
[0280] FIG. 33 Effect on islet Isolation on JNK/p38 activation.
That experiment was designed to identify any effect evoked by the
isolation process as such on JNK or p38. As control tubulin
detection was used. Western blot staining as a function of the
digestion time (min) is shown.
[0281] FIG. 34 By that figure the effect of XG-102 on JNK
activation during isolation is shown.
[0282] FIG. 35 Effect of XG-102 on JNK activation during
isolation
[0283] FIG. 36 Effect of XG-102 on OCR/DNA during islolation
[0284] FIG. 37 Effect of XG-102 (DJNK inhibitor) on ATP/proteinJ by
HPLC analysis
[0285] FIG. 38 shows that XG-103 increases significantly islet
viability (OCR/DNA) as measured after 7 days of culturing
[0286] FIG. 39 FIG. 41 (A): Fluorescein angiography evaluation
(mean score) ten minutes after fluorescein injection. The mean
score is presented for day 14 and day 21 for five groups (XG-102
300 microgramm/ml, XG-102 3 mg/ml, Kencort retard, 0.9% NaCl
solution, untreated)
[0287] FIG. 41 (B) Proportion of fluorescein angiography evaluation
(mean score) ten minutes after fluorescein injection, for five
groups (XG-102 300 microgramm/ml, XG-102 3 mg/ml, Kencort retard,
0.9% NaCl solution, untreated) at day 14 and day 21.
[0288] FIG. 41 (C) Incidence of ChNV formation ten minutes after
fluorescein injection at day 14 and 21, for five groups (XG-102 300
microgramm/ml, XG-102 3 mg/ml, Kencort retard, 0.9% NaCl solution,
untreated).
[0289] FIG. 41 (D) Incidence of fluorescein leakage extend at day
14 and day 21; for five groups (XG-102 300 microgramm/ml, XG-102 3
mg/ml, Kencort retard, 0.9% NaCl solution, untreated)
[0290] FIG. 40 The design of the experiment for assessing XG-102's
effect on kidney tissue upon adriamycin-induced induction of
nephropathy is shown. The rat groups and the and their treatment
regimen is shown.
[0291] FIG. 41 It is shown that XG-102 does not evoke any adverse
effect as to proteinuria. The ELISA assay was used to dertmine the
albumin concentration for group 1, group 4 and group 5 as a
function of the observation period (day 5, 8, 11, 14, 17, 20, 23,
26, 29, 32, 25, 38, 41)
[0292] FIG. 42 Histological analysis 8 days after the onset of the
experiment. Comparison of adriamycin treated rats of group 1 (left
hand) and adriamycin and XG-102 treated rats of group 4 (right
hand)
[0293] FIG. 43 Histological analysis 14 days after the onset of the
experiment. Comparison of adriamycin treated rats of group 1 (left
hand) and adriamycin and XG-102 treated rats of group 4 (right
hand)
[0294] FIG. 44 Histological analysis 19 days after the onset of the
experiment. Comparison of adriamycin treated rats of group 1 (left
hand) and adriamycin and XG-102 treated rats of group 4 (right
hand)
[0295] FIG. 45 Histological analysis 41 days after the onset of the
experiment. Comparison of adriamycin treated rats of group 1 (left
hand) and adriamycin and XG-102 treated rats of group 4 (right
hand)
[0296] FIG. 46 Histological analysis (staining) of c-jun expression
8 days after onset of the experiment. Left hand Adriamycin treated
histological preparation, in the middle: Adriamycin and XG-102
treated (resulting in a significant reduction of c-jun expression
in the interstitium) and control on the right.
[0297] FIG. 47 Histological analysis (staining) of c-jun expression
14 days after onset of the experiment. Left hand Adriamycin treated
histological preparation, in the middle: Adriamycin and XG-102
treated (resulting in a significant reduction of c-jun expression
in the interstitium) and control on the right.
[0298] FIG. 48 shows the renal function assessed by protidemia (A)
and urea level (B) of rats in an Adriamycin (ADR)-induced
nephropathy model on Days 8, 14, 29, 41 and 56 after ADR
administration. Groups No. 1 ("ADR") and No. 2 ("ADR+XG-102") have
been treated on Day 0 with ADR to induce necropathy, whereas groups
No. 3 ("NaCl") and No. 4 ("XG-102") received 0.9% NaCL. Moreover,
groups Nos. 2 and 4 have been treated on Day 0 with XG-102, whereas
groups Nos. 1 and 3 received vehicle (0.9% NaCl).
[0299] FIG. 49 shows kidney sections of the rats in the Adriamycin
(ADR)-induced nephropathy model stained with periodic acid-Schiff
(PAS) (original magnification .times.40). For the sections shown in
the left column, rats were sacrificed at Day 8 following ADR
administration, whereas for the sections shown in the left column,
rats were sacrificed at Day 56. ADR has been administered only to
the groups "ADR" and "ADR+XG102", whereas the group "NaCl" received
0.9% NaCL only. The group "ADR+XG102" has been treated on Day 0
with XG-102, whereas the other groups ("ADR" and "NaCl") received
vehicle (0.9% NaCl).
[0300] FIG. 50 shows the kidney fibrosis in ADR nephropathy
evaluated with Masson's trichrome (blue) on Days 8 (left four
panels) and 56 (right four panels) following ADR administration for
the group "ADR" (upper panel), which has been treated with ADR and
vehicle at Day 0 and for the group "ADR+XG102" (lower panel), which
has been treated with ADR and XG-102 at Day 0. The original
magnification .times.10 is depicted in the left panels for the
respective day and the original magnification .times.40 is depicted
in the right panels for the respective day.
[0301] FIG. 51 The study design of the experiment investigating the
effects of XG-102 on puromycine aminonucleoside (PAN)-induced
nephropathy. On day 0 and day 14 PAN or its vehicle have been
injected for induction of nephropathy. At day 0 and at day 14, PAN
has been administered first, followed by XG-102 administration.
From day 0 to day 42 XG-102 or its vehicle have been administered
once a week by i.v. route as described above. On day 56 animals
have been sacrificed and samples (blood and kidneys) have been
collected.
[0302] FIG. 52 shows the effects of XG-102 on the
glomerulosclerosis injury in puromycine aminonucleoside
(PAN)-induced nephropathy. XG-102 has been administered to Groups 3
to 6 (labelled as "cpd" in the legend). The Group 2 and the Group 6
are different in term of number of iv injections as stated in the
study plan of Example 20. Note that the score for Group 2 is very
similar to the one reported by Najakima et al. (2010) using the
same experimental protocol. ***P<0.001 versus Group 1 using
unpaired Student t-test; # P<0.05; ### P<0.001 versus Group 2
using one-way ANOVA followed by followed by Newman-Keuls test;
.sctn..sctn..sctn.P<0.001 versus Group 2 using unpaired Student
t-test.
[0303] FIG. 53 shows the effects of XG-102 on the glomerular damage
in puromycine aminonucleoside (PAN)-induced nephropathy. XG-102 has
been administered to Groups 3 to 6 (labelled as "cpd" in the
legend). The Group 2 and the Group 6 are different in term of
number of iv injections as stated in the study plan of Example 20.
***P<0.001 versus Group 1 using unpaired Student t-test;
###P<0.001 versus Group 2 using one-way ANOVA followed by
followed by Newman-Keuls test; .sctn..sctn..sctn.P<0.001 versus
Group 2 using unpaired Student t-test.
[0304] FIG. 54 shows the study schedule of Example 21 investigating
the effects of chronic administration of XG-102 in a rat model of
diabetic nephropathy. Animals were placed on high fat diet
immediately after arrival. Animals in groups E and F are dosed
daily each day from baseline phase onwards.
[0305] FIG. 55 shows the effects of chronic administration of
XG-102 in a rat model of diabetic nephropathy on the body weight of
the rats. Only non-STZ treated rats showed an increase in body
weight. Rats treated with XG-102 showed no differences in body
weight compared to vehicle-treated rats in the STZ model. The body
weight of rats treated with the positive reference (Losartan),
however, was significantly lower.
[0306] FIG. 56 shows that XG-102 dose-dependently decreased JNK (A)
and PAF2 (B) phosphorylation induced by 15-min ischemia in an
experiment evaluating the dose-response to XG-102 in islet
isolation/transplantation (Example 22). Isolation of rat islets has
been carried out either immediately after animal sacrifice or after
a 15-minute period of warm ischemia. JNK activation has been
assessed by western blot at the end of the isolation process. As
negative controls, JNK activation has been assessed on unprocessed
rat pancreases.
[0307] FIG. 57 shows the effects of XG-102 on function and
viability of rat pancreatic islets, whereby the islets have been
isolated islets from 15 min ischemia rat and from no ischemia rat.
A static insulin secretion test (basal or stimulated using glucose)
has been performed directly after islet isolation and 18 h after
culture at 37.degree. C. Isolation affected islet function, whereby
basal insulin secretion was higher in islets used directly after
isolation compared to islets incubated during 18 h whatever the
conditions. However after culture, ischemia and inhibitor XG-102
had no impact on islet function in this experiment.
[0308] FIG. 58 shows another experiment wherein ischemia was pushed
until 30 min and XG-102 was used at 100 microM. Still, a high basal
secretion is observed when insulin secretion test was performed
directly after isolation. Moreover, 30 min ischemia had a negative
impact on islet function. These preliminary results suggested that
30 min ischemia seems to be a better model than 15 min to induce
JNK activation. When islets from ischemic rats were isolated and
incubated with XG-102, glucose-induced insulin secretion was higher
as compared to ischemic rats.
[0309] FIG. 59 The disposition of patients included in the study of
Example 27, i.e. the randomized, double-blind, parallel group,
controlled, multicentre trial to assess the efficacy and safety of
a single sub-conjunctival injection of XG-102, compared to
dexamethasone eye drops in post-surgery intraocular inflammation
(Clinical Phase II).
[0310] FIG. 60 shows for the study of Example 27 the mean anterior
chamber cell grade up to 28 days after the administration of the
sub-conjunctival injection of study treatment for the PP analysis
population for the three treatment groups XG-102 90 Ng, XG-102 900
.mu.g and the dexamethasone. The vertical lines represents the
standard deviations (SD).
[0311] FIG. 61 shows for the study of Example 27 the results of the
primary outcome in addition to the first secondary outcome for both
the PP and FAS data sets regarding anterior chamber cell grade at
day 28: Confidence Intervals and the Non-inferiority margin.
[0312] FIG. 62 shows for the study of Example 27 the anterior
chamber flare grade (for the FAS) obtained up to day 28 after the
administration of the sub-conjunctival injection of study treatment
for the three treatment groups XG-102 90 .mu.g, XG-102 900 .mu.g
and the dexamethasone. The vertical lines represents the standard
deviations (SD).
[0313] FIG. 63 shows for the study of Example 27 the LFM (Laser
Flare Meter) measurements which were obtained at the defined time
points throughout the study up to day 28 for the FAS. The vertical
lines represents the standard deviations (SD).
[0314] FIG. 64 shows for the study of Example 27 the overview of
reported adverse events (serious and non-serious) by dose
group.
[0315] FIG. 65 shows for the study of Example 27 the summary of the
AEs (sorted by MedDRA SOC and PT term) which were reported for at
least 2% of patients randomized to either of the three study
groups.
[0316] FIG. 66 shows for the study of Example 27 the overview of
the reported serious adverse events (SAEs).
[0317] FIG. 67 shows for Example 28 the proliferation of
hepatocytes in XG-102 (in the figure referred to as "D-JNKI1") or
PBS treated Mapk14.sup.f/f and Mapk14.sup..DELTA.Ii* mice (left
panel) and in XG-102 (i.e. "D-JNKI1") treated Mapk14.sup.f/f
Jun.sup.f/f and Mapk14.sup..DELTA.Ii* Jun.sup..DELTA.Ii* mice
(right panel). Mice were injected with either XG-102 (20 mg per kg
body weight) or PBS, if applicable, before DEN treatment. The
proliferation of hepatocytes was analyzed by Ki67 staining 48 h
after DEN treatment. Quantification of Ki67-positive cells is
shown.
[0318] FIG. 68 3.times.10.sup.6 Huh7 human liver cancer cells were
injected subcutaneously to both flank area of nude mice at 4 weeks
of age (Example 29). Nude mice treated with XG-102
intraperitoneally twice a week at 5 mg/kg after Huh7 injection.
Tumor volumes were measured twice a week. Mice were killed 4 week
after xenograft. Dotted cycles indicate the xenografted tumors.
[0319] FIG. 69 shows for Example 30 the mean body weight and mean
body weight change curves of mice bearing orthotopically injected
HEP G2 tumor are shown. Mice were IV treated with XG-102 at 1
mg/kg/inj following the Q4D.times.4 treatment schedule repeated two
times, at D10 and D41. Accordingly, in FIG. 70 the respective
statistical data are presented.
[0320] FIG. 70 shows for Example 30 the tolerance of mice to
XG-102. Mean body weights and MBWC.+-.SD are indicated. MBWC %
corresponds to variation of mean body weight between the considered
day and day of first treatment (D10). Statistical analysis was
performed with the Bonferroni-Dunn test, taking vehicle treated
group as reference.
[0321] FIG. 71 shows for Example 30 the mice long survival curves,
whereby proportion of surviving mice per group until sacrifice day
(D185) is depicted. Mice were treated with XG-102 at the indicated
doses following the Q4D.times.4 treatment schedule repeated two
times, at D10 and D41.
[0322] FIG. 72 shows for Example 31 the tolerance of mice to XG-102
and XG-414 treatments, alone or in combination. Mean body weights
and mean body weight changes.+-.SD are indicated. MBWC %
corresponds to variation of mean body weight between the considered
day and day of first treatment (D10).
[0323] FIG. 73 shows for Example 31 the mice long survival curves,
whereby proportion of surviving mice per group until sacrifice day
(D171) is depicted. Mice sacrificed at D67 for autopsy were
excluded from calculation. Mice were treated with XG-102 at the
indicated doses following the Q4D.times.4 treatment schedule
repeated two timed, at D10 and D41.
[0324] FIG. 74 shows for Example 31 the tumor invasion observed by
microscopic evaluation of mice sacrificed at 067 or between D67 and
final sacrifice as histogram representations. The level of tumor
take was classified in 4 different categories specified in the
figure legend.
[0325] FIG. 75 shows for Example 32 the mean tumor volume of PC-3
tumor bearing mice during the antitumor activity experiment. At
D33, 3 groups of 5 animals were treated with vehicle and XG-102
(0.1 and 1 mg/kg/inj, Q4D.times.4).
[0326] FIG. 76 shows for Example 33 a histogram representation of
metastatic tumor invasion observed within liver or at its periphery
(hilus) twenty-six days after HCT 116 tumor xenografting on mice
caecum, in the different groups, PO or SC treated with vehicle or
XG-102 at 0.1 and 1 mgl/kg/adm. following the Q1D.times.14
treatment schedule. The classification of microscopic observations
was performed as described within the legend.
[0327] FIG. 77 shows for Example 34 the electroretinography (ERG)
measurements in right eyes of albino rats.
[0328] FIG. 78 Renal ischemia was induced in rats of group G2 and
group G3 by clamping both renal pedicles with atraumatic clamp for
40 min, whereas in group G1 rats no ischemia was induced. Rats of
group G3 received a single dose of 2 mg/kg XG-102 (in 0.9% NaCl as
vehicle) and rats of groups G1 and G2 received vehicle,
respectively, by IV injection in the tail vein on Day 0, one hour
after clamping period (after reperfusion) both renal pedicles with
atraumatic clamp. Serum creatinine (FIG. 78A) and urea (FIG. 78B)
were increased in vehicle-treated ischemic rats (G2) 24 h following
ischemia, as compared to vehicle-treated controls rats without
ischemia (G1). On the other hand, XG-102-treated-ischemic rats (G3)
exhibited lower serum creatinine, relatively to untreated ischemic
rats (G2).
[0329] FIG. 79 shows for Example 40 the impact of 30 min ischemia
and treatment with 100 .mu.M XG-102 on islet viability. Treatment
with XG-102 decreases apoptosis and necrosis. These results show
that XG-102 has a beneficial effect on islet viability
[0330] FIG. 80 shows for Example 40 a western blot. In these
experiments, 18 h after isolation, islets were pre-treated or not
with XG-102 100 .mu.M for 1 h and then submitted to hypoxia for 4
h, whereby XG-102 was still present (or not in control groups)
during the 4 hour hypoxia ("H4"). As expected, hypoxia ("H4")
induces JNK and JUN phosphorylation as compared to islets
maintained in normoxia conditions ("N4"). However, the JNK
inhibitor XG-102 did not inhibit phosphorylation of JNK and JUN
induced by hypoxia (cf. FIG. 80 "H4+XG102").
[0331] FIG. 81 shows for Example 40 the islet viability in the
hypoxia experiment. Hypoxia increased apoptosis and necrosis (H4
vs. N4). However, when islets were treated with XG-102, apoptosis
and necrosis were decreased either in normoxia and hypoxia
conditions. In conclusion XG102 had also a beneficial effect on
islet viability in this hypoxia model.
[0332] FIG. 82 shows the study design for Example 41.
[0333] FIG. 83 shows for Example 41 the effects of vehicle and
XG-102 (4 mg/kg, i.v.) on glomerular injury index at day 49 (Groups
1-5) and at day 77 (Groups 6-8) in a rat model of PAN-induced
nephropathy. ***P<0.001 group 2 and group 7 (PAN/vehicle) versus
group 1 and group 6 (Saline/vehicle) using unpaired Student t-test
(n=12-15/group). ## P<0.01; ### P<0.001 groups from 3 to 5
(PAN/XG-102) versus group 2 (PAN/vehicle) using one-way ANOVA
followed by Newman-Keuls test (n=15/group).
.sctn..sctn..sctn.P<0.001 group 8 (PAN/XG-102) versus group 7
(PAN/vehicle) using unpaired Student t-test (n=12-14/group).
[0334] FIG. 84 shows for Example 41 the effects of vehicle and
XG-102 (4 mg/kg, i.v.) on the percentage of injured glomeruli at
day 49 (Groups 1-5) and at day 77 (Groups 6-8) in a rat model of
PAN-induced nephropathy. ***P<0.001 group 2 and group 7
(PAN/vehicle) versus group 1 and group 6 (Saline/vehicle) using
unpaired Student t-test (n=12-15/group). ### P<0.001 groups from
3 to 5 (PAN/XG-102) versus group 2 (PAN/vehicle) using one-way
ANOVA followed by Newman-Keuls test (n=15/group).
.sctn..sctn..sctn.P<0.001 group 8 (PAN/XG-102) versus group 7
(PAN/vehicle) using unpaired Student t-test (n=12-14/group).
[0335] FIG. 85 shows for Example 41 representative images of
glomerulosclerosis injury from kidney at day 49 (groups 1-5; PAS,
40.times.) for exemplary animals 3 (A-C, 8 (D-F), 13 (G-I), 57
(J-L), and 63 (M-O). Group 1 (A-C): Normal glomeruli in A and B
(Grade 0) and focal segmental matrix deposition (Grade 1) (arrow)
in C. Group 2 (D-F): Grade 1 glomerulus (D), Grade 2 glomerulus (E)
and Grade 3 glomerulus (F). Matrix deposition and hypercellularity
are noted (arrows). Group 3 (G-I): Grade 0 glomerulus (G), Grade 1
glomerulus (H) and Grade 1 glomerulus (I). Matrix deposition and
hypercellularity are noted (arrows). Group 4 (J-L): Grade 1
glomerulus 0), Grade 1 glomerulus (K) and Grade 2 glomerulus (L).
Matrix deposition and hypercellularity are noted (arrows). Group 5
(M-O): Grade 1 glomerulus (D), Grade 2 glomerulus (E) and Grade 3
glomerulus (F). Matrix deposition and hypercellularity are noted
(arrows and circle).
[0336] FIG. 86 shows for Example 41 representative images of
glomerulosclerosis injury from kidney at day 77 (groups 6-8; PAS,
40.times.) for exemplary animals 28 (A-C), 34 (D-F), and 37 (G-I).
Group 6 (A-C): Normal glomeruli in A and B (Grade 0) and focal
segmental matrix deposition (Grade 1) (arrow) in C. Group 7 (D-F):
Grade 1 glomerulus (D), Grade 2 glomerulus (E) and Grade 3
glomerulus (F). Matrix deposition and hypercellularity are noted
(arrows and circle). Group 8 (G-I): Grade 0 glomerulus (G), Grade 1
glomerulus (H) and Grade 1 glomerulus (I). Matrix deposition and
hypercellularity are noted (arrows).
[0337] FIG. 87 shows for Example 42 the impact of hypxia and XG-102
on viability of human islets. FIG. 87A shows that XG-102 decreased
necrosis either in normoxic and hypoxic conditions. FIG. 87B shows
that XG-102 also decreases apoptosis induced by hypoxia. These
results show that XG-102 has a beneficial effect on islet viability
in the hypoxia model.
[0338] FIG. 88 shows for Example 43 the results of ocular
evaluation (A) and cellular infiltration in aqueous humor (B). FIG.
88A shows median values of ocular evaluation 24 h after induction.
FIG. 88B shows leucocyte counts (cells/.mu.l) in aqueous humor 24 h
after induction.
[0339] FIG. 89 shows for Example 39 SFT values (visual acuity) at
Day 71 (A), Day 85 (B), Day 99 (C) and Day 113 (D). *
p.ltoreq.0.05; ** p.ltoreq.0.01; *** p.ltoreq.0.001; Student's
t-test compared to vehicle group.
[0340] FIG. 90 shows for Example 39 Contrast Threshold values at
Day 71 (A), Day 85 (B), Day 99 (C) and Day 113 (D). *
p.ltoreq.0.05; ** p.ltoreq.0.01; *** p.ltoreq.0.001; Student's
t-test compared to vehicle group.
[0341] FIG. 91 shows for Example 39 the results of Multiplex
cytokine analysis of 23 unique cytokines of the retinal tissue.
STZ-induced diabetes raised retinal levels in vehicle treated
animals for 13 of the 23 cytokines observed. Seven of the 13
elevated cytokines were reduced in STZ-diabetic animals treated
with 2 .mu.g/eye XG-102. All cytokines were BLQ in the retinal
tissue collected from the groups of rats receiving either 20
.mu.g/eye, or 200 .mu.g/eye XG-102.
[0342] FIG. 92 shows for Example 24 the treatment effects on the
clinical parameters GI (gingival inflammation) and PP (periodontal
depth pocket). The first graph shows clinical parameters in the
negative control group (unligated rats). Results are expressed as
Mean.+-.SEM. n=10 rats per group. *p<0.05 day 10 vs 0. $
p<0.05 day 17 vs 10.
[0343] FIG. 93 shows for Example 24 the effects of placebo and
XG-102 administration on total bacterial flora. Group 3 (XG-102)
reduced significantly total bacterial flora at day 17 compared to
day 10. Results are expressed as Mean.+-.SEM. n=10 rats per group.
*p<0.05 day 17 vs day 10.
[0344] FIG. 94 shows for Example 24 IL1-.beta. quantification using
ELISA assay. IL1-.beta. was lower in group 3 than in placebo group.
"SDD-1002" refers to XG-102. Experiments were done in duplicate.
*p<0.05 ligated groups vs unligated group. .sctn.p<0.05
placebo group vs group 3.
[0345] FIG. 95 shows for Example 24 the effects of placebo and
XG-102 administration on ABHL. "SDD-1002" refers to XG-102. Each
measurement was done in duplicate. Results are expressed as
Mean.+-.SEM. n=6 rats per group. *p<0.05 ligated groups vs
unligated group.
[0346] FIG. 96 shows for Example 45 the study design.
[0347] FIG. 97 shows for Example 45 the effects of vehicle and
XG-102 (2 mg/kg, i.v.) on tubular damages in a rat model of
bilateral IR. ***P<0.001 versus Group 1 (Sham/Vehicle) by a
Student t-test ns; +P<0.05 versus Group 2 (IR/Vehicle) by a one
way ANOVA followed by a Bonferroni's post test.
[0348] FIG. 98 shows for Example 45 the effects of vehicle and
XG-102 (2 mg/kg, i.v.) on total tubular histological scores in a
rat model of bilateral IR. Total tubular score represents all
tubular changes including degeneration and necrosis, tubular cast,
tubular epithelial vacuolation and regeneration (basophil tubules).
***P<0.001 versus Group 1 (Sham/Vehicle) by a Student t-test;
+P<0.05 versus Group 2 (IR/Vehicle) by a one way ANOVA followed
by a Bonferroni's post test.
[0349] FIG. 99 shows for Example 45 representative images of
hematoxylin/eosin stained kidney sections: comparison between
Groups 2 (IR/Vehicle) and 3 (IR/XG-102). Animal 53 (Top Left),
Animal 15 (Top Right), Animal 17 (Bottom left), and Animal 33
(Bottom right): 10.times.. Representative photomicrographs of
tubular degeneration/necrosis and tubular casts in Group 2 and 3.
Animals having scores from 1 to 4 are represented. The main
difference between groups is that the severity of tubular necrosis
and cast in Group 2 is generally higher than that observed in Group
3. In Group 2, lesions are extended partially or to the majority of
the cortex. Comparatively, in Group 3 lesions are limited to the
cortico-medullary junction. Lesions consist of a mixture of active
necrosis, cellular tubular casts, hyaline casts, and occasional
basophilic tubules.
[0350] FIG. 100 shows for Example 46 the study design (A) and the
AUCs method to assess allodynia and hyperalgesia (B).
[0351] FIG. 101 shows for Example 46 the effect of XG-102 (50
mg/mL, i.ves.) and ibuprofen (50 mg/mL, i.ves.) treatments on
nociceptive parameters 24 h post-CYP injection. Nociceptive
threshold (A), nociceptive scores (B), AUC 1-8 g (C) or AUC 8-60 g
(D) 24 h after CYP injection. Results are expressed as
mean.+-.s.e.m. (n=10). * p<0.05, ** p<0.01, *** p<0.001 vs
Vehicletreated group, Mann Whitney test (A and C), Two-way RM ANOVA
(B), and Unpaired t test and Mann Whitney test (D).
[0352] FIG. 102 shows for Example 46 the effect of XG-102 (50
mg/mL, i.ves.) and ibuprofen (50 mg/mL, i.ves.) treatments on
urinary bladder wall thickness as well as haemorrhage scores 24 h
post-CYP injection. Urinary bladder wall thickness (A) or
haemorrhage scores (B) 24 h after CYP injection. Results are
expressed as mean.+-.s.e.m. (n=10). ns=p>0.05, ** p<0.01, ***
p<0.001 vs Vehicle-treated group, Mann Whitney test and Unpaired
t test (A) or Mann Whitney test (B).
[0353] FIG. 103 shows for Example 47 the effect of XG-102 (2 mg/kg,
i.v.) and ibuprofen (10 mg/kg, i.v.) treatments on nociceptive
parameters 24 h post-CYP injection. Nociceptive threshold (A),
nociceptive scores (B), AUC 1-8 g (C or AUC 8-60 g (D) 24 h after
CYP injection. Results are expressed as mean.+-.s.e.m. (n=10). **
p<0.01, *** p<0.001 vs Vehicle-treated group, Mann Whitney
test (A), Two-way RM ANOVA (B), Mann Whitney test and Unpaired t
test (C) and Unpaired t test (D).
[0354] FIG. 104 shows for Example 48 the study design (A) and the
cystometric parameters analysed (B).
[0355] FIG. 105 shows for Example 48 the effects of vehicle (i.v.)
on cystometric parameters in conscious female rats treated with
CYP. Not significant versus basal values with a one way ANOVA with
repeated measures, followed by a Dunnett's post-test.
[0356] FIG. 106 shows for Example 48 the effects of XG-102 (2
mg/kg, i.v.) on cystometric parameters in conscious female rats
treated with CYP. ** P<0.01 versus basal values with a one way
ANOVA with repeated measures, followed by a Dunnett's
post-test.
[0357] FIG. 107 shows for Example 37 the results of the
determination of the cytotoxic activity of XG-102 against HepG2 (A)
and PLC/PRF/5 (B) tumour cell lines using MTS assay.
[0358] FIG. 108 shows for Example 49 the effect of JNK inhibitor
XG-102 on JNK activation. (A) Immunoblot analysis of primary mouse
cortical neuron cultures exposed to 1 mM of hydrogen peroxide
(H.sub.2O.sub.2) during 15 minutes. Neurons were pre-treated or not
with 5 .mu.M or 10 .mu.M of the specific inhibitor of JNK, XG-102.
(B) Corresponding histogram showing an increase of 34% of JNK
activity, measured by the ratio of phosphorylated JNK on total JNK
(pJNK/JNK), after induction of the oxidative stress. Pre-treatment
of cortical neurons with the inhibitor XG-102 prevented JNK
activity when used at 5 .mu.M and decreased by 45% JNK activity at
a concentration of 10 .mu.M, in oxidative stress conditions. n=3
per condition. Error bars=standard error of the mean (SEM).
[0359] FIG. 109 shows for Example 49 the effect of JNK inhibition
on neuronal apoptosis. (A) Immunoblot analysis of primary mouse
cortical neuron cultures exposed to 2 .mu.M of A.beta.1-42
(A.beta..sub.42) during 5 hours. Neurons were pre-treated with or
without 10 .mu.M of the specific inhibitor, XG-102. (B)
Corresponding histograms showing no modification of JNK activity in
condition of A.beta..sub.42 cell stress. Pre-treatment of cortical
neurons with XG-102 did not modify JNK activity. (C) Neuronal
apoptosis is measured by the level of cleaved PARP
(poly(ADP-ribose) polymerase) protein, which is increased during
apoptosis. A.beta..sub.42 stress induced apoptosis, with an
increase of 40% of cleaved PARP, except if cultures were
pre-treated with XG-102. In that case, apoptosis is decreased by
37%. n=3 per condition. Error bars=standard error of the mean
(SEM).
[0360] FIG. 110 shows for Example 51 decrease of neuronal apoptosis
after PKR down-regulation and/or JNK inhibition with XG-102. (A)
Immunoblot results of the levels of JNK and c-Jun activation,
caspase 3 and PARP cleaved activated fragments in primary neuronal
cultures of WT and PKR.sup.-/- mice, treated by 2 .mu.M of
A.beta.42 after or not pre-inhibition of JNK with 10 .mu.M XG-102.
(B-D) Corresponding histograms of JNK activity (B), phospho c-Jun
(C), and total c-Jun (D). (E-G) Apoptosis is measured by the level
of cleaved caspase 3 (E), caspase 3 activity measured in the cell
culture supernatant (F) and cleaved PARP (G). Data are means.+-.SEM
(N.gtoreq.3 per condition). *P<0.05, **P<0.01, and
***P<0.001.
EXAMPLES
Example 1: Identification of JNK Inhibitor Sequences
[0361] Amino acid sequences important for efficient interaction
with JNK were identified by sequence alignments between known JNK
binding domain JBDs. A sequence comparison between the JBDs of IB1
[SEQ ID NO: 13], IB2 [SEQ ID NO: 14], c-Jun [SEQ ID NO: 15] and
ATF2 [SEQ ID NO: 16] defined a weakly conserved 8 amino acid
sequence (see FIG. 1A). Since the JBDs of IB1 and IB2 are
approximately 100 fold as efficient as c-Jun or ATF2 in binding JNK
(Dickens et al. Science 277: 693 (1997), it was reasoned that
conserved residues between IB1 and IB2 must be important to confer
maximal binding. The comparison between the JBDs of IB1 and IB2
defined two blocks of seven and three amino acids that are highly
conserved between the two sequences.
[0362] These two blocks are contained within a peptide sequence of
19 amino acids in L-IB1(s) [SEQ ID NO: 1] and are also shown for
comparative reasons in a 23 aa peptide sequence derived from IB1
[SEQ ID NO: 17]. These sequences are shown in FIG. 1B, dashes in
the L-IB1 sequence indicate a gap in the sequence in order to align
the conserved residues with L-IB1(s).
Example 2: Preparation of JNK Inhibitor Fusion Proteins
[0363] JNK inhibitor fusion proteins according to SEQ ID NO: 9 were
synthesized by covalently linking the C-terminal end of SEQ ID NO:
1 to a N-terminal 10 amino acid long carrier peptide derived from
the HIV-TAT4g 57 (Vives et al., J Biol. Chem. 272: 16010 (1997))
according to SEQ ID NO: 5 via a linker consisting of two proline
residues. This linker was used to allow for maximal flexibility and
prevent unwanted secondary structural changes. The basic constructs
were also prepared and designated L-IB1 (s) (SEQ ID NO: 1) and
L-TAT [SEQ ID NO: 5], respectively.
[0364] All-D retro-inverso peptides according to SEQ ID NO: 11 were
synthesized accordingly. The basic constructs were also prepared
and designated D-IB1 (s) [SEQ ID NO: 2] and D-TAT [SEQ ID NO: 6],
respectively.
[0365] All D and L fusion peptides according to SEQ ID NOs: 9, 10,
11 and 12 were produced by classical Fmock synthesis and further
analysed by Mass Spectrometry. They were finally purified by HPLC.
To determine the effects of the proline linker, two types of TAT
peptide were produced one with and one without two prolines. The
addition of the two prolines did not appear to modify the entry or
the localization of the TAT peptide inside cells. Generic peptides
showing the conserved amino acid residues are given in FIG. 2.
Example 3: Inhibition of Cell Death by JBD19
[0366] Effects of the 19 aa long JBD sequence of IB1(s) on JNK
biological activities were studied. The 19 aa sequence was linked
N-terminal to the Green Fluorescent Protein (GFP JBD19 construct),
and the effect of this construct on pancreatic beta-cell apoptosis
induced by IL1 was evaluated. This mode of apoptosis was previously
shown to be blocked by transfection with JBD.sub.1-280 whereas
specific inhibitors of ERK1/2 or p38 as known in the art did not
protect.
[0367] Oligonucleotides corresponding to JBD19 and comprising a
conserved sequence of 19 amino acids as well as a sequence mutated
at the fully conserved regions were synthesized and directionally
inserted into the EcoRI and SalI sites of the pEGFP-N1 vector
encoding the Green Fluorescent Protein (GFP) (from Clontech).
Insulin producing TC-3 cells were cultured in RPMI 1640 medium
supplemented with 10% Fetal Calf Serum, 100 .mu.g/mL Streptomycin,
100 units/mL Penicillin and 2 mM Glutamine. Insulin producing TC-3
cells were transfected with the indicated vectors and IL-1 (10
ng/mL) was added to the cell culture medium. The number of
apoptotic cells was counted at 48 hours after the addition of IL-1
using an inverted fluorescence microscope. Apoptotic cells were
discriminated from normal cells by the characteristic "blebbing
out" of the cytoplasm and were counted after two days.
[0368] GFP is Green Fluorescent protein expression vector used as a
control; JBD19 is the vector expressing a chimeric GFP linked to
the 19 aa sequence derived from the JBD of IB1; JBD19Mut is the
same vector as GFP-JBD19, but with a JBD mutated at four conserved
residues shown as FIG. 1B; and JBD.sub.1-280 is the GFP vector
linked to the entire JBD (aa 1-280). The GFP-JBD19 expressing
construct prevented IL-1 induced pancreatic -cell apoptosis as
efficiently as the entire JBD.sub.1-280.
[0369] As additional controls, sequences mutated at fully conserved
IB1(s) residues had greatly decreased ability to prevent
apoptosis.
Example 4: Cellular Import of TAT-IB1(s) Peptides
[0370] The ability of the L- and D-enantiomeric forms of TAT and
TAT-IB1(s) peptides ("TAT-IB peptides") to enter cells was
evaluated. L-TAT, D-TAT, L-TAT-IB1(s), and D-TAT-IB1(s) peptides
[SEQ ID NOs: 5, 6, 9 and 12, respectively] were labeled by
N-terminal addition of a glycine residue conjugated to fluorescein.
Labeled peptides (1 .mu.M) were added to TC-3 cell cultures, which
were maintained as described in Example 3. At predetermined times
cells were washed with PBS and fixed for five minutes in ice-cold
methanol-acetone (1:1) before being examined under a fluorescence
microscope. Fluorescein-labeled BSA (1 .mu.M, 12 moles/mole BSA)
was used as a control. Results demonstrated that all the above
fluorescein labeled peptides had efficiently and rapidly (less than
five minutes) entered cells once added to the culture medium.
Conversely, fluorescein labeled bovine serum albumin (1 .mu.M BSA,
12 moles fluorescein/mole BSA) did not enter the cells.
[0371] A time course study indicated that the intensity of the
fluorescent signal for the L-enantiomeric peptides decreased by 70%
following a 24 hours period. Little to no signal was present at 48
hours. In contrast, D-TAT and D-TAT-IB1(s) were extremely stable
inside the cells.
[0372] Fluorescent signals from these all-D retro-inverso peptides
were still very strong 1 week later, and the signal was only
slightly diminished at 2 weeks post treatment.
Example 5: In Vitro Inhibition of c-JUN, ATF2 and Elk1
Phosphorylation
[0373] The effects of the peptides on JNKs-mediated phosphorylation
of their target transcription factors were investigated in vitro.
Recombinant and non activated JNK1, JNK2 and JNK3 were produced
using a TRANSCRIPTION AND TRANSLATION rabbit reticulocyte lysate
kit (Promega) and used in solid phase kinase assays with c-jun,
ATF2 and Elk1, either alone or fused to glutathione-S-transferase
(GST), as substrates. Dose response studies were performed wherein
L-TAT or L-TAT-IB1 (s) peptides (0-25 .mu.M) were mixed with the
recombinant JNK1, JNK2, or JNK3 kinases in reaction buffer (20 mM
Tris-acetate, 1 mM EGTA, 10 mM p-nitrophenyl-phosphate (pNPP), 5 mM
sodium pyrophosphate, 10 mM p-glycerophosphate, 1 mM
dithiothreitol) for 20 minutes. The kinase reactions were then
initiated by the addition of 10 mM MgCl.sub.2 and 5 pCi
.sup.33P-gamma-dATP and 1 .mu.g of either GST-Jun (aa 1-89),
GST-AFT2 (aa 1-96) or GST-ELK1 (aa 307-428). GST-fusion proteins
were purchased from Stratagene (La Jolla, Calif.).
[0374] Ten .mu.L of glutathione-agarose beads were also added to
the mixture. Reaction products were then separated by SDS-PAGE on a
denaturing 10% polyacrylamide gel. Gels were dried and subsequently
exposed to X-ray films (Kodak). Nearly complete inhibition of
c-Jun, ATF2 and Elk1 phosphorylation by JNKs was observed at
TAT-IB(s) peptide doses as low as 2.5 .mu.M. However, a marked
exception was the absence of TAT-IB(s) inhibition of JNK3
phosphorylation of Elk1. Overall, the TAT-IB1(s) peptide showed
superior effects in inhibiting JNK family phosphorylation of their
target transcription factors. The ability of D-TAT, D-TAT-IB1(s)
and L-TAT-IB1(s) peptides (0-250 .mu.M dosage study) to inhibit
GST-Jun (aa 1-73) phosphorylation by recombinant JNK1, JNK2, and
JNK3 by were analyzed as described above. Overall, D-TAT-IB1(s)
peptide decreased JNK-mediated phosphorylation of c-Jun, but at
levels approximately 10-20 fold less efficiently than
L-TAT-IB1(s).
Example 6: Inhibition of c-JUN Phosphorylation by Activated
JNKs
[0375] The effects of the L-TAT or L-TAT-IB1(s) peptides as defined
herein on JNKs activated by stressful stimuli were evaluated using
GST-Jun to pull down JNKs from UV-light irradiated HeLa cells or
IL-1 treated PTC cells. PTC cells were cultured as described above.
HeLa cells were cultured in DMEM medium supplemented with 10% Fetal
Calf Serum, 100 .mu.g/mL Streptomycin, 100 units/ml Penicillin and
2 mM Glutamine. One hour prior to being used for cell extract
preparation, PTC cells were activated with IL-1 as described above,
whereas HeLa cells were activated by UV-light (20 J/m.sup.2). Cell
extracts were prepared from control, UV-light irradiated HeLa cells
and IL-1 treated TC-3 cells by scraping the cell cultures in lysis
buffer (20 mM Tris-acetate, 1 mM EGTA, 1% Triton X-100, 10 mM
p-nitrophenyl-phosphate, 5 mM sodium pyrophosphate, 10
mMP-glycerophosphate, 1 mM dithiothreitol). Debris was removed by
centrifugation for five minutes at 15,000 rpm in an SS-34 Beckman
rotor. One-hundred .mu.g extracts were incubated for one hour at
room temperature with one .mu.g GST-jun (amino acids 1-89) and 10
.mu.L of glutathione-agarose beads (Sigma). Following four washes
with the scraping buffer, the beads were resuspended in the same
buffer supplemented with L-TAT or L-TAT-IB1(s) peptides (25 .mu.M)
for 20 minutes. Kinase reactions were then initiated by addition of
10 mM MgCl.sub.2 and 5 pCi .sup.33P-gamma-dATP and incubated for 30
minutes at 30.degree. C.
[0376] Reaction products were then separated by SDS-PAGE on a
denaturing 10% polyacrylamide gel. Gels were dried and subsequently
exposed to X-ray films (Kodak). The TAT-IB(s) peptides efficiently
prevented phosphorylation of c-Jun by activated JNKs in these
experiments.
Example 7: In Vivo Inhibition of c-JUN Phosphorylation by TAT-IB(s)
Peptides as Defined Herein
[0377] To determine whether the cell-permeable peptides as defined
herein could block JNK signaling in vivo, we used a heterologous
GAL4 system. HeLa cells, cultured as described above, were
co-transfected with the 5.times.GAL-LUC reporter vector together
with the GAL-Jun expression construct (Stratagene) comprising the
activation domain of c-Jun (amino acids 1-89) linked to the GAL4
DNA-binding domain. Activation of JNK was achieved by the
co-transfection of vectors expressing the directly upstream kinases
MKK4 and MKK7 (see Whitmarsh et al., Science 285: 1573 (1999)).
Briefly, 3.times.10.sup.5 cells were transfected with the plasmids
in 3.5-cm dishes using DOTAP (Boehringer Mannheim) following
instructions from the manufacturer. For experiments involving
GAL-Jun, 20 ng of the plasmid was transfected with 1 .mu.g of the
reporter plasmid pFR-Luc (Stratagene) and 0.5 .mu.g of either MKK4
or MKK7 expressing plasmids. Three hours following transfection,
cell media were changed and TAT and TAT-IB1(s) peptides (1 .mu.M)
were added. The luciferase activities were measured 16 hours later
using the "Dual Reporter System" from Promega after normalization
to protein content. Addition of TAT-IB1(s) peptide blocked
activation of c-Jun following MKK4 and MKK7 mediated activation of
JNK. Because HeLa cells express JNK1 and JNK2 isoforms but not
JNK3, we transfected cells with JNK3. Again, the TAT-IB(s) peptide
inhibited JNK2 mediated activation of c-Jun.
Example 8: Synthesis of all-D Retro-Inverso IB(s) Peptides and
Variants Thereof
[0378] Peptides of the invention may be all-D amino acid peptides
synthesized in reverse to prevent natural proteolysis (i.e. all-D
retro-inverso peptides). An all-D retro-inverso peptide of the
invention would provide a peptide with functional properties
similar to the native peptide, wherein the side groups of the
component amino acids would correspond to the native peptide
alignment, but would retain a protease resistant backbone.
[0379] Retro-inverso peptides of the invention are analogs
synthesized using D-amino acids by attaching the amino acids in a
peptide chain such that the sequence of amino acids in the
retro-inverso peptide analog is exactly opposite of that in the
selected peptide which serves as the model. To illustrate, if the
naturally occurring TAT protein (formed of L-amino acids) has the
sequence GRKKRRQRRR [SEQ ID NO: 5], the retro-inverso peptide
analog of this peptide (formed of D-amino acids) would have the
sequence RRRQRRKKRG [SEQ ID NO: 6]. The procedures for synthesizing
a chain of D-amino acids to form the retro-inverso peptides are
known in the art (see e.g. Jameson et al., Nature, 368,744-746
(1994); Brady et al., Nature, 368,692-693 (1994); Guichard et al.,
J. Med. Chem. 39,2030-2039 (1996)). Specifically, the
retro-peptides according to SEQ ID NOs 2, 4, 6, 8, 11-12, 18, 20,
22 and 25-26, were produced by classical F-mock synthesis and
further analyzed by Mass Spectrometry. They were finally purified
by HPLC.
[0380] Since an inherent problem with native peptides is
degradation by natural proteases and inherent immunogenicity, the
heterobivalent or heteromultivalent compounds of this invention
will be prepared to include the "retro-inverso isomer" of the
desired peptide. Protecting the peptide from natural proteolysis
should therefore increase the effectiveness of the specific
heterobivalent or heteromultivalent compound, both by prolonging
half-life and decreasing the extent of the immune response aimed at
actively destroying the peptides.
Example 9: Long Term Biological Activity of all-D Retro-Inverso
IB(s) Peptides and Variants Thereof
[0381] Long term biological activity is predicted for the
D-TAT-IB(s) retro-inverso containing peptide heteroconjugate (see
chimeric sequences above) when compared to the native L-amino acid
analog owing to protection of the D-TAT-IB(s) peptide from
degradation by native proteases, as shown in Example 5.
[0382] Inhibition of IL-1 induced pancreatic beta-cell death by the
D-TAT-IB1(s) peptide was analyzed. TC-3 cells were incubated as
described above for 30 minutes with one single addition of the
indicated peptides (1 .mu.M), then IL-1 (10 ng/ml) was added.
[0383] Apoptotic cells were then counted after two days of
incubation with IL-1 by use of Propidium Iodide and Hoechst 33342
nuclear staining. A minimum of 1,000 cells were counted for each
experiment. Standard Error of the Means (SEM) are indicated, n=5.
The D-TAT-IB1 peptide decreased IL-1 induced apoptosis to a similar
extent as L-TAT-IB peptides.
[0384] Long term inhibition of IL-1 P induced cell-death by the
D-TAT-IB1 peptide was also analyzed. TC-3 cells were incubated as
above for 30 minutes with one single addition of the indicated
peptides (1 .mu.M), then IL-1 (10 ng/ml) was added, followed by
addition of the cytokine every two days. Apoptotic cells were then
counted after 15 days of incubation with IL-1 by use of propidium
iodide and Hoechst 33342 nuclear staining. Note that one single
addition of the TAT-IB1 peptide does not confer long-term
protection. A minimum of 1.000 cells were counted for each
experiment. As a result, D-TAT-IB1(s), but not L-TAT-IB1 (s), was
able to confer long term (15 day) protection.
Example 10: Suppression of JNK Transcription Factors by
L-TAT-IB1(s) Peptides as Used According to the Present
Invention
[0385] Gel retardation assays were carried out with an AP-1 doubled
labeled probe (5'-CGC TTG ATG AGT CAG CCG GAA-3' (SEQ ID NO: 101).
HeLa cell nuclear extracts that were treated or not for one hour
with 5 ng/mITNF-.alpha., as indicated. TAT and L-TAT-IB1(s)
peptides as used according to the present invention were added 30
minutes before TNF-alpha. Only the part of the gel with the
specific AP-1 DNA complex (as demonstrated by competition
experiments with non-labeled specific and non-specific competitors)
is shown.
[0386] L-TAT-IB1(s) peptides as used according to the present
invention decrease the formation of the AP-1 DNA binding complex in
the presence of TNF-alpha.
Example 11: Inhibition of Endogenous JNK Activity in HepG2 Cells
Using an all-in One Well Approach (See FIG. 3)
[0387] HepG2 cells were seeded at 3'000 cells/well the day prior
the experiment. Then, increasing concentrations of either
interleukin-1 [IL-1beta .nu.)] or tumor necrosis factor [TNFalpha]
(a) were added to activate JNK for 30 minutes. Cells were lysed in
20 mM Hepes, 0.5% Tween pH 7.4 and processed for AlphaScreen JNK.
(b) Z' for the JNK activity induced by 10 ng/ml IL-1 and measured
in 384 wells/plate (n=96). (c) Inhibition of endogenous IL-1
beta-induced JNK activity with chemical JNK inhibitors
[staurosporin and SP600125]. (d) Effect of peptidic inhibitors
L-TAT-IB1(s) according to SEQ ID NO: 9 [here abbreviated as L-JNKi
(.nu.)) and D-TAT-IB1(s) according to SEQ ID NO: 11 (here
abbreviated as D-JNKi) and JBDs (corresponds to L-JNKI without the
TAT sequence)] on IL-1 dependent JNK activity. All panels are
representative of three independent experiments (n=3).
[0388] Methods: Alphascreen Kinase Assay
[0389] Principle: AlphaScreen is a non-radioactive bead-based
technology used to study biomolecular interactions in a microplate
format. The acronym ALPHA stands for Amplified Luminescence
Proximity Homogenous Assay. It involves a biological interaction
that brings a "donor" and an "acceptor" beads in close proximity,
then a cascade of chemical reactions acts to produce an amplified
signal. Upon laser excitation at 680 nm, a photosensitizer
(phthalocyanine) in the "donor" bead converts ambient oxygen to an
excited singlet state. Within its 4 .mu.sec half-life, the singlet
oxygen molecule can diffuse up to approximately 200 nm in solution
and if an acceptor bead is within that proximity, the singlet
oxygen reacts with a thioxene derivative in the "acceptor" bead,
generating chemiluminescence at 370 nm that further activates
fluorophores contained in the same "acceptor" bead. The excited
fluorophores subsequently emit light at 520-620 nm. In the absence
of an acceptor bead, singlet oxygen falls to ground state and no
signal is produced.
[0390] Kinase reagents (B-GST-cJun, anti P-cJun antibody and active
JNK3) were first diluted in kinase buffer (20 mM Tris-HCl pH 7.6,
10 mM MgCl.sub.2, 1 mM DTT, 100 .mu.M Na.sub.3VO.sub.4, 0.01%
Tween-20) and added to wells (15 .mu.l). Reactions were then
incubated in presence of 10 .mu.M of ATP for 1 h at 23.degree. C.
Detection was performed by an addition of 10 .mu.l of beads mix
(Protein A acceptor 20 .mu.g/ml and Streptavidin donor 20
.mu.g/ml), diluted in detection buffer (20 mM Tris-HCl pH 7.4, 20
mM NaCl, 80 mM EDTA, 0.3% BSA), followed by an another one-hour
incubation at 23.degree. C. in the dark. For measurement of JNK
endogenous activity, kinase assays were performed as described
above except active JNK3 was replaced by cells lysates and reaction
kinase components were added after the cells lysis. B-GST-cjun and
P-cJun antibody were used at the same concentrations whereas ATP
was used at 50 .mu.M instead of 10 .mu.M. AlphaScreen signal was
analyzed directly on the Fusion or En Vision apparatus.
Example 12: Determining the Activity of all-D Retro-Inverso IB(s)
Peptides and Variants Thereof in the Treatment of Viral
Infections-Varicella-Zoster Virus (VZV)
[0391] Determination of the activity of IB(s) peptides and all-D
retro-inverso IB(s) peptides as used according to the present
invention was tested using the JNK inhibitor peptide XG-102 (SEQ ID
NO: 11) as a test compound in cultured host cells (human foreskin
fibroblasts (HFFs)). Viruses are obligate intracellular parasites
that require a functional cell environment to complete their
lifecycle; dying cells do not support virus replication.
Additionally, inhibitors of cell functions may be toxic to cells,
which could non-specifically prevent virus growth. Thus, sick or
dying host cells could exhibit nonspecifically reduced virus
titers. Since this may falsify the results, a cytotoxicity assay
was carried out first, determining the tolerance of the cultured
cells to the test compound. Subsequently, a plaque reduction assay
was carried out and then activity of the JNK inhibitor peptide
XG-102 (SEQ ID NO: 11) was tested with respect to Viral Zoster
Virus (VZV) in infected cells.
[0392] A) Determination of the Cytotoxicity of all-D Retro-Inverso
IB(s) Peptides: [0393] For determination of toxicity, cultured
cells (human foreskin fibroblasts (HFFs)) were seeded in 96-well
tissue culture plates. Medium containing DMSO (same level as 5
.mu.M XG-102 (SEQ ID NO: 11)), or XG-102 (SEQ ID NO: 11) was added
at several concentrations of (1, 2, and 5 .mu.M) for 24 h.
Subsequently, a Neutral Red assay was carried out. Neutral Red
colorimetric assays for cytotoxicity assays (in sets of 6
replicates) were used to set the maximum dose for subsequent
efficacy assays (as performed in Taylor et al, 2004, J. Virology,
78:2853-2862). Live cells absorb Neutral Red and, accordingly, the
level of absorbance is a quantitative measure of cell viability and
number. Neutral Red uptake is directly proportional to the number
of cells and also reflects normal endocytosis. Therefore, a brief
pulse of Neutral Red was added to the medium at 0 or 24 hours.
After fixation and extraction, dye was added and the amount of dye
in each sample was measured in an ELISA plate reader at 540 nm (see
FIG. 4). No cytotoxicity was observed with any amount of XG-102
(SEQ ID NO: 11), and cell growth was not restricted compared to the
DMSO diluent alone (control). Thus the standard concentration of 1
.mu.M had no evident effects on HFF cells, and higher doses would
also be well tolerated.
[0394] B) Plaque Reduction Assay to Evaluate the Antiviral Effects
of XG-102 (SEQ ID NO: 11) Against Varicella-Zoster Virus (VZV)
[0395] To determine whether XG-102 (SEQ ID NO: 11) had a
dose-dependent antiviral effect, a range of concentrations
surrounding the standard 1 .mu.M dose were tested. In this plaque
reduction assay, confluent human foreskin fibroblasts (HFFs) in
24-well plates were inoculated with VZV-infected HFFs at a ratio of
1:100 (multiplicity of infection MOI=0.01) and adsorbed to the
cells for 2 hours. The excess virus was washed out, and medium
containing 0 (DMSO only), 0.5, 1, or 2 .mu.M XG-102 (SEQ ID NO: 11)
was added. One sample was taken at this time to measure the initial
level of infection; wherein each well contained -150 pfu. After 24
hours, duplicate wells were trypsinized, and then the cell
suspensions were titered on MeWo cell monolayers in triplicate to
determine the number of VZV-infected cells in each sample. During
unrestricted growth, VZV usually increases by 10-fold over 1 day
because it propagates by cell-cell spread. This is what was
observed for the cultures treated with DMSO alone, which yielded
1200.+-.430 pfu (FIG. 5). The results of the determination were as
follows:
TABLE-US-00003 [0395] XG-102 (SEQ ID NO: 11) Spread of VZV (pfu)
.+-. SD 0 .mu.M (DMSO) 1233 .+-. 432 0.5 .mu.M 260 .+-. 53 1.0
.mu.M 212 .+-. 48 2.0 .mu.M 312 .+-. 79
[0396] XG-102 (SEQ ID NO: 11) had thus a strong antiviral effect at
all the concentrations tested, with VZV yields near 200-300 pfu.
Using the graph of these results to interpolate the EC.sub.50, it
was calculated that approximately 0.3 .mu.M XG-102 (SEQ ID NO: 11)
caused VZV yield to decrease by 50%. [0397] From the cytotoxicity
and efficacy data, a preliminary Selective Index (Tox/EC.sub.50) of
5.0 .mu.M/0.3 .mu.M was calculated, which gives a value of
.about.17, wherein the true SI is considered even higher, since the
concentration of XG-102 (SEQ ID NO: 11) was not yet approached that
would kill 50% of the cells.
[0398] C) Measurement of Varicella-Zoster Virus (VZV) Replication
in Human Foreskin Fibroblasts (HFFs) with XG-102 (SEQ ID NO: 11)
[0399] To determine the minimum effective dose of XG-102 that
prevents varicella-zoster virus (VZV) replication in human foreskin
fibroblasts (HFFs) with XG-102 (SEQ ID NO: 11) confluent monolayers
of HFFs were inoculated with VZV-BAC-Luc strain for 2 h, then
treated for 24 h with XG-102 (SEQ ID NO: 11) in concentrations of
0.25, 0.5, or 1.0 .mu.M or with the negative control (XG-100, 1.0
.mu.M). Virus yield was measured by luciferase assay. Samples were
in triplicate and the average luminescence is shown; error bars
represent the standard deviation of the mean. [0400] As a result,
VZV replication was normal in the presence of the negative control
(the Tat peptide alone). XG-102 (SEQ ID NO: 11) prevented VZV
replication at the lowest concentration tested, 0.25 .mu.M. The
minimum effective dose could not be determined in this experiment.
While it is not yet known why VZV depends on JNK activity during
infection, there appears to be a critical requirement for this
enzyme. A low concentration (0.25 .mu.M) of XG-102 (SEQ ID NO: 11)
is thus sufficient to completely block VZV spread in culture. One
possible explanation for this effect is that this amount of XG-102
(SEQ ID NO: 11) binds to all the JNK molecules in the infected
cells. Alternatively, 0.25 .mu.M XG-102 (SEQ ID NO: 11) may reduce
JNK activity below a threshold level that is optimal for VZV
replication. The results of this experiment are summarized in FIG.
6.
Example 13: Determining the Activity of all-D Retro-Inverso IB(s)
Peptides and Variants Thereof in the Treatment of Chronic
Obstructive Pulmonary Disease (COPD)
[0401] In order to determine the activity of the exemplary all-D
retro-inverso IB(s) peptide XG-102 (SEQ ID NO: 11) in the treatment
of Chronic Obstructive Pulmonary Disease (COPD) XG-102 (SEQ ID NO:
11) is used in an animal model of Bleomycin induced acute lung
inflammation and fibrosis. The protocol of bleomycin induced
inflammation and fibrosis has been described before in the
literature. The aim of the Experiment was to investigate the effect
of XG-102 (SEQ ID NO: 11) by subcutaneous (s.c.) route on
neutrophil recruitment in broncho alveolar lavage (BAL) and lung in
bleomycin induced inflammation and fibrosis: [0402] at 1 day after
a single bleomycin administration (10 mg/kg) [0403] and at day 10
with the development of fibrosis
[0404] 1) Method and Experimental Approach [0405] The test compound
XG-102 (SEQ ID NO: 11) at two doses and vehicle control were given
s.c. with a single intranasal administration of bleomycin and mice
were analyzed after 1 and 10 days. The animals used in the model
were 10 C57BL/6 mice (8 weeks old) per group. The experimental
groups included vehicle, 0.001 mg/kg XG-102 (SEQ ID NO: 11) and 0.1
mg/kg XG-102 (SEQ ID NO: 11), and the treatment consisted of
repeated sub-cutaneous administration of XG-102 (SEQ ID NO: 11),
prior to bleomycin administration then every 3 days. Acute lung
inflammation at 24 h was monitored by BAL lavage, cytology, cell
counts, and lung myeloperoxidase activity. The effect of the
compound was compared with vehicle controls. Lung fibrosis was
assessed histologically using hematoxylin and eosin staining at day
10 after the single dose of bleomycin.
[0406] 1.1) Bleomycin Administration [0407] Bleomycin sulfate in
saline (10 mg/kg body weight) from Bellon Laboratories (Montrouge,
France) or saline were given through the airways by nasal
instillation in a volume of 40 .mu.L under light ketamine-xylasine
anesthesia. The groups for Bleomycin administration for both
bleomycin induced inflammation and fibrosis included: Vehicle,
0.001 mg/kg XG-102 (SEQ ID NO: 11) and 0.1 mg/kg XG-102 (SEQ ID NO:
11). The route for bleomycin induced inflammation was subcutaneous
(s.c.) route, and administration occurred as a single dose. The
route for bleomycin induced fibrosis was subcutaneous (s.c.) route,
and administration occurred 3 times in 10 days.
[0408] 1.2) Bronchoalveolar Lavage Fluid (BALF) [0409] After
incision of the trachea, a plastic cannula was inserted and
airspaces were washed using 0.3 ml of PBS solution, heated to
37.degree. C. The samples collected were dispatched in 2 fractions:
the first one (1 ml corresponding to the 2 first lavages) was used
for mediator measurement and the second one for the cell
determination (4 ml). The first fraction was centrifuged (600 g for
10 min) and supernatant was fractionated and kept at -80.degree. C.
until mediator determination. The cell pellet was then resuspended
in 0.4 ml sterile NaCl, 0.9%, and pooled with the second fraction
and was used for cell counts.
[0410] 1.3) Lung Homogenization [0411] After BAL the whole lung was
removed and placed inside a microtube (Lysing matrix D, Q Bio Gene,
Illkrich, France) with 1 ml of PBS, total lung tissue extract was
prepared using a Fastprepm system (FP120, Q Bio Gene, Ilkrich,
France), the extract was then centrifuged and the supernatant
stored at -80.degree. C. before mediator measurement and collagen
assay with Sircol Collagen Assay (France Biochem Division,
France).
[0412] 1.4) Cell Count and Determination [0413] Total cell count
was determined in BAL fluid using a Malassez hemocytometer.
Differential cell counts were performed on cytospin preparations
(Cytospin 3, Thermo Shandon) after staining with MGG Diff-quick
(Dade Behring AG). Differential cell counts were made on 200 cells
using standard morphological criteria.
[0414] 1.5) TNF Measurement [0415] TNF level in BALF was determined
using ELISA assay kits (Mouse DuoSet, R&D system, Minneapolis,
USA) according to manufacturer's instructions. Results are reported
as pg/ml.
[0416] 1.6) MPO-Measurement [0417] MPO-levels were measured upon
administration of XG-102. MPO was not significantly induced after
bleomycin in this experiment. Furthermore, XG-102 had no effect on
MPO levels in the lung.
[0418] 1.7) Histology [0419] After BAL and lung perfusion, the
large lobe was fixed in 4% buffered formaldehyde for standard
microscopic analysis. 3-m sections were stained with hematoxylin
and eosin (H&E).
[0420] 2.) Results
[0421] A) First Study: Bleomycin (BLM) Induced Acute Lung
Inflammation
[0422] Groups: Vehicle, XG-102 (SEQ ID NO: 11) 0.001 mg/kg and
XG-102 (SEQ ID NO: 11) 0.1 mg/kg
[0423] Route: s.c. route, single dose
[0424] a) Cell Recruitment in Bronchoalveolar Lavage Space [0425]
At 0.1 mg/kg, XG-102 (SEQ ID NO: 11) reduces significantly the
neutrophil recruitment and the number of total cells recruited
during the inflammatory stage. At 0.001 mg/kg, XG-102 (SEQ ID NO:
11) has no effect on neutrophil recruitment or other cell types
into the bronchoalveolar space (one representative experiment with
n=5 mice per group; *, p<0.05; **, p<0.001).
[0426] b) Cell Recruitment in Lung Using MPO in Lung Homogenization
[0427] Myeloperoxidase (MPO) plays an important role in host
defense systems. This 140 kDa protein, composed of two heavy chains
of 53 kDa and two light chains of 15 kDa, was first discovered in
the 1960s. The release of MPO from the granules of neutrophils and
monocytes in response to the activation of leukocytes allows the
conversion of hydrogen peroxide and chloride ions into hypochlorous
acid (HOCl), a strong oxidizing agent. Although MPO serves an
important purpose in the defense system, various studies show that
MPO also plays a role in several inflammatory conditions, wherein
an elevated MPO level e.g. has been linked to coronary artery
diseases. Furthermore, tissue MPO levels reflect the state of
activation of neutrophils and gives an indication on neutrophil
tissue infiltration. [0428] In the present experiment, MPO was not
significantly induced after bleomycin administration. XG-102 (SEQ
ID NO: 11) had thus no effect on the MPO levels in the lung (see
FIG. 7).
[0429] c) TNF Measurement [0430] When measuring TNF levels, a trend
to reduction of the TNF level in BALF after administration of
XG-102 (SEQ ID NO: 11) was observed, although TNF levels were very
low after BLM administration (see FIG. 8).
[0431] d) Conclusion [0432] It could be observed that at 0.1 mg/kg,
XG-102 (SEQ ID NO: 11) decreases the neutrophil and total cell
recruitment into the bronchoalveolar space and induces a trend to
decrease the TNF level. Moreover, the study of the histological
slides showed a decrease of the inflammatory cell accumulation in
the peribronchial space. It can thus be concluded that XG-102 (SEQ
ID NO: 11) reduces the Bleomycin-induced inflammation. [0433]
According to the acquired results, the experiment was additionally
performed in a fibrosis model.
[0434] B) Second Study: Bleomycin (BLM) Induced Lung Fibrosis
[0435] Groups: Vehicle, XG-102 (SEQ ID NO: 11) 0.001 mg/kg and
XG-102 (SEQ ID NO: 11) 0.1 mg/kg
[0436] Route: s.c. route, 3 times in 10 days
[0437] a) Cell Recruitment in Bronchoalveolar Lavage Space [0438]
At 0.001 mg/kg, XG-102 (SEQ ID NO: 11) reduced significantly the
lymphocyte recruitment and the number of total cells recruited
during the inflammatory stage characterised at this point by the
lymphocytes recruitment. At 0.1 mg/kg, XG-102 (SEQ ID NO: 11) had
no effect (n=5 mice per group; *, p<0.05; **, p<0.001) (see
FIG. 9).
[0439] a) Histology [0440] 3 .mu.m sections of lungs were stained
with haematoxylin and eosin. Inflammatory cells accumulation,
fibrotic areas, loss of lung architecture were observed 10 days
after BLM administration. A decrease of these parameters was
observed after administration of XG-102 at the low dose (0.001
mg/kg) but not with the high dose (0.1 mg/kg) (see FIG. 10).
[0441] b) Conclusion: [0442] It can be concluded that XG-102 (SEQ
ID NO: 11) administered 3 times at the low dose of 0.001 mg/kg
decreases the Bleomycin-induced later inflammation, in particular
the lymphocytes recruitment observed at this time. Moreover, the
test substance administered 3 times at this dose attenuates the
Bleomycin-induced fibrosis. Less extended fibrotic areas with a
more conserved lung structure could be observed.
Example 14: Determining the Activity of all-D Retro-Inverso IB(s)
Peptides and Variants Thereof in the Treatment of Alzheimer's
Disease
[0443] In order to determine the activity of the exemplary all-D
retro-inverso IB(s) peptide XG-102 (SEQ ID NO: 11) in Alzheimer's
disease, XG-102 (SEQ ID NO: 11) was evaluated in the
hAPP-transgenic mice model overexpressing APP751 with London and
Swedish mutations using the behavioral Morris Water Maze test as
well as immunohistological tests measuring plaque load and ELISA
tests measuring .beta.-amyloid.sub.1-40 and .beta.-amyloid.sub.1-42
levels in the brain of mice.
[0444] a) METHODS [0445] i) Introduction [0446] The study was
designed to evaluate the efficacy of the test substance (XG-102,
SEQ ID NO: 11) on behavioral, biochemical and histological markers
using 5 months (.+-.2 weeks) old female hAPP Tg mice. Therefore,
mice were treated every two or three weeks up to 4 months and in
the end of the treatment period behavior was evaluated in the
Morris Water Maze. At sacrifice brain, CSF and blood were
collected. A.beta.40 and A.beta.42 levels were determined in four
different brain homogenate fractions as well as in CSF of Tg mice.
Plaque load was quantified in the cortex and the hippocampus of 8
Tg animals per treatment group. [0447] ii) Animals
[0448] Female Tg mice with a C57BL/6.times.DBA background and an
age of 5 months (.+-.2 week) were randomly assigned to treatment
groups 1 to 3 (n=12). Animals were subjected to administration of
vehicle or XG-102 (SEQ ID NO: 11) in two different concentrations
beginning at 5 months of age and continued for up to 4 months with
subcutaneous (s.c.) applications every second or third week. All
animals which were used for the present study had dark eyes and
were likely to perceive the landmarks outside the MWM pool.
However, it had to be excluded that seeing abilities of an animal
were poor, which was controlled in the visible platform training,
the so called pretest, before treatment start for all animals
including reserves enclosed to the study. In case a seeing handicap
for a specific animal would have been affirmed, the mouse would
have been excluded from the study. [0449] iii) Animal
Identification and Housing [0450] Mice were individually identified
by ear markings. They were housed in individual ventilated cages
(IVCs) on standardized rodent bedding supplied by Rettenmaier.RTM..
Each cage contained a maximum of five mice. Mice were kept
according to the JSW Standard Operating Procedures (SOP GEN011)
written on the basis of international standards. Each cage was
identified by a colored card indicating the study number, sex, the
individual registration numbers (IRN) of the animals, date of
birth, as well as the screening date and the treatment group
allocation. The temperature during the study was maintained at
approximately 24.degree. C. and the relative humidity was
maintained at approximately 40-70%. Animals were housed under a
constant light-cycle (12 hours light/dark). Normal tap water was
available to the animals ad libitum. [0451] iv) Treatment [0452]
Forty female hAPP transgenic mice were treated with either 0.1
mg/kg b.w./every two weeks or 10 mg/kg b.w./every three weeks of
the test substance XG-102 (SEQ ID NO: 11) in two different dosages
(n=12/group) or treated with the vehicle (n=12) s.c. once every
three weeks over four months. [0453] v) Morris Water Maze (MWM)
[0454] The Morris Water Maze (MWM) task was conducted in a black
circular pool of a diameter of 100 cm. Tap water was filled in with
a temperature of 22.+-.1.degree. C. and the pool was virtually
divided into four sectors. A transparent platform (8 cm diameter)
was placed about 0.5 cm beneath the water surface. During the whole
test session, except the pretest, the platform was located in the
southwest quadrant of the pool. One day before the 4 days lasting
training session animals had to perform a so called "pre-test" (two
60 sec lasting trials) to ensure that the seeing abilities of each
animal were normal. Only animals that fulfilled this task were
enclosed to the MWM testing. In the MWM task each mouse had to
perform three trials on four consecutive days. A single trial
lasted for a maximum of maximum one minute. During this time, the
mouse had the chance to find the hidden, diaphanous target. If the
animal could not find a "way" out of the water, the investigator
guided to or placed the mouse on the platform. After each trial
mice were allowed to rest on the platform for 10-15 sec. During
this time, the mice had the possibility to orientate in the
surrounding. Investigations took place under dimmed light
conditions, to prevent the tracking system from negative influences
(Kaminski; PCS, Biomedical Research Systems). On the walls
surrounding the pool, posters with black, bold geometric symbols
(e.g. a circle and a square) were fixed which the mice could use
the symbols as landmarks for their orientation. One swimming group
per trial consisted of five to six mice, so that an intertrial time
of about five to ten minutes was ensured. For the quantification of
escape latency (the time [second]-the mouse needed to find the
hidden platform and therefore to escape from the water), of pathway
(the length of the trajectory [meter] to reach the target) and of
the abidance in the goal quadrant a computerized tracking system
was used. The computer was connected to a camera placed above the
centre of the pool. The camera detected the signal of the light
emitting diode (LED), which was fixed with a little hairgrip on the
mouse's tail. One hour after the last trial on day 4 the mice had
to fulfill a so-called probe trial. At this time, the platform was
removed from the pool and during the one-minute probe trial; the
experimenter counted the number of crossings over the former target
position. Additionally the abidance in this quadrant as well as the
three other quadrants was calculated. Through out this trial a
mouse could not get any, howsoever-natured, clue from the platform.
[0455] vi) Tissue Sampling [0456] At the end of the treatment
period, and following all behavioral testing, all remaining mice
(n=28) were sacrificed. Therefore, all mice were sedated by
standard inhalation anesthesia (Isofluran, Baxter) as described in
SOP MET030. Cerebrospinal fluid (CSF) was obtained by blunt
dissection and exposure of the foramen magnum. Upon exposure, a
Pasteur pipette was inserted to the approximate depth of 0.3-1 mm
into the foramen magnum. CSF was collected by suctioning and
capillary action until flow fully ceases. Two aliquots of each
sample were immediately frozen and kept at -80.degree. C. until
ready for further analysis with ELISA technique. After CSF
sampling, each mouse was placed in dorsal recumbence, thorax was
opened and a 26-gauge needle attached to a 1 cc syringe was
inserted into the right cardiac ventricular chamber. Light suction
was applied to the needle and blood was collected into EDTA and
consequently used to obtain plasma. To get plasma, blood samples
from each mouse were spun at 1,750 rpm (700 g) for 10 minutes in a
centrifuge (GS-6R Beckman) using a rotor with swing buckets
(GH--3.8 Beckman). Plasma was frozen and stored at -20.degree. C.
until further analysis. After blood sampling transgenic mice were
intracardially perfused with 0.9% sodium chloride. Brains were
rapidly removed the cerebellum was cut off. The right hemispheres
of all mice were immersion fixed in freshly produced 4%
Paraformaldehyde/PBS (pH 7.4) for one hour at room temperature.
Thereafter brains were transferred to a 15% sucrose PBS solution
for 24 hours to ensure cryoprotection. On the next day brains were
frozen in isopentane and stored at -80.degree. C. until used for
histological investigations (SOP MET042). The left hemispheres were
weighed and frozen in liquid nitrogen and stored at -80.degree. C.
for biochemical analysis. [0457] vii) Determination of
A.beta..sub.1-40 and A.beta..sub.1-42 [0458] In four different
brain homogenate fractions of each Tg mouse as well as in CSF
samples the A.beta..sub.1-40 and A.beta..sub.1-42 levels were
evaluated with ELISA technique. Highly sensitive A.beta..sub.1-40
and A.beta..sub.1-42 ELISA test kits were purchased from The
Genetics Company.TM., Switzerland (SOP MET058). CSF was prepared as
described above. For the brain homogenates frozen hemispheres were
homogenized in TRIS buffered saline (TBS)-buffer (5 ml) containing
protease inhibitor cocktail. 1.25 ml of this initial brain TBS
homogenate was stored at -80.degree. C., 1.25 ml have been further
investigatated. The remaining brain homogenate (2.5 ml) was
centrifuged and the resulting supernatant (=TBS fraction) was
aliquoted and kept at -20.degree. C. until ELISA determination. The
pellet was suspended in Triton X-100 (2.5 ml), centrifuged and the
supernatant (=Triton X-100 fraction) was aliquoted and kept at
-20.degree. C. These steps were repeated with SDS (2.5 ml). The
pellet out of the SDS fraction was suspended in 70% formic acid
(0.5 ml) prior to subsequent centrifugation. The obtained
supernatant was neutralized with 1 M TRIS (9.5 ml) aliquoted and
kept at -20.degree. C. (=FA fraction). Samples of the four brain
homogenate fraction (TBS, Triton X-100, SDS, and FA) were used for
A.beta..sub.1-40 and A.beta..sub.1-42 determination with ELISA
technique. ELISA test kits were purchased from The Genetics
Company.TM., Switzerland (SOP MET062). It could be assumed that TBS
and Triton X-100 solubilize monomeric to oligomeric structures.
Polymers like protofibrils and water insoluble fibrils could be
dissolved in SDS and FA. In this regard the investigation of all
four fractions also provides insight in A polymerization status.
[0459] viii) Evaluation of Brain Morphology [0460] Brain tissues of
all Tg animals investigated were handled in exactly the same way to
avoid bias due to variation of this procedure. From brain halves of
24 Tg mice (8 of each group) 20 cryo-sections per layer (altogether
5 layers), each 10 .mu.m thick (Leica CM 3050S) were sagittally cut
and 5 (one from each layer) were processed and evaluated for
quantification of plaque load. The five sagittal layers
corresponded with the FIGS. 104 to 105, 107 to 108, 111 to 112, 115
to 116 and 118 to 119 according to the morphology atlas "The Mouse
Brain" from Paxinos and Franklin (2nd edition). The first layer was
specified by the requirement to include the whole hippocampus with
it's regions CA1, CA2, CA3, GDIb and GDmb. Immunoreactivity was
quantitatively evaluated in the hippocampus and in the cortex using
the monoclonal human A.beta.-specific antibody 6E10 (Signet) as
well as ThioflavinS staining. Remaining brain hemispheres or tissue
not used were saved and stored at JSW CNS until the end of the
project.
[0461] B) Evaluation [0462] i) Behavior [0463] In the Morris Water
Maze trials length of swimming path, escape latencies, swimming
speed and in the probe trial crossings over the former platform
position and the time spent in each quadrant of the pool were
measured for each Tg animal with a special computer software.
[0464] ii) Biochemical Evaluation [0465] From all Tg mice CSF
samples as well as samples from the brain preparations were
analyzed with commercially available A.beta..sub.1-40 and
A.beta..sub.1-42 ELISAs. Measurements of adequate standards were
performed concurrently. Samples from brain preparations were
analyzed in duplicates. Due to the small sample amount CSF samples
were analyzed in a single measurement only. [0466] iii) Histology
[0467] Measurement of Amyloid Depositions and Plaque Load [0468]
For 6E10 immunohistochemistry the following evaluation procedure
was used: [0469] aa) Contrasting the image for visualization of
slice borders without applying the contrast on the image. [0470]
bb) Interactive drawing of the cortical outlines and the following
measurement of the cortical area (=region area). [0471] cc)
Interactive drawing of the area of interest (AOI), in which stained
objects are detected over a certain intensity based threshold level
(the same for each image) and above a size of 8 .mu.m.sup.2. [0472]
dd) Measurement of the area of each object, the sum of stained area
in the AOI as well as the number of objects after a smooth
contrasting to enhance signal/noise ratio (the same for each
image). [0473] ee) Repetition of aa)-dd) for the hippocampus.
[0474] ff) Calculation of the mean plaque size (="sum area of
plaques/number of plaques"), the relative plaque number and area
(="number of plaques/region area" and "sum area of plaques/region
area*100"). [0475] gg) Automated data export into an Excel spread
sheet, including the parameters "image title, region area, number
of plaques, sum of plaque area, relative plaque number, relative
plaque area and mean plaque size. A field for remarks was used to
record image quality and exclusion criteria, respectively.
Exclusion criteria were missing parts of the slice, many wrinkles,
dominant flaws or staining inconsistencies (e.g. due to bulges,
which can impede the full reaction of the blocking reagent). [0476]
hh) Closing the image without saving (to keep raw data raw).
[0477] c) Results [0478] i) General Observations [0479] In total 40
female hAPP Tg mice were enclosed to study. From these mice 12
animals died due to unknown reason before the treatment period was
finished. [0480] ii) Behavioral Results [0481] Spatial learning in
the MWM remained widely uninfluenced by XG-102 (SEQ ID NO: 11)
treatment. 0.1 mg/kg treated mice showed a tendency to have worse
learning performance between day 1 and day 4. A two-way ANOVA of
the mean performance on day 1 and 4 revealed highly significant
learning for all groups (p<0.001), but also a significant
influence of factor treatment (p=0.045). However, Bonferroni's post
tests did not reach significance. [0482] iii) Biochemical Results
[0483] aa) A.beta. Levels in the Brain Homogenate Fractions [0484]
A treatment with the test compound XG-102 (SEQ ID NO: 11) did not
affect brain homogenate A.beta..sub.1-40 levels (see FIG. 11).
Group differences in A.beta..sub.1-42 levels appeared in Triton
X-100 fraction, only. There, animals treated with the low dose of
the test compound XG-102 (SEQ ID NO: 11) (0.1 mg/kg) featured a
significant reduction compared to the vehicle group (p<0.05) as
well as compared to the high dose group (p<0.01). [0485] bb) CSF
A.beta. Levels [0486] After treatment with the test substance
XG-102 (SEQ ID NO: 2) A.beta..sub.1-40 and A.beta..sub.1-42 levels
were significantly decreased in CSF compared to vehicle group. For
both, A.beta..sub.1-40 and A.beta..sub.1-42 p-values were p<0.01
for the high dosage (10 mg/kg) and <0.05 for the lose dosage of
XG-102 (SEQ ID NO: 2) (see FIG. 12). [0487] iv) Results of Brain
Histology and Immunohistochemistry [0488] aa) Amyloid Depositions
and Plaque Load [0489] Plaque load was quantified with two
different methods. On the one hand an IHC staining with 6E10
primary directed against AA1-17 of the human amyloid peptide was
performed, on the other hand a ThioflavinS staining marking
beta-sheet structures and cores of mature, neuritic plaques was
carried out. First of all, measured region areas, cortex and
hippocampus, were highly constant throughout all groups, indicating
that problems in the cutting and IHC procedures can be excluded and
to a certain degree also a treatment induced atrophy (changes of
>5% would be detectable with this method). 6E10 and ThioflavinS
quantifications revealed a selective reduction of beta-sheet
structures in the center of the plaques after XG-102 (SEQ ID NO:
11) treatment, whereas human amyloid remained uninfluenced from
treatment or slightly increased. In detail cortical 6E10 IR plaque
load was increased versus vehicle in the 10 mg/kg XG-102 (SEQ ID
NO: 11) treated mice, however, significance level was reached for
the number of hippocampal plaques. FIGS. 13 and 14 show, in
contrast to 6E10 IHC, that XG-102 (SEQ ID NO: 11) treatment led to
a negatively dose dependent reduction of the number of hippocampal
ThioflavinS positive plaques, as well as area percentage (number of
plaques: p<0.05 for 10 mg/kg, p<0.01 for 0.1 mg/kg XG-102
(SEQ ID NO: 11)). 0.1 mg/kg XG-102 (SEQ ID NO: 11) treatment also
reduced mean plaque size, however this effect did not reach
significance level in the ANOVA (unpaired, two-tailed T-test:
p=0.074) These effects were not given for cortical plaques, a
circumstance which is most probably due to the later onset of
plaque pathology in the hippocampus than in the cortex. Treatment
start at five months of age exactly hits the time point of plaque
deposition in the hippocampus, whereas cortical plaques start to
become visible at the used magnification for quantification at the
age of three months. Qualitatively the proportion of 6E10 to
ThioflavinS stained plaques increase and the beta-sheet plaque
cores, as seen in doubly labeled slices, become smaller in size.
Summarized, these data support that XG-102 (SEQ ID NO: 11)
treatment acts against beta-sheet formation in the early phase of
plaque deposition and beta sheet formation in plaque cores,
respectively.
[0490] D) Summary of Effects and Conclusions [0491] Spatial
navigation measured in the Morris water maze remained widely
uninfluenced from treatment. 0.1 mg/kg XG-102 (SEQ ID NO: 11)
treatment resulted in a slightly poorer learning performance
between the first and the last training day. [0492] Except a
decrease in the Triton X-100 fraction in the 0.1 mg/kg XG-102 (SEQ
ID NO: 11) group A.beta..sub.1-40 and A.beta..sub.1-42 brain levels
stayed stable. [0493] A decrease of A.beta. levels was detectable
in CSF for both dosages and fragments. [0494] XG-102 (SEQ ID NO:
11) treatment led to a tendentious increase of human amyloid beta
in the higher dosed group in the 6E10 quantifications, which is in
compliance with data obtained in A.beta. ELISA. [0495] In contrast
to that hippocampal beta-sheet load detected by ThioflavinS
staining was dose dependently reduced after XG-102 (SEQ ID NO: 11)
treatment, to a higher degree at lower dose 0.1 mg/kg XG-102 (SEQ
ID NO: 11), whereas cortical plaque load remained unchanged. In
accordance with the age-dependent onset of plaque deposition in the
hippocampus at treatment start this hints at an early action on
beta-sheet formation in the early phase of plaque deposition.
Example 15: Determining the Activity of all-D Retro-Inverso IB(s)
Peptides and Variants Thereof in the Treatment of Diabetes Type
2
[0496] Example 15 is designed to determine the activity of IB(s)
peptides and all-D retro-inverso IB(s) peptides and variants
thereof in the treatment of Diabetes Type 2, particularly to
determine the effect of chronic treatment with XG-102 (SEQ ID NO:
11) in the db/db mice model of type 2 diabetes by evaluating
fasting blood glucose levels every third day (28 days)
[0497] a) Materials and Methods [0498] i) Animals [0499] A total of
twenty (20) male db/db mice (8 weeks old) were obtained from
Charles River (Germany). Upon arrival, animals were group housed
(n=6-7/group) and offered regular rodent chow (Altromin standard
#1324 chow; C. Petersen, Ringsted, Denmark) and water ad libitum
unless otherwise stated. The mice were housed under a 12:12 L/D
cycle (lights on at 4:00 and lights off at 16:00) and in
temperature and humidity controlled rooms. [0500] ii) Groups and
Randomization [0501] On day-4, mice were randomized according to
blood glucose level (fasted; blood glucose measured on Biosen S
line analyzer (EKF diagnostic, Germany) to participate in one of
the following drug treatment groups (n=6): [0502] 1) Vehicle
control, S.C. (physiological saline) [0503] 2) XG-102 (SEQ ID NO:
11); 1 mg/kg; s.c. [0504] 3) XG-102 (SEQ ID NO: 11); 10 mg/kg; s.c
[0505] All doses listed were calculated for the free-base. Drug
purity: 95.28%, peptide content: 78.0%. All compounds were
administered sub-cutaneously (s.c.) in a volume of 3 ml/kg. The
formulation instructions for vehicle control and XG-102 (SEQ ID NO:
11) were as follows: [0506] First, XG-102 (SEQ ID NO: 11) was
dissolved in the vehicle. The formulations (concentrations of 0.33
and 3.3 mg/ml, corresponding to the doses of 1 and 10 mg/kg,
respectively) were prepared according to the procedure detailed
below. Concentrations were calculated and expressed taking into
account test items purity and peptide content (multiplier
coefficient was 1.346). [0507] Preparation of a stock solution: the
freeze-dried test compound XG-102 (SEQ ID NO: 11) is thawed for one
hour minimum and prepared as a stock solution in the vehicle at 1
mM (corresponding to 3.823 mg/mL). Aliquots are prepared for each
treatment day and stored at approximately -80.degree. C. Dilutions
of this stock solution to the required concentrations are performed
on each treatment day; [0508] Storage of the stock solution: at
approximately -80.degree. C.; [0509] Storage of the diluted
preparations: at room temperature for 24 hours maximum. [0510]
Prior to solubilisation, the powder was stored at -20.degree. C.
The stability of the stock solution is 3 months at approximately
-80.degree. C.; the stability of the diluted formulations for
animal dosing is 24 hours at room temperature. Unused diluted
material could be stored for up to 7 days if kept at 4-8.degree.
C.
[0511] c) Experimental Procedure [0512] Following 8 days of
acclimatization the mice were treated daily at 08.00 AM for 21 days
by SC dosing 8 hours prior to lights out at 04.00 PM according to
the outline groups. Then, on study day 21 dosing of the highest
concentration of XG-102 (SEQ ID NO: 2) (10 mg/kg) was stopped,
whereas daily dosing of vehicle control and XG-102 (SEQ ID NO: 2)
(1 mg/kg) were continued until day study 28. From day 28 until
termination at day 111 the vehicle and XG-102 (SEQ ID NO: 2) (10
mg/kg) treated mice were observed in a wash-out period (no dosing),
whereas the mice treated with XG-102 (SEQ ID NO: 2) (1 mg/kg) was
terminated after 28 days of treatment [0513] i) Blood glucose
[0514] Blood glucose was measured from 7 hour fasted animals 6
hours post dosing by collection of 10 .mu.l blood samples from the
tail-vein in hematocrite tubes and subsequent analysis on a Biosen
s-line analyzer (EKF-diagnostic; Germany). [0515] ii) Metabolic
cages [0516] Groups 1+3: Mice were placed in metabolic cages for
the recording of 24-hour food and water intake as well as 24-hour
urine and faeces production.
[0517] Mice were stratified into two sub-teams of n=6-7 and
subsequently the metabolic characterisation were performed on study
days 71-72. [0518] iii) Adipokine panel [0519] Groups 1+3: On three
occasions (study days 57, 66 and 108) blood was collected from the
tail vein using EDTA coated hematocrite tubes (100 .mu.l).
Following centrifugation of blood the plasma was collected and
stored at -20.degree. C. until measurement. Then, the following
panel of adipokines/cytokines was determined using Luminex based
7-plex: leptin, resistin, MCP-1, PAI-1, TNF, insulin and
interleukin-6 (IL-6). [0520] iv) Termination [0521] Groups 1+3 (day
111): The following organs were excised and weighed: [0522]
inguinal subcutaneous fat, epididymal fat, retroperitoneal fat,
brain, liver, kidney, spleen and heart. All organs described above
were samples in 4% PFA for possible future histo-pathological
examination. Also, pancreas (en bloc) was sampled for possible
stereological and imunohistochemical analysis, and eyes were
sampled for possible later analysis of retinopathy. Group 2 (day
28): No tissues or plasma were collected.
[0523] c) Results [0524] i) General observations [0525] During the
acute dosing period animals showed normal levels of alertness and
activity and there were no signs of sedation in the drug treated
animals. Food and water intake were within normal ranges among
vehicle treated animals.
[0526] However, after approximately two weeks dosing, nodular
fibrosis was observed in the subcutaneous tissue as a reaction to
the XG-102 (SEQ ID NO: 2) compound in the high dose, these
progressed into open wounds all of the mice from group C. In group
B mild nodular fibrosis was observed. As a consequence an
alternation of injection sites were used. Following the end of
dosing of the animals the animals healed and the nodular fibrosis
was gradually disappearing. We observed no clinical effects in the
vehicle treated animals. [0527] ii) Blood Glucose [0528] Fasting
blood glucose levels (absolute and relative) are shown in FIG. 15.
Fasting blood glucose was measured every third day until day 68 and
on a regular basis until termination at day 111 in groups A and C.
We observed a clear and significant (p<0.001) decrease in the
level of fasting blood glucose of the diabetic db/db mice treated
with XG-102 (SEQ ID NO: 2) (10 mg/kg) as compared to vehicle
control. The fasting blood glucose levels of the mice treated with
XG-102 (SEQ ID NO: 2) (10 mg/kg) reached a low plateau of
approximately 5 mmol/L. This effect was evident after 14 days of
dosing and persisted throughout the study, thus during the entire
wash-out period from day 21 to day 111. In contrast, we observed no
effect of low dose of XG-102 (SEQ ID NO: 2) (1 mg/kg) during 28
days of dosing. [0529] iii) Body Weight [0530] Body weight
determinations (absolute and relative) are shown in FIG. 16. We
observed a clear and significant (p<0.001) prevention of body
weight increase in mice treated with XG-102 (SEQ ID NO: 2) (10
mg/kg) as compared to vehicle control. This effect was evident from
day 28 of dosing and remained until the day of termination day 111.
In contrast, we observed no effect of low dose of XG-102 (SEQ ID
NO: 2) (1 mg/kg) on body weight during 28 days of dosing. [0531]
iv) Metabolic cages [0532] The effect of vehicle or XG-102 (SEQ ID
NO: 2) (10 mg/kg) on 24 hour food and water intake, and urine and
faeces production as measured in metabolic cages on study day 68
are shown in FIGS. 17 (g) and 18 (normalized to g of body weight).
We observed no significant effects of XG-102 (SEQ ID NO: 2) (10
mg/kg) on any of the measured parameters as compared to vehicle
control though a trend towards a decrease in food intake and urine
production was observed. [0533] v) Adipokines [0534] The effect of
vehicle or XG-102 (SEQ ID NO: 2) (10 mg/kg) as measured on day 57,
77 and 108 on plasma levels of insulin, MCP-1 and IL-6 are shown in
FIG. 19; on plasma levels of tPAI-1, TNF and resistin in FIG. 20;
We observed no significant effects of XG-102 (SEQ ID NO: 2) (10
mg/kg) on any of the measured parameters as compared to vehicle
control except the levels of plasma resistin, which was
significantly higher in XG-102 (SEQ ID NO: 2) treated animals at
day 77 and 108. [0535] vi) Tissue weight at termination [0536] The
effect of vehicle or XG-102 (SEQ ID NO: 2) (10 mg/kg) on tissue
weight of epididymal, inguinal subcutaneous, and retroperitoneal
fat pads are shown in FIG. 21. We observed a significant decrease
of epididymal (p<0.05) and retroperitoneal (p<0.01) fat mass
in the mice treated with XG-102 as compared to vehicle control. The
effect of vehicle or XG-102 (SEQ ID NO: 2) (10 mg/kg) on tissue
weight of brain, spleen and heart is shown in FIG. 22. We observed
no significant effects of XG-102 (SEQ ID NO: 2) (10 mg/kg) on these
parameters as compared to vehicle control. Finally, the effect of
vehicle or XG-102 (SEQ ID NO: 2) (10 mg/kg) on tissue weight of
kidney and liver is shown in FIG. 23. We observed a significant
decrease of kidney (p<0.05) and liver (p<0.01) mass in the
mice treated with XG-102 (SEQ ID NO: 2) as compared to vehicle
control.
[0537] Summarizing the results, administration of XG-102 (SEQ ID
NO: 11), 10 mg/kg, appears to lead to a significant decrease in
blood glucose levels and therefore, XG-102 (SEQ ID NO: 11) appears
to be a promising new tool for treating diabetes and elevated blood
glucose levels.
Example 16: Safety. Tolerability and Pharmacokinetics of a Single
Intravenous Infusion of 10, 40 and 80 .mu.g/kg XG-102 (SEQ ID No.:
11) Administered to Healthy Male Volunteers in a Randomized, Double
Blind, Placebo Controlled, Dose Escalating Phase I Study
[0538] The primary objective of the study was to assess the safety
and tolerability of XG-102 following intravenous (iv) infusion of
single escalating doses of XG-102 to healthy male volunteers. The
secondary objective of the study was to assess the pharmacokinetics
of XG-102 following iv infusion of single escalating doses of
XG-102 to healthy male volunteers. Doses were administered as a 60
minute iv infusion. For control purposes, placebo iv infusion was
administered to control subjects.
[0539] This was a single-centre, randomized, double blind, placebo
controlled, ascending single dose, sequential group study. Three
dose levels of XG-102 (10, 40 and 80 .mu.g/kg) were studied in
ascending order of dose, within each group subjects were randomized
such that 6 subjects received XG-102, and 2 subjects received
placebo. Screening was performed in the 3-week period prior to
dosing. Dosing occurred on Day 0 for each subject. The Investigator
checked on all subjects' well-being prior to their discharge from
the CRU (at 24 hours after dosing). Subjects returned to the CRU
8.+-.2 days and 28.+-.5 days after dosing for post study
assessments.
[0540] A total of 24 subjects (healthy male subjects in the age of
18 to 45), in 3 groups of 8. 24 subjects entered and completed the
study. Data for all subjects were included in the safety analyses;
data for all subjects who received XG-102 were included in the
pharmacokinetic analyses.
TABLE-US-00004 Summary: Pharmacokinetic results: The
pharmacokinetic parameters of XG-102 are presented in the following
table: 10 .mu.g/kg 40 .mu.g/kg 80 .mu.g/kg Parameter (N = 6) (N =
6) (N = 6) AUC.sub.0-last 24.7 (26.1) 134 (15.2) 431 (41.0) (ng
h/mL) AUC.sub.0-.infin. 36.8 (23.4) 146 (17.5) 443 (41.0) (ng h/mL)
AUCextrap.sup.a (%) .sup. 34.1 (18.6-49.7) .sup. 6.7 (4.2-12.9)
.sup. 2.9 (1.9-3.4) C.sub.max (ng/mL) 31.3 (24.4) 146 (16.7) 362
(34.9) t.sub.max.sup.a (h) .sup. 1.00 (1.00-1.05) .sup. 1.00
(1.00-1.00) .sup. 1.00 (1.00-1.00) AUC.sub.0-last(norm) 3.10 (29.3)
3.64 (13.8) 5.91 (41.8) (ng h/mL)/(.mu.g/kg)
AUC.sub.0-.infin.(norm) 4.61 (24.8) 3.96 (15.7) 6.07 (41.8) (ng
h/mL)/(.mu.g/kg) C.sub.max(norm) 3.93 (28.0) 3.98 (15.9) 4.97
(35.6) (ng/mL)/(.mu.g/kg) MRT (h) 1.00 (29.9) 0.76 (11.0) 1.02
(14.7) t.sub.1/2 (h) 0.57 (44.6) 0.36 (22.3) 0.65 (38.8) CL (mL/h)
17537 (23.9).sup. 18399 (16.4).sup. 13217 (43.5).sup. CL (mL/h/kg)
217 (24.8) 253 (15.7) 165 (41.8) V.sub.ss (mL) 17536 (36.8).sup.
14040 (15.7).sup. 13500 (30.5).sup. V.sub.ss (mL/kg) 217 (27.5) 193
(13.7) 168 (29.8) Geometric mean (CV %) data are presented N =
Number of subjects studied (norm) = Normalized for dose and body
weight .sup.aMedian (min max)
[0541] The observed values of tin were short. Both peak exposure as
measured by C.sub.max and cumulative exposure as measured by
AUC.sub.0-last increased with dose. The increase with dose of
C.sub.max appears to be more than linearly proportional on the
basis of graphical examinations and of the geometric mean of its
dose normalized values which after the highest 80 .mu.g/kg dose are
above the 90% confidence intervals for the other doses. The
increase with dose of AUC.sub.0-last is clearly more than linearly
proportional from 40 to 80 .mu.g/kg as the 90% confidence intervals
for its geometric mean dose normalized value does not overlap with
those after the other tested doses; whereas when comparing values
after 10 and 40 .mu.g/kg the 90% confidence intervals overlap, but
its geometric mean dose normalized value after the 10 .mu.g/kg dose
is lower than all values in the corresponding 90% confidence
interval after the 40 .mu.g/kg dose.
[0542] XG-102 was safe and well tolerated when administered as
single iv doses of 10, 40 or 80 .mu.g/kg to healthy male subjects.
The incidence of adverse events in subjects who received XG-102 was
similar to the incidence in subjects who received placebo. There
were no clinically significant findings in clinical laboratory
data, vital signs, ECGs, physical examinations or ocular
examinations (fundus and IOP).
[0543] After the end of XG-102 intravenous infusion, its plasma
concentrations quickly decreased, leading to values below the lower
limit of quantification by at most 2 hours after the start of 10
.mu.g/kg XG-102 iv infusions, 3 hours after the start of 40
.mu.g/kg XG-102 iv infusions and by at most 7 hours after the start
of 80 .mu.g/kg XG-102 intravenous infusions. The measured tin and
MRT values are short, with geometric mean values per dose level
ranging from 0.36 to 0.65 hours and from 0.76 to 1.02 hours,
respectively.
[0544] The AUC.sub.0-last of XG-102 increases in a more than linear
proportion with dose in the tested dose range, with non-overlapping
90% confidence intervals for its geometric mean dose normalized
values between the 40 .mu.g/kg and the 80 .mu.g/kg dose and only
limited overlap between the 90% confidence intervals for its
geometric mean dose normalized values between the 10 .mu.g/kg and
the 40 .mu.g/kg.
[0545] The C.sub.max of XG-102 appears to increase in a more than
linear proportion with dose from 40 to 80 .mu.g/kg. The geometric
mean dose normalized C.sub.max in the 80 .mu.g/kg dose group is
higher than and outside the 90% confidence intervals for the
geometric mean dose normalized C.sub.max in the other dose groups,
but the 90% confidence intervals for the geometric mean dose
normalized C.sub.max overlap among all dose levels.
[0546] The intersubject variability of XG-102 pharmacokinetic
parameters was moderate in subjects treated with 10 and 40 .mu.g/kg
doses (CV % of the geometric mean for most parameters approximately
in the 15-30% range, exception was tin and total V.sub.ss at the 10
.mu.g/kg dose group), but higher in the 80 .mu.g/kg dose group, in
the approximately 29-44% range, other than for MRT (14.7%). This
higher variability may be either an effect of the low sample size
or a consequence of the observed non-linearities which are clearer
at this dose.
Example 17: Use of XG-102 (SEQ ID No.: 11) to Improve Porcine Islet
Isolation Outcomes
[0547] The object was to evaluate the ability of XG-102 to (a)
block the massive activation of JNK that occurs during islet
isolation leading to cell stress and death; (b) reduce islet death,
resulting to improvements in islet viability post-isolation, using
the porcine model.
[0548] Porcine islet isolation results in a dramatic activation of
JNK first observed in tissue samples .about.20 min after the
initiation of the islet isolation procedure (FIG. 33). Analysis of
existing data demonstrates that the addition of the XG-102 JNK
inhibitor at the pancreas level during procurement and transfer to
the isolation lab and in islet isolation solutions (10 micromolar
concentration) during isolation blocks the activation of JNK (FIG.
34), reduces the relative expression of the c-fos gene (FIG. 35),
and has a statistically significant and important effect on the
viability of freshly isolated islets as measured by OCR/DNA (FIG.
36) and ATP/protein [total cell protein] (FIG. 37). Comparisons
were always made with paired untreated controls originating from
the same pancreas donor. The data on islet viability presented in
FIGS. 36 and 37 is consistent with a reduction in the activation of
JNK typically observed during isolation (FIG. 33) and a reduction
in resulting c-fos gene expression (FIG. 35). The differences in
viability, JNK activation and c-fos expression became smaller after
7 days of culture.
[0549] 6/6 (100%) of the isolations resulted in OCR/DNA values
above the cut-off and were successfully transplanted in NHPs (FIG.
38). This confirms that in this model even modest improvements in
viability can have a profound impact on the transplantability of
preparations. Based on the available data. XG-102 turned out to be
an excellent agent to be used for clinical human or porcine islet
isolations.
[0550] The porcine model is relevant for the following reasons: (1)
The size of the porcine pancreas is closer to that of a human
pancreas than a rat or canine pancreas; (2) Porcine islets are
considered a viable option for future clinical islet
xenotransplantation-therefore improvements in porcine islet
isolation, which are critically needed can ultimately be clinically
relevant.
[0551] Human pancreata for clinical islet allo-transplantation
originating from brain-dead donors are typically not subjected to
WIT but have 8-12 hrs of CIT (time needed for transportation from
the procurement hospital to the isolation lab).
[0552] Human pancreata from non-heart beating donors are exposed to
-'15 min of WIT and are not currently utilized routinely) because
of concerns about damage due to the WIT and they would also
experience 8-12 hrs of CIT.
[0553] Organs removed from chronic pancreatitis patients for islet
auto-transplantation may experience WIT and limited (1-2 hrs CIT).
It is anticipated that improvements reported with the porcine model
below would be even bigger in the clinical auto-transplant case
because the pancreata from chronic pancreatitis patients are
typically inflamed and already stressed. This is also expected to
be true in the clinical allo-cases with prolonged cold ischemia
time and it has been reported by other groups using different JNK
inhibitors. JNK activation increases with CIT from the time of
pancreas procurement; Blocking JNK activation with a JNK inhibitor
improves islet yield, viability and transplant outcomes and that is
most pronounced at the longest cold ischemia time tested.
Example 18: Efficacy of XG-102 (SEQ ID No. 11) in Reducing the
Choroidal Neovascularization Using the Rat Argon Laser-Induced
Choroidal Neovascularization Model
[0554] The aim of this example was to determine whether two
intravitreous administrations of XG-102 at two doses resulted in a
decrease of choroidal neovascularization in a rat model of
laser-induced choroidal neovascularization (ChNV). That model
allows to make predections on the potential use of a test compound
for the treatment of age-related macular degeneration.
[0555] Forty (40) (+10 reserve) pigmented Brown Norway rats were
divided into five (5) groups of eight (8) animals each. Choroidal
neovascularization was induced using a 532 nm argon laser
photocoagulator (six (6) 75 .mu.m-sized spots at 150 mW for 100 ms)
in the right eyes. Test, reference and control items were
administered by intravitreous injection on Days 0 (just after
induction) and 7. Angiography was performed 10 min after
fluorescein (tracer) subcutaneous injection, on Days 14 and 21
after induction on treated and untreated animals.
[0556] After sacrifice on Day 23, the right treated eye from all
animals was sampled and the choroid was flat mounted. On sponsor's
request, no quantification of the volume of the ChNV was
performed.
[0557] Experimental Set-Up:
[0558] XG-102: 3 000 .mu.g/ml (equivalent to 15 .mu.g/eye) and 300
.mu.g/ml (equivalent to 1.5 .mu.g/eye). Kenacort.RTM. Retard (4%
triamcinolone acetonide) as control reference. Control Vehicle:
Saline (0.9% NaCl).
[0559] Animals [0560] Species: Rat. This is the species most
commonly used in this experimental model [0561] Strain: Brown
Norway (pigmented). [0562] Age: Approximately 8 weeks. [0563]
Weight: 175-200 g (on ordering). [0564] Number/sex: 50 males (study
40; reserve 10). [0565] Breeder: "HARLAN FRANCE"--FR-03800
GANNAT.
[0566] Study Design [0567] Forty (40) (+ten (10) reserve) pigmented
rats from the Brown Norway strain were divided into five (5) groups
of eight (8) (+2 reserve) animals. Choroidal neovascularization was
induced using a 532 nm argon laser photocoagulator (six (6) 75
.mu.m-sized spots at 150 mW for 100 ms) in the right eyes. [0568]
Test item (two doses, groups 1-2), vehicle and reference (5 .mu.l)
were administered by intravitreous injection in right eyes at Day 0
(after induction of neovascularization under the same anesthesia)
and Day 7. Fundus neovessels were evaluated on Days 14 and 21 using
Heidelberg's Retinal Angiography (HRA) in right eyes for treated
and untreated animals. [0569] The table below summarizes the
allocation of animals in treatment groups:
TABLE-US-00005 [0569] Route of Group Adminis- Number of No.
Treatment Dose tration animals 1 XG-102 3 000 .mu.g/ml IVT 14, 17,
38, 26, (5 .mu.l in 28, 31, 23, S8 2 300 .mu.g/ml right eye 24, 40,
19, 21, at Day 0 S5, 6, 39, 18 3 Saline -- and Day 7) 37, 12, 22,
S3, 4, 3, 33, 35 4 Kenacort .RTM. 4% triamcinolone 10, 3, 15, S1,
Retard acetonide 32, 8, 16, 9 5 Untreated -- -- 29, 7, 20, 36, S9,
27, 1, 11
[0570] Selection of the Animals
[0571] Forty (40)+ten (10) reserve animals were involved in this
study. Only animals with no visible sign of ocular defect were
selected. Then, the allocation in the treatment groups was done by
a random function in Excel.RTM. software. Fifty (50) animals were
induced and followed. The random allocation in the treatment groups
determined the eight animals and the reserve animals per group.
These latter animals were included in the calculations of results
only if one or two animals normally involved died, had impact on
lens during administration procedure or a corneal opacity (due to
repetitive anesthesia).
[0572] Induction of Neovascularization
[0573] On Day 0, animals were anesthetized by an intramuscular
injection of a mix xylazine/ketamine. Pupils from the right eyes
were dilated by instillation of one drop of 0.5% tropicamide. Then,
six (6) choroidal burns (75 .mu.m spot size) were done through a
slit lamp, with a contact lens, around the optic disc, between the
main vessel branches using an argon laser photocoagulator (532 nm;
150 mW; 100 ms). Production of a bubble at the time of laser
treatment confirmed the rupture of Bruch's membrane.
[0574] Route and Method of Administration
[0575] Animals were anesthetized by intramuscular injection of a
mix xylazine/ketamine. Test item, reference and vehicle (5 .mu.l)
were intravitreously injected in the right eyes dose regimen was on
Day 0 and Day 7. The injection was performed under an operating
microscope.
[0576] The intravitreal injections scheduled on Day 0 were done
following the induction of neovascularization, under the same
anesthesia.
[0577] The intravitreal injection was located in the supratemporal
area at pars plana and performed using a 30G-needle mounted on a 10
.mu.l Hamilton. The filled syringe was then mounted into the
UltraMicroPump III to achieve accurate injection in microliter
range.
[0578] Body Weights
[0579] The body weight of all animals was recorded before the start
of study then once a week. The animal body weights, recorded before
induction and treatment (baseline), then on Days 7, 14 and 21 were
all within a normal range at the baseline: 180.6.+-.12.3 g
(mean.+-.SD, n=40). At Day 21, no relevant difference between test
item, vehicle and untreated groups was observed. The animals
gained: +53 g (+29%) and +62 g (+34%) for XG-102 at 300 .mu.g/ml
and 3000 .mu.g/ml, respectively, versus +56 g (+31%) and +59 g
(+34%) for the vehicle group and untreated group, respectively.
[0580] Animals treated with Kenacort.RTM. retard gained +21 g
(+12%) between the baseline and Day 21 after induction.
[0581] Fluorescein Angiography
[0582] Fluorescein angiography was performed on Days 14 and 21
using an HRA. After anesthesia by an intramuscular injection of a
mix xylazine/ketamine and pupillary dilation, 250 .mu.l/100 g (body
weight) of a 10% sodium fluorescein was injected subcutaneously
using a 26G insulin syringe, and fluorescein photos were recorded
10 minutes after dye injection.
[0583] This study was carried out on forty (40) Brown Norway rats.
Argon laser was used to induce ChNV in the right eyes. The
development of ChNV was evaluated by fluorescein angiography (FA).
Treatments (test, reference and control items) were made by
intravitreous administration on Days 0 and 7 after induction.
Angiography was performed 10 min after fluorescein (tracer)
injection, on Days 14 and 21 after induction. The grading was based
on the highest intensity of fluorescein in each lesion and it was
not determined by the size of the leakage area.
[0584] Results were expressed as the group mean score per
time-point and by incidence of the number of spots at a given
intensity score for each treatment and at each of both time-points.
The Mann and Whitney test was used to determine if there was a
statistically significant difference in the FA score between
treated and control group. The statistical significance was
attributed when p<0.05 was obtained with Mann and Whitney-U
test.
[0585] FIG. 39 A shows the intensity of fluorescein leakage (mean
score.+-.SD). and FIG. 39 B illustrates the proportion of leaking
spots in test item-treated eyes at both time-points. FIGS. 39 C and
39 D illustrate the percentage of leaking spots (score >0) and
of maximum leaking spot (score of 3) respectively
[0586] Evaluation by Fluorescein Angiography
[0587] The leakage of fluorescein on the angiograms was evaluated
by two examiners in a masked fashion and graded as follows: Score
0, no leakage; Score 1, slightly stained; Score 2, moderate
stained; Score 3, strongly stained. If the two scores assigned to a
particular lesion did not coincide, the higher score was used for
analysis.
[0588] Evaluation with Isolectin B.sub.4 of ChNV by Labelling on
Flat Mount Preparation (Quantification in Option)
[0589] On Day 23, after euthanasia by an i.p. injection of
Dolethal.RTM., the treated right eyes were harvested and fixed 4%
paraformaldehyde solution 1 hour at room temperature. After
washing, retina, choroid and sclera were dissected. The retina was
carefully peeled. The sclera-choroid was flat mounted and incubated
after blocking with FITC-isolectin B.sub.4.sup.i antibody.
[0590] Statistical Analyses
[0591] Group mean values and standard deviation were calculated for
all parameters. To assess the statistical significance of
differences between the various concentration of the test item and
the vehicle, a Mann and Whitney U test was used. [0592] Results
[0593] (1) Reference Compound Kenacort Vs Vehicle and Untreated
Groups [0594] The following table summarizes the results of FA at
10 min on Days 14 and 21 (n=8 animals per group, right eyes)
TABLE-US-00006 [0594] Mean score of fluorescein leakage % of
Incidence reduction (% spots with score x) Treatment Dose
Time-point Mean .+-. SD vs Vehicle Score 0 Score 1 Score 2 Score 3
Untreated -- Day 14 2.1 .+-. 1.0 -- 10 19 25 46 (n = 48) Day 21 2.1
.+-. 0.9 -- 4 21 38 38 (n = 48) Vehicle (NaCl) 0.9% Day 14 2.9 .+-.
0.5 -- 0 4 6 90 IVT D0, D7 (n = 48) Day 21 2.2 .+-. 1.0 -- 6 21 23
50 (n = 48) Kenacort .RTM. 4% Day 14 0.1 .+-. 0.3 97% 87 13 0 0
retard (n = 46) (p < 0.001) (triamcinolone Day 21 0.3 .+-. 0.5
86% 69 31 0 0 acetonide) (n = 45) (p < 0.001) IVT D0, D7 Please
note that numerical data may have been rounded for presentation,
therefore, manual recalculation may result in slightly different
values.
[0595] At Day 14, 90% of the spots were leaking in the untreated
right eyes indicating the formation of ChNV. The mean score was
2.1.+-.1.0 (n=48). At Day 21, the untreated animals showed 96% of
leaking spots and mean score at 2.1.+-.0.9 (n=48) indicating the
persistence of the ChNV. [0596] At Day 14, 100% of the spots were
leaking in vehicle treated eyes with a mean score of 2.9.+-.0.5
(n=48) indicating the formation and the severity of the ChNV. By
Day 21, no relevant change in the incidence of leaking spots with
94% of the spots that were leaking and a mean score of 2.2.+-.1.0
(n=48), indicating the persistance of the ChNV. [0597] Scoring of
FA revealed that Kenacort.RTM. retard following two intravitreal
administrations at Days 0 and 7 significantly reduced the
fluorescein leakage by 97% (p<0.001, Mann & Whitney-U test)
compared to the vehicle at Day 14 as shown by a mean score of
0.1.+-.0.3 (n=46) vs 2.9.+-.0.5 for vehicle group. [0598] The
incidence of the leaking spots were reduced in Kenacort.RTM. retard
group with 13% of the leaking spots compared to the vehicle-treated
animals which showed 100% of the leaking spots at Day 14. [0599] By
Day 21, animals treated twice with Kenacort.RTM. retard showed a
relevant reduction by 86% of the vascular leakage compared to
vehicle-treated animals (p<0.001, Mann & Whitney test) as
shown by a mean score of 0.3.+-.0.5 (n=45) vs 2.2.+-.1.0,
respectively. [0600] The proportion of leaking spots compared to
vehicle group at Day 21 was unchanged as shown by 31% of leaking
spots for Kenacort.RTM. retard versus 94% for vehicle.
[0601] (2) XG-102-Treated Groups Vs Vehicle Group [0602] The
following table summarizes the results of FA at 10 min on Days 14
and 21 (n=8 animals per group, right eyes).
TABLE-US-00007 [0602] Mean score of fluorescein leakage Incidence %
of reduction (% spots with score x) Treatment Dose Time-point Mean
.+-. SD vs Vehicle Score 0 Score 1 Score 2 Score 3 XG-102 300
.mu.g/ml Day 14 2.4 .+-. 0.9 17% 7 9 18 66 IVT D0, D7 (n = 44) (p
< 0.05) Day 21 2.4 .+-. 0.8 -9% 5 9 32 55 (n = 44) 3000 .mu.g/ml
Day 14 1.7 .+-. 0.7 41% 0 44 44 12 (n = 43) (p < 0.001) Day 21
2.3 .+-. 0.7 -5% 0 14 47 40 (n = 43) Vehicle 0.9% Day 14 2.9 .+-.
0.5 -- 0 4 6 90 NaCl (n = 48) IVT D0, D7 Day 21 2.2 .+-. 1.0 -- 6
21 23 50 (n = 48) Please note that numerical data may have been
rounded for presentation, therefore, manual recalculation may
result in slightly different values.
[0603] A summary of the results is provided in FIG. 39.
[0604] The general behaviour of animals was not altered following
intravitreous administrations of XG-102 at both doses. No relevant
complications were found during the clinical follow-up.
[0605] The animal body weight increased during the study period:
+53 g (+29%) and +62 g (+34%) for XG-102 at 300 .mu.g/ml and 3000
.mu.g/ml, respectively, versus +56 g (+31%) and +59 g (+34%) for
the vehicle group and untreated group, respectively. Animals
treated with Kenacort.RTM. showed a weight gain of 21 g (+12%).
[0606] In the vehicle group, the induced eyes showed consistent
fluorescein leakage 14 and 21 Days after laser injury. The mean
fluorescein leakage was 2.9.+-.0.5 (n=48 impacts) at Day 14 with
100% of leaking spot indicating the formation and the severity of
the ChNV. At Day 21, formation of the ChNV remained consistent with
94% of the leaking spots and a mean fluorescein leakage of
2.2.+-.1.0 (n=48 impacts).
[0607] Two intravitreous administrations at Days 0 and 7 of
Kenacort.RTM. (200 .mu.g/administration) inhibited the incidence of
ChNV formation at Days 14 and 21 after induction with a mean score
of 0.1.+-.0.3 (p<0.001) and 0.3.+-.0.5 (p<0.001) for
Kenacort.RTM. retard versus 2.9.+-.0.5 and 2.2.+-.1.0 for vehicle,
on Days 14 and 21, respectively. On day 14, 13% of the lesions
showed leakage in the reference-treated group while 100% showed
leakage in vehicle group. By Day 21, the incidence of the leaking
spots remained reduced with Kenacort.RTM. retard (31%) in
comparison to vehicle (94%).
[0608] Animals treated with XG-102 at 300 .mu.g/mL and 3000
.mu.g/mL showed a significant reduction of the vascular leakage at
Day 14 by 17% (p<0.05) with a mean score of 2.4.+-.0.9 for low
dose, and by 41% (p<0.001) with a mean score of 1.7.+-.0.7 for
high dose of XG-102, compared to vehicle. At Day 21, XG-102 at both
doses did not show a relevant reduction of the vascular leakage
compared to vehicle.
[0609] A reduction of the proportion of spots with a score 3 was
recorded for 300 .mu.g/ml and 3000 .mu.g/ml XG-102 groups on Day 14
as shown by 66% and 12% of score 3 for low and high XG-102
concentration respectively, compared to 90% of spots scored by 3
for vehicle group.
[0610] Using anatomic and functional metrics of measuring ChNV and
under the given experimental conditions, XG-102 intravitreously
administered at 300 and 3000 .mu.g/ml inhibited the vascular
leakage 7 days (Day 14 of the study) after the last
administration.
Example 19: Effects of XG-102 on Adriamycin-Induced Nephropathy
[0611] The object of that example was to study the effects of
XG-102 on inflammatory kidney disease, nephropathy. Adriamycin
treatment induces glomerular disease in rat and mice mimicking
human focal segmental and glomerular sclerosis (FSGS). In this
model, tubular and interstitial inflammatory lesions occur during
the disease course, partly due to heavy proteinuria. In the absence
of therapy, kidney disease progresses to terminal renal failure
within eight weeks. Podocyte injury is one of the initial steps in
the sequences leading to glomerulosclerosis. The aim of the study
was to investigate whether XG-102 could prevent the development of
renal lesions and the renal failure.
[0612] XG-102 (control NaCl 0.9%) were administered to rats i.v. In
total 50 rats were treated, whereby 3 groups (of 10 rats) received
XG-102 (low dose (20 .mu.g/kg), medium dose (200 .mu.g/kg) and high
dose (2000 .mu.g/kg). All of these three groups (and the placebo
group) were treated with 10 mg/kg Adriamycin on day 0. A fifth
group of 10 animals did not receive any adriamycin and was treated
by the NaCl control. Histological preparations were provided at day
8, 14, 29 and 41.
[0613] These histological preparations clearly indicated that
XG-102 has-over the entire observation period--a significantly
positive effect on adriamycin-induced nephropathy. The
nephrological tissue is significantly rescued from cell loss, see
FIGS. 42 to 45). The effect on c-jun expression without treatment
by XG-102 or with treatment by XG-102 is provided in FIGS. 46 and
47, respectively.
[0614] In a further study 40 male Sprague-Dawley rats (Charles
River) were used (divided into 4 groups of ten rats). Nephropathy
has been induced by a single intravenous injection of Adriamycin 10
mg/kg on Day 0. XG-102 (SEQ ID NO: 11; 2 mg/kg; in NaCl 0.9%) was
administered intravenously in the tail vein on Day 0. The
administration volume has been 0.2 ml.
[0615] The table below summarizes the random allocation:
TABLE-US-00008 Dose volume/ ADR Treatment Route of Dose Number of
Group N.sup.o (Day 0) (Day 0) administration concentration animals
1 10 mg/kg NaCl 0.9% 0.2 ml, IV 0 10 2 10 mg/kg XG-102 0.2 ml, IV 1
mg/ml 10 2 mg/kg 3 NaCl 0.9% NaCl 0.9% 0.2 ml, IV 0 10 4 NaCl 0.9%
XG-102 0.2 ml, IV 1 mg/ml 10 2 mg/kg
[0616] Each day, the general behavior and the appearance of all
animals were observed. The health of the animals was monitored
(moribund animals, abnormal important loss of weight, major
intolerance of the substance, etc. . . . ). No rats were
removed.
[0617] Blood was collected from the tail vein at Days 7, 14, 28, 42
and 56 from 4 rats per group. Serum creatinine concentrations,
blood urea and protidemia were measured using appropriate kits from
Advia Chemistry 1650 (Bayer Healthcare AG, Leverkusen, Germany).
Two rats per group were sacrificed on Days 7, 14, 28, 42 and 56
after anesthesia. After animal sacrifice, both kidneys were
collected. For histopathological examination fixed tissue specimens
were dehydrated in graded alcohol solutions, cleared in toluene,
and embedded in paraffin. Sections (4 .mu.m) were stained with
periodic acid-Schiff (PAS), and Masson's trichrome staining was
performed to detect collagen deposition. Glomerular and
tubulointerstitial sclerosis were quantified under microscope.
[0618] Results were expressed in the form of individual and
summarized data tables using Microsoft Excel.RTM. Software.
Numerical results were expressed as mean.+-.standard error of the
mean (SEM). Due to the small number of animal tested, no
statistical analyses was performed.
[0619] Effect of XG-102 on renal function during the progression of
the disease:
[0620] Urea and creatinine serum levels were measured to study the
renal function during the kidney disease course. Because creatinine
interferes with the calorimetric dosage, only urea that is a fine
indicator of renal function was analyzed. Whereas urea serum levels
were remarkably stable in untreated rats (below 5 mmol/l), ADR
induced progressive increase of urea levels, which sharply raised
from Day 28 up to 25 mmol/l at Day 41, then 48 mmol/l at Day 56
reflecting terminal renal failure (FIG. 38 B). On the other hand,
XG-102-treated rats exhibited an urea serum level below 10 mmol/l
throughout the course of the disease (FIG. 48 B). On the other
hand, XG-102-treated rats exhibited an urea serum level below 10
mmol/l throughout the course of the disease (FIG. 48 B). The renal
function of rats treated with XG-102 alone was similar to 0.9%
NaCl-treated rats. These results suggest that XG-102 prevents the
progression to renal disease and renal failure.
[0621] Histopathological Findings (PAS and Masson Trichrome
Staining):
[0622] ADR-induced structural changes were evaluated under light
microscope. Saline-treated control rats showed morphologically
normal glomeruli and tubules. On Day 8, light microscopic
examination showed some areas with focal segmental
glomerulosclerosis and proteinaceous casts in the ADR nephrosis
group. In contrast, although some tubules were filled with proteins
in XG-102-treated rats, glomeruli exhibited a normal architecture
with absence or discrete mesangial hypercellularity, while the
tubular structures and interstitium did not display pathological
changes (FIG. 49). By Day 14, ADR treated rats exhibited
progressive glomerulosclerosis, hyaline deposits, tubular dilation
and cast formation. The degree of glomerulosclerosis was
dramatically worsened in this group and became diffuse with obvious
adhesion between the glomerular tufts and the Bowman's space in
most glomeruli by Day 29 and 41, associated with severe tubular
atrophy and interstitial fibrosis. At Day 56, diffuse glomerular
sclerosis was observed in all glomeruli (FIG. 50). However,
XG-102-treated rats had a relatively normal appearance at Day 8,
and develop few focal and segmental glomerulosclerosis and
tubulointerstitial fibrosis at Day 56 compared with ADR-treated
rats. Altogether, these results strongly suggest that XG-102
prevents the development of glomerular and tubulointerstitial
fibrosis and may explain the preservation of renal function in this
group.
[0623] The study results provide evidence that XG-102 prevents the
progression of glomerular and tubulointerstitial injuries induced
by ADR. Moreover, this molecule preserves renal function.
Example 20: Effects of XG-102 on Puromycine Aminonucleoside
(PAN)-Induced Nephropathy
[0624] The aim of this study was to evaluate the effects of XG-102
on chronic puromycine aminonucleoside-induced nephropathy in rats
during 56 days. Puromycin aminonucleoside (PAN) is a podocyte toxin
inducing a loss and fusion of podocytes foot processes. PAN-induced
nephropathy is a well-described model of human idiopathic nephritic
syndrome and focal segmental glomerulosclerosis (Pippin J W, 2008).
The glomerular morphologic changes seen in rats with PAN nephrosis
closely resemble those in human minimal change disease (MCD) and
focal segmental glomerulosclerosis (FSGS). Intraperitoneal
administration of PAN in rats results in a rapid development of
nephritic syndrome, characterized by proteinuria, hypoalbuminemia
and hypercholesterolemia (acute phase). This is a well-established
animal model of human MCD. The pathological lesions of focal
segmental glomerulosclerosis have been observed in chronic PAN
nephrosis induced by repeated intraperitoneal PAN injections
(Nakajima, T., Kanozawa, K., & Mitarai, T. (2010). Effects of
edaravone against glomerular injury in rats with chronic puromycin
aminonucleoside nephrosis. J Saitama medical university, 37(1)). In
accordance with the mechanism of injury, PAN causes direct DNA
damage via the production of reactive oxygen species (ROS) and
tissue damages, including glomerulosclerosis and interstitial
fibrosis (Hewitson T D, 2012) in the chronic phase.
[0625] In this experiment 90 male Wistar rats (Charles River,
France) were used (divided into 6 groups of 15 rats). To induce
nephropathy puromycin aminonucleoside (PAN) was intraperitoneally
administered at the dose of 130 mg/kg (5 ml/kg) at day 0 and at the
dose of 60 mg/kg (5 ml/kg) at day 14 (Nakajima, T., Kanozawa, K.,
& Mitarai, T. (2010). Effects of edaravone against glomerular
injury in rats with chronic puromycin aminonucleoside nephrosis. J
Saitama medical university, 37(1)). Control rats (Group 1) received
an equal amount of saline i.p at day 0 and at day 14. XG-102 or its
vehicle (NaCl 0.9%) were administered into the tail vein (i.v.)
once a week (Groups 1 to 5) starting from first PAN injection at
day 0 for a total of 7 injections at day 0, 7, 14, 21, 28, 35 and
42. In a separate experimental group (Group 6), XG-102 was
administered into the tail vein (i.v.) once a week starting from
day 21 for a total of 4 injections at day 21, 28, 35 and 42 after
PAN injection at day 0.
[0626] For XG-102 administration XG-102 powder has been dissolved
in the vehicle NaCl 0.9% at the highest concentration to be tested.
The highest concentration then represented the stock solution for
the lower concentrations. Each stock solution has been filter (0.2
.mu.m) sterilized. The lower concentration solutions to be
administered were prepared by diluting the filtered stock solution
in saline (0.9% NaCl) depending on the volume for i.v.
injection.
[0627] The table below summarizes the experimental groups:
TABLE-US-00009 PAN Number of i.v. Number of Group (i.p.) Treatment
(i.v.) administrations animals/group 1 no vehicle 7 15 2 yes
vehicle 7 15 3 yes XG-102 (1 mg/kg) 7 15 4 yes XG-102 (2 mg/kg) 7
15 5 yes XG-102 (4 mg/kg) 7 15 6 yes XG-102 (4 mg/kg) 4 15
[0628] The study design is shown in FIG. 51. Briefly, on day 0 and
day 14 PAN or its vehicle (saline) have been injected for induction
of nephropathy. At day 0 and at day 14, PAN has been administered
first, followed by XG-102 administration. From day 0 to day 42
XG-102 or its vehicle (NaCl 0.9%) have been administered once a
week by i.v. route as described above.
[0629] Animals were weighted once a week. All PAN-treated animals
showed a decrease of body weight. However, all PAN-treated animals
were homogeneous for body weight, i.e. no effects of XG-102 were
observed compared to PAN/saline group (Group 2) on body weight. On
day 56 animals have been sacrificed and samples (blood and kidneys)
have been collected.
[0630] In particular, for blood and kidney sampling animals have
been anesthetized by injection of pentobarbital (60 mg/kg; Ceva
Sante Animale; Libourne, France). Blood samples have been collected
from abdominal vein, transferred into tubes for coagulation (EDTA
3K; 30 minutes, 4.degree. C.) then centrifuged (10 minutes, 3000
rpm, 4.degree. C.) for plasma collection. Plasma has been stored at
-20.degree. C. until use for biomarkers assay, e.g. creatinine and
urea assays.
[0631] For quantification of biomarkers, Plasma LDL levels were
quantified using an ABX Pentra 400 Clinical Chemistry analyzer
(HORIBA) by the Phenotypage platform of Genotoul (Rangueil
Hospital, Toulouse, France).
[0632] Kidneys have been removed, cleaned from all connective
tissue and capsule and weighted on an electronic microbalance
(Mettler, Toledo). Kidney samples have been fixed in formalin
solution 10% (Sigma Aldrich, France) for 24-72 h, in particular 48
h, then embedded in paraffin. Three sections (3 to 5 .mu.m) were
made per block. The slides were stained by hematoxylin/eosin (HE),
PAS-methenamine silver and Sirius Red for histological evaluation
of morphological alterations, glomerulosclerosis and interstitial
fibrosis quantification, respectively. All the slides were
digitalized at .times.20 using Nanozoomer 2.0 HT from Hamamatsu
(Japan). Histological preparation and imaging has been performed by
Histalim (Montpellier, France). Plasma creatinine and urea have
been quantified using an ABX Pentra 400 Clinical Chemistry analyzer
(HORIBA) by the Phenotypage platform of Genotoul (Rangueil
Hospital, Toulouse, France).
[0633] Results are expressed by semi-quantitative scoring following
to expert histopathologist evaluation. For the histological
examination of glomerulosclerosis glomerular changes have been
evaluated using a semi quantitative scoring system as described by
Nakajima, T., Kanozawa, K., & Mitarai, T. (2010). Effects of
edaravone against glomerular injury in rats with chronic puromycin
aminonucleoside nephrosis. J Saitama medical university, 37(1),
which is hereby incorporated by reference. In brief, the degree of
glomerular injury was assessed in 25 glomeruli per kidney section
(2 sections per animal) for a total of 50 glomeruli per animal.
Degree of injury in individual glomeruli was graded using a scale
from 0 to 4, based on the percentage of glomerular involvement.
[0634] Score 0: normal,
[0635] Score 1: lesions in up to 25% of the glomerulus,
[0636] Score 2: lesions between 25-50% of the glomerulus,
[0637] Score 3: lesions between 50-75% of the glomerulus, and
[0638] Score 4: lesions between 75-100% of the glomerulus
[0639] All data have been calculated as mean values.+-.standard
error of the mean (s.e.m.). Statistical analysis has been performed
using GraphPad Prism, version 4 (GraphPad Software Inc., LaJolla,
USA). The comparison of all the groups using two-way ANOVA followed
by Bonferroni's post-test for body weight results. Comparison
between group 1 (Saline/saline) and group 2 (PAN/saline) was
performed using unpaired Student t-test. The effects of vehicle and
XG-102 were compared using one way ANOVA followed by Newman-Keuls
test. A P<0.05 value was accepted as statistical significance.
Comparison between group 2 (PAN/vehicle) and group 6 (PAN/XG-102 4
mg/kg, 4.times. iv) was performed using unpaired Student
t-test.
[0640] The results of the glomerulosclerosis injury are shown in
FIG. 52. One of the main objectives of this study was to evaluate
the glomerulosclerosis injury in a well-established model of focal
segmental glomerulosceloris (FSGS) induced by repeated puromycin
aminonucleoside injections in rats. The results showed that 7 iv
injections of XG-102 significantly reduced PAN-induced
glomerulosclerosis in a dose dependent manner. However, the dose of
1 mg/kg had no effect on this pathological feature. 4 iv injections
of XG-102 at the dose of 4 mg/kg, starting from day 21 resulted in
a strong effect of XG-102 in reducing glomerulosclerosis induced by
PAN (FIG. 52).
[0641] The results of the glomerular damage are shown in FIG. 53.
One of the main objective of this study was to evaluate the effect
of XG-102 on the glomerular damage induced by repeated PAN
injections in rats. The results showed that XG-102 has (i) a
preventive effect in that 7 iv injections at the dose of 2 and 4
mg/kg significantly reduced PAN-induced glomerulosclerosis in term
of severity of lesions (glomerular injury score) but also
significantly decreased glomerular damage incidence (percentage of
injured glomeruli) and that (ii) XG-102 has a curative effect in
that 4 iv injections of XG-102 at the dose of 4 mg/kg, starting
from day 21 post-PAN administration lead to a strong effect on
glomerulosclerosis in term of both severity of lesions (glomerular
injury score) and of glomerular damage incidence (percentage of
injured glomeruli). Taken together, XG-102 showed a dose-response
effect on glomerulosclersosis injury, namely a preventive and a
curative effect on the severity of lesions and glomerular damage
incidence.
[0642] Regarding the analysis of biomarkers, serum LDL represents a
good marker of the progression of FSGS and oxidative stress in this
model. Serum levels of LDL increase and peak between day 21 and day
28 after PAN injection, remaining still high in the chronic phases
(cf. Nakajima et al., 2010). Accordingly, in the present study
PAN-treated animals showed a significant increase of LDL plasma
levels compared to Saline-treated animals (Group 1). In XG-102
treated animals a decrease in Plasma LDL was observed in particular
for the 4 mg/kg groups (Group 5 and 6), although it was not
significant. Thus, XG-102 tends to decrease oxidative stress as
shown by the decreases in serum LDL and by decreases in major lipid
peroxidation product (4-HNE: 4-hydroxy-2-nonenal). Moreover,
results obtained regarding the biomarkers ED-1 (rat CD-68) with
Anti-CD68 showed that XG-102 also tends to decrease infiltrating
macrophages.
Example 21: Effects of Chronic Administration of XG-102 in a Rat
Model of Diabetic Nephropathy
[0643] The aim of this study has been to evaluate the effects of
chronic administration of the JNK inhibitor peptide, XG-102 (1, 2,
4 mg/kg, weekly intravenous administration for 9 weeks), in a rat
model of diabetic nephropathy. Losartan has been used as a positive
control.
[0644] Seventy-four male Sprague-Dawley rats (200-250 g; including
4 spare animals) from Charles River (Margate, Kent) were used. Rats
were housed in pairs in polypropylene cages with free access to a
high fat diet (D12492 60% of kcal derived from fat) and tap water
at all times. The diet has been purchased from Research Diets, New
Jersey, USA. All animals have been maintained at 21.+-.4.degree. C.
and 55.+-.20% humidity on a normal light (lights on:
07:00-19:00).
[0645] The study schedule is shown in FIG. 54. Animals have been
housed in pairs throughout the study. For a 3-week period, during
which time they have been weighed weekly (food and water will be
weighed twice during the third week only (i.e. the week prior to
STZ dosing on a Monday and a Thursday). During the third week of
habituation, a blood sample has been taken from the lateral tail
vein in the freely fed state using a hand-held glucose meter (One
Touch Ultra 2). Blood sampling began at approximately 09:00.
[0646] Due to the size of the study, the animals have been run as
two separate cohorts (each n=4 or 6 as far as possible due to
paired housing) 72 hours out of phase (see FIG. 54). About 40
animals have been assigned to Cohort A and the remaining 30 to
Cohort B, balanced as far as possible for body weight, plasma
glucose and food and water intake. For induction of diabetes
streptozotocin (STZ) has been used. Since the diabetic phenotype of
animals dosed with STZ is highly dependent on the batch of STZ, a
pilot study has been undertaken in order to confirm the optimal STZ
dose (35 or 45 mg/kg ip). STZ or vehicle has been given after the
animals have been maintained on the diet for approximately 3 weeks
as detailed in FIG. 54. The spare animals will be dosed with STZ
(one pair per cohort).
TABLE-US-00010 Group Dose (ip) Cohort A Cohort B A vehicle 0.05M
citric acid pH 4.5 ip 4 6 B-G STZ (selected from pilot) ip 36
24
[0647] Each pair of animals has been administered the same
treatment (i.e. both vehicle-treated or both will be STZ-treated).
For the 7-day period post STZ dose, animals have been weighed daily
and food and water intake determined twice weekly. For the
remaining study duration, animals have been weighed and water and
food intake assessed twice weekly (always on the day of intravenous
dosing and typically on water refill day(s)). Subsequently, based
on body weight and available food and water intake post STZ,
animals have been allocated in groups B-F as detailed below in
light of differences in dosing regimen.
TABLE-US-00011 Group Dose Cohort A Cohort B B-E IV dosing 24 16 F-G
PO dosing 12 8
[0648] One week after STZ (or vehicle) treatment a blood sample has
been taken from the lateral tail vein using a glucometer (One Touch
Ultra2) in the freely fed state (blood samples taken beginning at
approx. 09:00). Subsequently, animals in groups A-E have been dosed
with vehicle by the intravenous route and animals in Groups F-G
have been dosed with 1% methyl cellulose by the oral route. Animals
in groups F-G continued to be dosed once daily beginning at
approximately 09:00 each day. Animals have been weighed prior to
dosing (this weight was recorded). Food and water have been
recorded on the same days as the intravenous groups (A-E) only.
[0649] This baseline phase lasted for one week. Towards the end of
the week animals have been allocated to drug treatments on the
basis of blood glucose, and available body weight and food and
water intake data. The allocation has been as detailed in the table
below:
TABLE-US-00012 Cohort Cohort Total Group Group STZ A B N A Vehicle
(saline) - NON-STZ NO 4 6 9-10 B Vehicle (saline iv weekly) STZ 6 4
9-10 C XG-102 (1 mg/kg iv weekly) STZ 6 4 9-10 D XG-102 (2 mg/kg iv
weekly) STZ 6 4 9-10 E XG-102 (4 mg/kg iv weekly) STZ 6 4 9-10 F
Vehicle (methyl cellulose STZ 6 4 9-10 po daily) G Losartan (25
mg/kg po daily) STZ 6 4 9-10
[0650] Dosing has been for 9 weeks in duration (9 administrations
in total, see FIG. 54). Animals in groups F and G have been weighed
and dosed daily at approximately 09:00. Animals in groups A-E have
been dosed once weekly by the intravenous route (as detailed on
FIG. 54). In all groups, food and water intake have been determined
twice weekly (on the day of iv dosing and on water refill days.
Blood glucose has been determined monthly. Samples were collected
as detailed previously by glucometer (One Touch Ultra2). Blood
samples have been taken in the freely fed state (beginning at
approx. 09:00). Animals have been dosed immediately afterwards by
the respective route to a timed schedule. Subsequent to dosing,
each animal has been placed in a metabolism cage with free access
to food and water for a 24 h period. To reduce evaporation, the
glass urine collectors have been placed in a polystyrene container
(Sca-online, UK) which was filled with ice. Due to the anticipated
increase in daily urine volume with STZ, urine has been collected
(and stored refrigerated) at intervals (e.g. 8 hourly) to ensure
that twenty four hours total urine volume for each metabolic cage
can be recorded. The aliquots at each time point have been pooled
so that a single 24 h sample per animal is collected. Ten aliquots
of 300 .mu.l of pooled 24 h urine have been taken and frozen at
-80.degree. C. Creatinine, glucose, urea, total protein and
electrolytes (Na, K, Cl and Ca) have been determined on urine
samples using a COBAS C111 and associated reagents (n=2 for all
urine analyses). For urine collection sessions, the rats have been
weighed at the time of placement in the cage and upon removal. Food
consumed and water drunk has also been calculated. Blood glucose
and urine parameters (creatinine, glucose, urea, total protein and
electrolytes) have been determined again after a further month of
dosing as previously described (see FIG. 54).
[0651] During week 8 of treatment (see FIG. 54) the glomerular
filtration rate (GFR) of the animals has been assessed using the
FITC-inulin method. This was performed based on the method of
Stridh, S., Sallstrom, J. et al (2009): "C-Peptide Normalizes
Glomerular Filtration Rate in Hyperfiltrating Conscious Diabetic
Rats" Oxygen Transport to tissue XXX. Advances in experimental
medicical and biology. 645:219-25, which is hereby incorporated by
reference. Specifically, FITC-inulin (1.5%) has been dissolved in
saline and filtered through a 0.45 .mu.m syringe filter. In order
to remove residual free FITC, the solution has been dialysed in
2000 ml of saline at 4.degree. C. overnight using a 1000 Da cut-off
dialysis membrane (Spectra Por 6 from Fisher UK) and protected from
light. The dialysed inulin has been filtered through a 0.22 .mu.m
syringe filter before use. Each animal has been dosed with 1 ml (15
mg) of FITC-inulin via the tail vein (i.e. intravenously). At 2, 5,
9, 15, 24, 35, 55, 80 minutes post dose a blood sample (80 .mu.l)
has been taken into a lithium-heparin collection tube (Sarstedt
CB300LH). Each blood sample underwent centrifugation in a cooled
centrifuge and the plasma sample dispensed into a clean aliquot
vial for subsequent determination of fluorescence at 496 nm
excitation and 520 nm emission.
[0652] At termination, animals and food and water have been
weighed. Animals have then been killed and a terminal blood sample
(approx. 4.5 mL in an EDTA-coated tube) has been taken via cardiac
puncture). The blood sample has been spun in a cooled centrifuge
and aliquots (5 aliquots of 0.5 mL) stored frozen (-80.degree. C.).
At necropsy, the left and right kidneys have been removed and
weighed. Each kidney was cut sagittally into two halves and placed
into a pot of 10% neutral buffered formalin to fix for
approximately 5 days. The kidneys have then been wax embedded and
one half from each kidney placed into each cassette to produce one
wax block for subsequent processing (i.e. one block with one half
right kidney and one half left kidney) The remaining kidney halves
have been disposed of. For the wax blocks, all tissues have been
prepared using a Tissue Tek VIP processor (using graded alcohols to
dehydrate and xylene as a clearant). The blocks have then been
impregnated with paraffin histo-wax prior to embedding in fresh
histo-wax. Kidney tissues were sectioned at approximately 4-51
.mu.m and stained using methods for Haematoxylin and Eosin
(H&E) and periodic acid Schiff (PAS). Subsequently, slides will
be sent for assessment by a pathologist (e.g. to Harlan
Laboratories Ltd. UK). The pathologist evaluated all slides stained
by H&E and PAS for glomerular sclerosis, tubule atrophy and
interstitial expansion semi-quantitatively using a "+, ++, +++"
system (or similar).
[0653] XG-102 has been dosed in the volume 1 ml/kg in commercially
available sterile saline. To this end, XG-102 has been formulated
prior to the first dosing by the addition of sterile saline,
whereby the highest dose has been formulated (4 mg/ml) and the
lower doses were prepared by dilution of this 4 mg/ml stock.
Aliquots were then prepared for each dosing session and stored
frozen (-80.degree. C., stability 3 months at -80.degree. C.) until
use. On the morning of dosing each aliquot has been removed from
the freezer and allowed to thaw at room temperature prior to dosing
(e.g. 30 minutes). The thawed solution has been mixed by inversion
prior to dosing. All dosing was completed as soon as possible after
thawing but in all cases within 8 hours since the test item is
stable in saline at room temperature at concentrations of 10
.mu.g/ml-50 mg/ml for 8 hours. Sterile polypropylene plastics
(including pipette tips) have been used. The stock solution will be
filter sterilised (0.2 .mu.m) prior to use and prior to dilution to
lower doses. Losartan potassium has been purchased from a Chemical
supplier (e.g. Tocris UK) and prepared for dosing each morning in a
vehicle of 1% methyl cellulose at a volume of 5 ml/kg. Dosing
factors have been applied where appropriate.
[0654] At the end of the study, body weights and weight of food and
water bottles have been analysed. Results have been expressed as
body weights, change in body weight per week for the first 4 weeks
and per 4 weeks thereafter, and over the entire drug administration
period, % reduction in body weight at the end of the study and drug
treatment compared to the control group, food and water intakes,
cumulative food intake and average food and water intakes per week
for the first 4 weeks and per 4 weeks thereafter and over the
duration of the feeding study. The effects of different treatments
on body weight and food, cumulative food and water intake have been
analysed by two-way analysis of covariance with treatment and
cohort as factors and baseline (Day 1 body weight or the average
food or water consumption from days-6 to 0) as the covariate,
followed by appropriate multiple comparisons tests (two-tailed) to
compare each group to the appropriate STZ vehicle group. Blood
glucose has been analysed by general linear model with treatment
and cohort as factors and baseline body weight, bleeding order and
pre-study plasma level as covariates. Appropriate transformations
and/or robust regression techniques may have been used to reduce
the influence of outliers. Suitable multiple comparison tests
(two-tailed) have been used to compare each group to the
appropriate STZ vehicle group. Urine creatinine, glucose, urea,
total protein and electrolytes have been expressed as treatment
group means.+-.SEM. Analysis has been by general linear model with
treatment and cohort as factors. Appropriate transformations and/or
robust regression techniques may have been used to reduce the
influence of outliers. Suitable multiple comparison tests
(two-tailed) have been used to compare each group to the
appropriate STZ vehicle group. Kidney weights have been analysed by
general linear model with treatment and cohort as factors and Day 1
body weight as a covariate. To determine effects in addition to
effects caused by changes in body weight, analysis has been by
general linear model with treatment and cohort as factors and
terminal body weight as a covariate. A log transformation and/or
robust regression techniques has been used if appropriate.
Appropriate multiple comparison techniques has been used to compare
each group to the appropriate STZ vehicle group. For the pathology
assessment, each treatment has been compared to the appropriate STZ
vehicle group by exact Wilcoxon rank sum tests.
[0655] GFR has been calculated as Dose of FITC inulin/AUC. The AUC
(of FITC inulin concentration) has been calculated by the
log-linear trapezoidal rule (Stridh) with extrapolation of the 2 to
5 min line to 0 min and linear regression of log-transformed data
during a terminal phase from 24 to 80 min. Calculated GFR values
were analysed by two-way analysis of variance with treatment and
cohort as factors. A log transformation and/or robust regression
techniques has been used if appropriate.
[0656] In all analyses except GFR, animals dosed iv have been
analysed separately from animals dosed po, as dosing by different
routes during the baseline week may affect the baseline values used
as covariates. The non-STZ group has been excluded from all
analyses described above. Separate analyses have been performed for
comparisons to the non-STZ group, including all groups in the
analysis, but using baseline covariates before treatment with STZ,
rather than those during the week before dosing. In all analyses, a
p value of less than 0.05 will be considered to be statistically
significant.
[0657] The effects of chronic administration of XG-102 in this rat
model of diabetic nephropathy on the body weight of the rats are
shown in FIG. 55. Only non-STZ treated rats showed an increase in
body weight. Rats treated with XG-102 showed no differences in body
weight compared to vehicle-treated rats in the STZ model. The body
weight of rats treated with the positive reference Losartan,
however, has been significantly lower. These results indicate that
XG-102 is well-tolerated, whereas the positive reference Losartan
resulted in a significant decrease of the body weight.
Example 22: Evaluation of the Dose-Response to XG-102 in Islet
Isolation/Transplantation
[0658] This study is based on the previous study on islet isolation
(cf. Example 17) and on the publication by Noguchi et al. (Noguchi,
H., S. Matsumoto, et al. (2009). "Ductal injection of JNK
inhibitors before pancreas preservation prevents islet apoptosis
and improves islet graft function." Hum Gene Ther 20(1): 73-85.).
These studies have shown, in a porcine islet isolation model that
islets undergo a dramatic activation of JNK starting as early as 20
minutes after the initiation of the islet isolation procedure. This
activation is the result of the method that combines warm ischemia,
enzymatic digestion and mechanic stress on an already fragile
tissue. The study of Example 17 it has shown that intravascular
addition of XG-102 (10 .mu.M) to the preservation solution flushed
into the porcine pancreas at the time of procurement has a
significant impact on islet cell viability and functionality,
assessed by oxygen consumption rate (OCR), and ATP concentration,
and correlates with a decrease in JNK activation and c-fos gene
expression. Noguchi et al have used a different inhibitor and added
it at the same molar concentration into the pancreatic duct
immediately after procurement. Porcine and human pancreases were
used. They showed a similar effect on islet viability assessed by
ATP concentration, but also an impact in vivo on diabetes reversal
after transplantation under the kidney capsule of diabetic mice.
The purpose of the present set of experiments has been to determine
the dose-response curve of XG-102 and the optimal concentration at
which to utilize it in islet isolation. In order to answer this
question, a rodent model has been utilized. While differences
between human and rodent pancreas and islets are acknowledged, this
model was selected because of its straightforwardness and high
cost-efficiency. The purpose of these experiments being solely the
determination of the optimal dose of XG-102 required, the rat model
appears as valid. Since the major purpose is JNK inhibition in
human pancreases for the improvement of clinical allogeneic islet
transplantation outcome, intraductal injection of the inhibitor has
been done in these experiments. This is in effect the most likely
way that the compound will be used in the clinical setting.
[0659] To assess the JNK activation in rat islets after isolation,
islets of Langerhans have been isolated from Lewis rats by a
classic enzymatic method using collagenase. Isolation has been
carried out either immediately after animal sacrifice or after a
15-minute period of warm ischemia. JNK activation has been assessed
by western blot at the end of the isolation process. JNK activation
has been assessed on unprocessed rat pancreases as negative
controls. Experiments have been done on 3 rats for each condition
of ischemia plus 1 for the negative control, and repeated 3 times.
This represents a total of 21 Lewis rats. The results shown in FIG.
56 show that XG-102 dose-dependently decreased JNK (FIG. 56 A) and
PAF2 (FIG. 56 B) phosphorylation induced by 15-min ischemia.
[0660] To study the effects of XG-102 on islet viability, the best
model in terms of duration of ischemia (no warm ischemia vs
15-minute warm ischemia), i.e. the model most likely to show
differences after JNK inhibition, has been selected based on the
results of the previous experiments. Isolation has been carried out
using XG-102 at a set concentration or vehicle, diluted in the
collagenase solution and injected into the pancreatic duct prior to
enzymatic digestion of the pancreas. XG-102 at the same molar
concentration or vehicle has been used throughout the isolation
procedure in the various washing or purification solutions
utilized, and in the culture medium. Isolated islets have been
cultured overnight in RPMI-based culture medium. For each set of
experiments, the following XG-102 concentrations have been
utilized: 1 .mu.M, 3 .mu.M, 10 .mu.M, 50 .mu.M and 100 .mu.M. Three
animals have been utilized in each group for each concentration,
and experiments have been repeated 2-3 times depending on results.
This represents a total of 60-90 Lewis rats. Islet yields have been
determined. The following assessments of islet viability has been
performed: JNK activation, OCR, ATP concentration, caspase release,
etc.
[0661] To study the effect of XG-102 on islet function in vivo
supplementary isolations have been done in order to assess the
effect of JNK inhibition on in vivo islet function. In vivo
experiments have been done only with islets isolated using the most
effective XG-102 molar concentration in the in vitro experiments
detailed above or with vehicle. Islet isolation has been performed
as above. For each isolation, 1000 and 2000 IEQ have been
transplanted under the kidney capsule of streptozotocin-induced
diabetic immunodeficient mice. Proportion of animals reversing
diabetes and time necessary for reversal of diabetes have been
compared between animals transplanted with XG-102-treated or
control islets. Transplants have been repeated 3 times. Number of
animals required is approximately 30 Lewis rats and 24 NOD-scid
mice.
[0662] As shown in FIG. 57, to study the effects of XG-102 on
function and viability of rat pancreatic islets have been isolated
islets from 15 min ischemia rat and from no ischemia rat. A static
insulin secretion test (basal or stimulated using glucose) has been
performed directly after islet isolation and 18 h after culture at
37.degree. C. It can be observed that isolation affects islet
function. Indeed basal insulin secretion was higher in islets used
directly after isolation compared to islets incubated during 18 h
whatever the conditions. These high basal levels reflect a distress
of islet. However after culture, ischemia and inhibitor XG-102 had
no impact on islet function in this experiment.
[0663] Because in the previous experiment it has been shown that
islet from 15 min ischemia rats secreted same amount of insulin
than islet from control rats in response to glucose, a new
experiment has been performed, wherein ischemia was pushed until 30
min and JNK inhibitor XG-102 was used at 100 microM (FIG. 58). In
this experiment a high basal secretion when insulin secretion test
was performed directly after isolation is still observed. Moreover,
30 min ischemia had a negative impact on islet function. These
preliminary results suggested that 30 min ischemia seems to be a
better model than 15 min to induce JNK activation. When islets from
ischemic rats were isolated and incubated with XG-102,
glucose-induced insulin secretion was higher as compared to
ischemic rats (FIG. 58), suggesting a positive effect of XG-102 on
the islet function.
Example 23: Efficacy of XG-102 (SEQ ID No. 11) in a Rat
Laser-Induced Choroidal Neovascularization (CNV) Model Following
Subconjunctival Injections
[0664] The objectives of this study were to determine the efficacy
of XG-102, a JNK-inhibitor, when administered by subconjunctival
injections to rats in a model of laser-induced choroidal
neovascularization (CNV). As outlined in the context of Example 18,
this model allows predictions about a potential use of a compound
for the treatment of age-related macular degeneration (AMD). In
contrast to the study described in Example 18, the subconjunctival
route of administration has been selected for the present study,
because it is another preferred route for the administration in
humans.
[0665] The following experimental groups have been assigned:
TABLE-US-00013 Dose Dose Number of Group Level Volume Dose Animals
No. Test Material (.mu.g/eye) (.mu.L/eye) Concentration Males 1
Vehicle 0 5 0 mg/mL 8 Control 2 XG-102 0.15 5 0.03 mg/mL 8 3 XG-102
1.5 5 0.3 mg/mL 8 4 XG-102 15 5 3 mg/mL 8 5 Reference 200 5 4% 8
Item 2
[0666] The vehicle control, 0.9% NaCl, has been administered as
received. Triamcinolone acetonide 4% serves as "Reference Item 2"
and has also been administered as received. For XG-102 preparation,
a stock solution equal to the highest dose level has been prepared
in vehicle, 0.9% Sodium Chloride for Injection, and sterile
filtered through a 0.22 .mu.m polyvinylidene difluoride (PVDF)
filter. The lower dose levels have been prepared by directly
diluting the stock solution. Dose formulations have been prepared
once at appropriate concentrations to meet dosage level
requirements. All dilutions have been prepared by directly diluting
the stock solution with vehicle. Two dosing aliquots (Days 1 and 8)
have been prepared and stored in a freezer set to maintain
-20.degree. C. Aliquot(s) of each dose level have been thawed at
ambient temperature on each day of dosing and the solution
maintained at room temperature for no longer than 6 hours.
[0667] 44 male Brown Norway rats (Charles River; age 10 weeks) have
been used. A minimum acclimation period of 14 days has been allowed
between animal receipt and the start of treatment in order to
accustom the animals to the laboratory environment. Animals have
been assigned to groups by a stratified randomization scheme
designed to achieve similar group mean body weights. Animals in
poor health or at extremes of body weight range were not assigned
to groups. Before the initiation of dosing, any assigned animals
considered unsuitable for use in the study has been replaced by
alternate animals obtained from the same shipment and maintained
under the same environmental conditions. After initiation of
dosing, study animals have been replaced during the replacement
period with alternate animals in the event of accidental injury,
non-test article-related health issues, or similar circumstances.
The alternate animals have been used as replacements on the study
within 3 days. On arrival, animals have been individually housed
until randomization. Following randomization, animals have been
group housed (up to 3 animals of the same dosing group together) in
stainless steel perforated floor cages equipped with an automatic
watering valve. Animals have been separated during designated
procedures/activities. PMI Nutrition International Certified Rodent
Chow No. 5CR4 (14% protein) has been provided ad libitum throughout
the study, except during designated procedures. Municipal tap water
after treatment by reverse osmosis and ultraviolet irradiation has
been freely available to each animal via an automatic watering
system (except during designated procedures). Animals have been
socially housed for psychological/environmental enrichment and
provided with items such as a hiding tube and a chewing object,
except during study procedures/activities.
[0668] On day 1 of the study Laser-Induced Choroidal
Neovascularization (CNV) Procedure has been performed. Prior to the
CNV procedure, mydriatic drops (1% tropicamide) were applied to
both eyes. Further applications have been performed as considered
appropriate by the veterinary ophthalmologist. The animals have
been anesthetized an isoflurane/oxygen mix prior to and during the
procedure. Under anesthesia, a 4-spot pattern have been made
between the major retinal vessels around the optic disc of each eye
using an 810 nm diode laser at an initial power setting of 300 mW
(laser power may be increased for bubble formation), an initial
spot size of 80 .mu.m and a duration of 0.1 seconds. Laser
parameters have been adjusted as required to ensure rupture of
Bruch's membrane (correlated with bubble formation). In the event
that rupture of Bruch's membrane is not confirmed for a particular
spot, this has been documented. In this case or in the case of
hemorrhage, an additional spot may be added if considered
appropriate by the veterinary ophthalmologist. Any notable events,
such as retinal hemorrhage were documented for each laser spot. If
hemorrhage is too severe, the animal has been excluded from the
study and replaced. Hydration of the eyes has been maintained with
a saline solution and/or carboxymethylcellulose sodium 1.0% during
the procedure, as necessary.
[0669] Vehicle control, test item or reference item will be
administered by subconjunctival injection to the left and right
eyes of each animal on Days 1 and 8 as indicated in the
Experimental Design above. The animals have been anesthetized
(isoflurane) for the dose administration, which has been performed
by a board-certified veterinary ophthalmologist. Topical
antibiotics (gentamicin ophthalmic solution) have been applied to
both eyes twice on the day before treatment, following the
injection and at least once on the day following the injection.
Prior to dosing, mydriatic drops (1% tropicamide and/or 2.5%
phenylephrine) have been applied to each eye (further applications
may be performed as considered appropriate by the veterinary
ophthalmologist). During dosing, animals are maintained under
anesthesia with isoflurane/oxygen gas. The conjunctivae has been
flushed with 0.9% Sodium Chloride for Injection USP. A 29-gauge,
1/2-inch needle attached to a 0.5 cc Terumo insulin syringe has
been used for each subconjunctival injection (one
syringe/group/treatment). XG-102, vehicle control or reference item
has been administered into the eyes of each animal at a dose volume
of 50 .mu.L/eye on Days 1 and 8. Both eyes have been examined
immediately following each treatment to document any abnormalities
caused by the administration procedure.
[0670] The in-life procedures, observations, and measurements
listed below have been performed. More frequent observations may be
undertaken if considered appropriate. Twice daily, once in the
morning and once in the afternoon, throughout the study
Mortality/Moribundity Checks have been performed, whereby the
animals were observed for general health/mortality and moribundity.
Animals have not been removed from cage during observation, unless
necessary for identification or confirmation of possible findings.
Once daily, beginning Week-1, Cageside Observations have been
performed, whereby animals have not been removed from cage during
observation, unless necessary for identification or confirmation of
possible findings. Weekly, beginning Week-1, Detailed Clinical
Observations have been performed, whereby the animals were removed
from the cage for examination. Weekly, starting Week-2, Body
Weights have been recorded for health monitoring purposes only
whereby animals were individually weighed. Weekly, starting during
the last week of the pre-treatment period, Food consumption has
been quantitatively measured except on the day of scheduled
euthanasia for health monitoring purposes only. Once prestudy for
screening purposes, Ophthalmic Examinations have been performed,
whereby all animals were subjected to funduscopic (indirect
ophthalmoscopy) and biomicroscopic (slit lamp) examinations. The
mydriatic used was 1% tropicamide. Once prestudy and at the end of
Weeks 1, 2 and 3, Fluorescein Angiography has been performed,
whereby mydriatic drops (1% tropicamide) have been applied to each
eye at least 10 minutes prior to the test (further applications may
be administered if considered necessary). Hydration of the eyes has
been maintained by frequent irrigation with saline solution. The
animals have been maintained under isoflurane/oxygen mix and/or
with a sedative cocktail (ketamine 75 mg/kg; xylazine 7.5 g/kg), as
necessary. Single and/or ART fundus images in infrared and/or red
free modes have been obtained to serve as reference images for the
angiographies. 0.2 ml of 10% Sodium Fluorescein Injection USP has
been administered via rapid tail vein injection (via an abbocath),
followed by a 0.5 ml saline flush. Still images have been recorded
from both eyes at least 2 minutes following the fluorescein
injection and no later than 5 minutes following the fluorescein
injection. For evaluation the individual laser spots on the still
images have been evaluated for leakage semiquantitatively on a
scale of 0-4 by 2 independent readers, who will subsequently
determine a consensus score.
[0671] In the fluorescein angiogram scoring procedure, firstly
Angiography images (JPEG or BMP) have been exported from the HRA2
and copied on a CD or other appropriate medium and reviewed on a
suitable computer. In the Grading Procedure the Images have been
selected at an appropriate focus level for grading. (More than 1
image/eye may be needed in order to grade all laser spots.) The
angiograms have been graded independently by 2 scientific personnel
and the grade for each of the laser spots has been recorded.
Following completion of the grading by each person, the grades have
been compared and any discrepancy has been reviewed by both
parties, and a grade agreed upon and documented. The grading scale
will be from 0-4 as indicated below:
[0672] 0=no leakage (only laser scar or very diffuse small
hyper-fluorescent area visible).
[0673] 1=minimal leakage (small areas of diffuse or solid
hyper-fluorescence generally remaining within the laser-induced
defect region).
[0674] 2=slight leakage (semisolid hyperfluorescence generally
remaining within the boundary of the laser-induced defect
region).
[0675] 3=moderate leakage (semisolid to solid hyper-fluorescence
generally remaining within the boundary of the laser-induced defect
region).
[0676] 4=Substantial leakage (solid hyper-fluorescent region
extending beyond the boundary of the laser-induced defect
region).
[0677] If an animal dies or is euthanized during the study, a
necropsy has not been conducted and the carcass discarded. Animals
surviving until scheduled euthanasia have a terminal body weight
recorded. The animals will undergo exsanguination from the
abdominal aorta after isoflurane anesthesia. When possible, the
animals have been euthanized rotating across dose groups such that
similar numbers of animals from each group, including controls,
have been necropsied throughout the day(s). Representative samples
of the tissues identified in the Tissue Collection and Preservation
table below have been collected from all animals and preserved in
10% neutral buffered formalin, unless otherwise indicated:
TABLE-US-00014 Microscopic Tissue Weight Collect Evaluation Comment
Animal -- X -- -- identification Eye -- X -- Bilateral; fixed 24 to
48 hrs in Davidson's fixative and transferred in ethanol 70% for at
least 18 hrs, stored in 70% ethanol until processing. (euthanized
animals only) Nerve, optic -- X -- Bilateral; fixed 24 to 48 hrs in
Davidson's fixative and transferred in ethanol 70% for at least 18
hrs, stored in 70% ethanol until processing (euthanized animals
only) X = procedure to be conducted; -- = not applicable.
[0678] The following critical computerized systems have been used
in the study:
TABLE-US-00015 System Name Description of Data Collected and/or
Analyzed Provantis Dose administration, bodyweight, food
consumption, clinical observations, incidence of clinical
observations, clinical biochemistry, hematology, coagulation,
urinalysis, ophthalmology and gross pathology Dispense Test Item
receipt and/or accountability of Test Item and/or vehicle and/or
Reference Item(s) SRS (PCS-MTL in- Statistical analyses of
numerical in-life and house application built terminal data with
SAS) and SAS system for Windows Heidelberg HRA 2/ Fluorescein
angiography Heidelberg Spectralis with EyeExplorer
[0679] Means and standard deviations have been calculated for body
weight, food consumption and fluorescein angiography. Other data
have been reported on an individual basis.
Example 24: Inhibitory Effects of the JNK Inhibitor XG-102 on the
Inflammatory Response in a Rat Periodontitis Model
[0680] The aim of this study is to investigate the influence of
XG-102 (SEQ ID NO: 11) on inflammation induced in a periodontitis
model in the rat.
[0681] 30 Wistar rats (male, 6-8 weeks old) are used in this study
(divided into 3 groups of ten rats).
[0682] Experimental periodontitis is induced by a ligature placed
around the 1.sup.st molar (one molar per animal) on Day 0. One of
the mandibular first molars of each animal was randomly assigned
(left/right) to receive a 4/0 silk ligature in a cervical position.
In order to immobilize the ligature, two knots were made at the
mesial aspect of the first molars. The ligatures were kept in
position in order to allow biofilm accumulation over 10 days. This
procedure was performed under general anesthesia by intraperitoneal
injection of ketamine hydrochloride (80 mg/kg) and xylazine
hydrochloride (10 mg/kg).
[0683] One dose of 1 mg/kg XG-102 (dissolved in 0.9% NaCl as
vehicle) is administered intragingivally (IGV) on day 10. In Group
2, vehicle was administered IGV on day 10. The administration
volume is 10 .mu.l. Administrations are performed IGV in the
attached gingiva surrounding the first molar, whereby a fine
hypodermic needle (Terumo, Myjector) was inserted in the buccal
attached gingiva of the first molar. The total volume of injection
was successfully introduced in gingival tissue.
[0684] The table below summarizes the random allocation:
TABLE-US-00016 Group Route of Number of N.sup.o Ligature (Day 0)
Treatment administration animals 1 -- -- IGV 10 2 Yes NaCl 0.9% IGV
10 3 Yes XG-102 1 mg/ IGV 10 injection
[0685] Each day, the general behavior and the appearance of all
animals is observed. If animal health is not compatible with the
continuation of the study (moribund animals, abnormal important
loss of weight, major intolerance of the substance, etc. . . . ),
animals are ethically sacrificed under the responsibility of the
Study Director. Periodontitis inflammation aspect are analyzed by
macroscopic observation of gingival tissue on days 0, 10 and 17,
whereby the gingival inflammation (GI), periodontal depth pocket
(PP) and dental plaque index (IP) were noted blindly by an
experimented dentist on days 0, 10 and 17 as periodontal clinical
indices. Periodontitis inflammation was assessed by means of
macroscopic observation of gingival index using a clinical scoring:
0) no gingival inflammation, 1) slight inflammation, 2) moderate
inflammation, 3) severe inflammation. The depth pocket was
estimated using a graduated probe (HU-Friedy, USA). Finally dental
plaque index was estimated using a 0 to 3 score grade 0) no plaque
formation, 1) thin biofilm dental plaque 2) visible dental plaque,
3) thick dental plaque.
[0686] For the identification of oral bacteria, bacterial
population in dental pockets are identified by DNA probes (real
time PCR) on 9 periodontopathogens (Aa: Aggregatibacter
actinomycetemcomitan, Pg: Porphyromonas gingivalis, Tf: Tannerella
forsythensis, Td: Treponema denticola, Pi: Prevotella intermedia,
Pm: Peptostreptococcus micros, Fn: Fusobacterium nucleatum, Cr:
Campylobacter rectus, Ec: Eikenella corrodens) on days 0, 10 and 17
as well as total bacterial flora (Perio-analyses, Institut
Clinident). For the collagen framework, measurements of total
collagen amount are performed using Polarized-light microscopy. The
collagen I/collagen III ratio is evaluated by histomorphometrical
analysis.
[0687] On day 17 the animals are sacrificed and samples are
collected. Gingival tissue will be excised for bio-molecular
analysis on all animals. After euthanasia, mandibles will be
excised for histological evaluation. Buccolingual serial sections
were stained with a Modified Goldner's Masson Trichrome solution
for measurement of bone loss and to evaluate inflammatory
score.
[0688] For the evaluation of inflammatory cells, quantification of
inflammatory cells is performed by histomorphometric measurements.
To evaluate inflammatory score, slides were observed under an
optical microscope (Zeiss, Axioskop, Germany). The areas between
the first and second molars, where the ligature was placed, were
analyzed under light microscopy using on a 0 to 3 score grade,
considering the inflammatory cell influx, as described previously
[Bitto A, Oteri G, Pisano M, Polito F, Irrera N, Minutoli L,
Squadrito F, Altavilla D. Adenosine receptor stimulation by
polynucleotides (PDRN) reduces inflammation in experimental
periodontitis. J Clin Periodontol. 2013; 40(1):26-32]: Score 0:
absence of or only discrete cellular infiltration (inflammatory
cell infiltration is sparse and restricted to the region of the
marginal gingival). Score 1: minimal cellular infiltration
(inflammatory cellular infiltration present all over the insert
gingival). Score 2: moderate cellular infiltration (inflammatory
cellular infiltration present in both gingival and periodontal
ligament). Score 3: accentuated cellular infiltrate. A single
examiner, who was not aware of the experimental data, carried out
the histomorphometric measurements.
[0689] For the evaluation of tissue destruction, bone tissue
destruction is evaluated on 3 animals per group by radiological
analysis (micro-CT). Periodontal complex destruction is evaluated
by histological analysis. The images were digitized at a
magnification of .times.2.5 (Explora-Nova Morpho-Expert, software).
The influence of treatments on periodontal bone loss was
histometrically assessed by measuring the alveolar bone height loss
(ABHL). Measurements were taken (in millimeters) from the
cementenamel junction (CEJ) to the alveolar bone crest (ABC) along
the buccal and lingual sides of the root of the first molars (FIG.
6), according to a method previously reported [Bitto A, Oteri G,
Pisano M, Polito F, Irrera N, Minutoli L, Squadrito F, Altavilla D.
Adenosine receptor stimulation by polynucleotides (PDRN) reduces
inflammation in experimental periodontitis. J Clin Periodontol.
2013; 40(1):26-32]. Alveolar bone specimens from control group
(unligated) were also measured to compare the results from both
ligature groups. The mean amelo-cemental junction to alveolar bone
height was calculated for each group of animals. To validate
measurement conversions, a millimeter ruler was photographed and
used as a calibrator. Evaluations was performed by a two examiners
blind to the treatment assignment using an image analysis system
(Image J, USA) and then mean values from the two observers were
averaged.
[0690] For the evaluation of inflammatory markers, the level of
inflammatory proteins (p-JNK, TNF-.alpha., IL-1.beta., IL-10,
MMP-8, MMP-9) are measured from gingival tissue homogenates by
ELISA using commercially available kits (Biorad, Bioplex Pro
Cytokine Assays, France for TNF-.alpha., IL-1.beta., IL-10; Uscn
Life Science, USA for MMP-8, MMP-9, and Novateinbio, USA for JNK),
according to the manufacturer's instructions.
[0691] For the evaluation of bone microarchitecture, bone
trabecular measurements (thickness, separation) are evaluated by
radiological analysis (micro-CT) on 3 animals per group on days 0,
10 and 17.
[0692] Results:
[0693] Only one dose of XG-102 treatment was given on day 10. The
experimental periodontal disease induced by the placement of a silk
thread around the cervix of first lower molars caused a significant
increase (p<0.05) in GI for the two ligated groups, and in both
GI and PP only in group 3 (XG-102) as shown in FIG. 92. No
significant effect of placebo on clinical parameters at day 17 was
found. In group 3, one week after XG-102 injection (day 17), the
treatment robustly decreased GI level (FIG. 92).
[0694] Regarding the microbiological quantification, the results
showed an increase in total bacterial flora in all groups that did
not reach significance value at day 10 (p>0.05). Interestingly,
only XG-102 had diminished significantly (p<0.05) the total
bacterial flora at day 17 compared to day 10 (FIG. 93). This change
coincided with the administration of the experimental treatment.
For group 3, XG-102 achieved to significantly decrease the total
bacterial flora until the baseline level.
[0695] For the expression of IL1-.beta. the XG-102 treated group
(group 3) reduced significantly IL1-.beta. expression compared to
placebo group. This points out the beneficial effect of the XG-102
treatment for periodontitis obtained by decreasing pro-inflammatory
cytokine expression (FIG. 94).
[0696] In addition, periodontal bone loss/Alveolar bone height loss
(ABHL) was assessed on day 17. The ABHL is an indicative not only
of histological change/remodeling but also of bone resorption. The
results showed that ligation significantly increased the ABHL of
the molar in ligated group 2 compared with the control group
(p<0.05). Intergroup analysis revealed that bone destruction was
less severe in the XG-102 treated animals (FIG. 95). In fact, the
group 3 had an ABHL level statistically comparable with negative
control group. Thus, XG-102 administration prevents bone
degradation and avoids bone loss. These data confirm the
anti-inflammatory property (protective effect) of XG-102 against
periodontitis. Intergroup analysis revealed that all ligatured
groups had approximately the same levels of ABHL (p>0.05)
validating the rat periodontitis model.
[0697] Thus, the data of this study show a protective effect of
XG-102 against experimental periodontitis.
Example 25: Effects of XG-102 (SEQ ID No. 11) in a Diabetic
Retinopathy Prevention Study in the Streptozotocin Treated Rat
(IVT)
[0698] The objective of this study was to determine the ability of
XG-102 to prevent diabetic retinopathy when administered by
intravitreal injections to streptozotocin (STZ)-treated
(hyperglycemic) rats.
[0699] The study design was as follows:
TABLE-US-00017 XG-102 Dose Level Number STZ (.mu.g/eye) Dose Dose
of Group No./ (mg/kg) Days 1, 8, Volume Concentration Animals
Identification Day -7 15 (.mu.L) (mg/mL) Males 1/Not induced, 0 0 5
0 3 Vehicle 2/XG-102 - 55 0.2 5 0.04 8 0.2 .mu.g/eye 3/XG-102 - 55
2 5 0.4 8 2 .mu.g/eye 4/Vehicle 55 0 5 0 5
[0700] All animals from Groups 2, 3, 4 received a 55 mg/kg
intravenous (IV) dose of STZ on Day-7.
[0701] Sterile vials containing 0.0412 g of inducing agent (STZ)
were pre-weighed, sealed and transferred to the dosing room for
administration to Groups 2 to 4 animals and Spares on Day-7. A
duplicate set of empty, appropriately labeled sterile vials were
provided. The reconstituted STZ solution was filtered into these
vials for dosing. The Reference Item, 0.9% NaCl, was administered
as received. XG-102 was prepared using the correction factor 1.383.
A stock solution equal to the highest dose level was prepared in
vehicle, 0.9% Sodium Chloride for Injection, and sterile filtered
through a 0.22 .mu.m polyvinylidene difluoride (PVDF) filter. The
lower dose levels were prepared by directly diluting the stock
solution. Dose formulations were prepared once at appropriate
concentrations to meet dosage level requirements. All dilutions
were prepared by directly diluting the stock solution with vehicle.
Three dosing aliquots (Days 1, 8 and 15) were prepared and stored
in a freezer set to maintain -20.degree. C. Aliquot(s) of each dose
level were thawed at ambient temperature on each day of dosing and
the solution maintained at room temperature for no longer than 6
hours.
[0702] 60 male Brown Norway rats were received from Charles River
Labs, Inc., Portage, Ill. The animals were approximately 8 weeks
old and weighed between 166 and 228 g. The Brown Norway rat was
chosen as the animal model for this study as it is an accepted
species for use in the STZ-induced diabetic retinopathy model. The
total number of animals used in this study was considered to be the
minimum required to properly characterize the effects of the Test
Items. This study has been designed such that it didnot require an
unnecessary number of animals to accomplish its objectives. A
minimum acclimation period of 20 days was allowed between animal
receipt and the start of treatment in order to accustom the animals
to the laboratory environment. Animals were assigned to groups by a
stratified randomization scheme designed to achieve similar group
mean body weights. Animals in poor health or at extremes of body
weight range were not assigned to groups. Before the initiation of
dosing, any assigned animals considered unsuitable for use in the
study were replaced by alternate animals obtained from the same
shipment and maintained under the same environmental conditions.
The alternate animals were used as replacements on the study within
3 days of initiation. On arrival, animals were individually housed
until randomization. Following randomization, animals were group
housed (up to 3 animals of the same dosing group together) in
stainless steel perforated floor cages equipped with an automatic
watering valve. The room in which the animals were kept was
documented in the study records. Animals were separated during
designated procedures/activities. Temperatures of 19.degree. C. to
25.degree. C. with a relative humidity of 30% to 70% were
maintained. A 12-hour light/12-hour dark cycle was maintained,
except when interrupted for designated procedures. PMI Nutrition
International Certified Rodent Chow No. 5CR4 (14% protein) was
provided ad libitum throughout the study, except during designated
procedures. Municipal tap water after treatment by reverse osmosis
and ultraviolet irradiation was freely available to each animal via
an automatic watering system (except during designated procedures).
Animals were socially housed for psychological/environmental
enrichment and were provided with items such as a hiding device and
a chewing object, except when interrupted by study
procedures/activities.
[0703] For administration of Inducing Agent (Groups 2 to 4, Day-7),
one vial of STZ per animal (including spares) was reconstituted
within 3 minutes of injection with 1.5 mL of Sterile Water for
Injection, USP, to provide a concentration of 27.5 mg/mL. The vial
was inverted or swirled to dissolve STZ. The resultant solution was
filtered via a 0.22 .mu.m Millex-GV filter into a empty sterile
appropriately labeled vial. The STZ (55 mg/kg) was administered by
intravenous injection on Day-7, within 3 minutes of formulation via
a syringe. The dose volume was 2 mL/kg and the actual dose
administration was based on the most recent practical body weight
of each animal. The animals were restrained during the
injection.
[0704] Test items or reference item were administered by
intravitreal injection to the left and right eyes of each animal on
Days 1, 8 and 15 as indicated in the Experimental Design table. The
animals were anesthetized (isoflurane) for the dose administration,
which was performed by a board-certified veterinary
ophthalmologist. Topical antibiotics (gentamicin ophthalmic
solution) were applied to both eyes twice on the day before
treatment, following the injection and at least once on the day
following the injection. Prior to dosing, mydriatic drops (1%
tropicamide and/or 2.5% phenylephrine) were applied to each eye
(further applications were performed when considered appropriate by
the veterinary ophthalmologist). During dosing, animals were
maintained under anesthesia with isoflurane/oxygen gas. The
conjunctivae were flushed with 0.9% Sodium Chloride for Injection
USP. A 10 .mu.L Hamilton syringe with 32-gauge, 1/2-inch needle was
used for each intravitreal injection (one syringe/group/treatment).
The dose volume was 5 L/eye. Both eyes were examined by slit-lamp
biomicroscopy and/or indirect ophthalmoscopy immediately following
each treatment to document any abnormalities (especially to the
lens, vitreous and retina) caused by the administration procedure.
Corneal opacities were considered secondary to experimental
procedures involving anesthesia. Some of these opacities were
associated also with corneal vascularization. Other ocular findings
were noted, but were generally of low incidence or sporadic across
groups, and/or did not persist. These findings included, but were
not limited to: multifocal/diffuse corneal opacities, vitreous air
bubbles, focal/diffuse/multifocal vitreous opacities, and focal
retina opacities.
[0705] Streptozotocin was administered by intravenous injection to
induce diabetic retinopathy in the rat. The intravitreal injection
route was selected for the Test Items because this is the intended
route of administration in humans. The dose levels were selected
based on information obtained with previous proof of concept
studies as well as MTD and toxicity studies using the IVT route of
administration.
[0706] The in-life procedures, observations, and measurements
listed below were performed for study animals. Throughout the
study, animals were observed for general health/mortality and
moribundity twice daily, once in the morning and once in the
afternoon. Animals were not removed from cage during observation,
unless necessary for identification or confirmation of possible
findings. The animals were removed from the cage, and a detailed
clinical observation was performed weekly, beginning during Week-1.
Animals were weighed individually twice weekly, starting during
Week-1. Food consumption was quantitatively measured weekly
starting during the last week of the pretreatment period. All
animals were subjected to funduscopic (indirect ophthalmoscopy) and
biomicroscopic (slit lamp) examinations once pre-treatment and
again on Day 22. The mydriatic used was 1% tropicamide. Intraocular
pressure was measured following each ophthalmology examination,
once prestudy and on Day 22, using a TonoVet.TM. rebound tonometer.
The pre-treatment tonometry readings were performed at the same
times as anticipated for the final measurements to reduce diurnal
variability.
[0707] Electroretinogram evaluations were performed once
pretreatment and on Days 6, 13, and 20, prior to fluorescein
angiography Animals were dark-adapted overnight prior to ERG
recording and then anesthetized with an intramuscular injection of
75 mg/kg ketamine and 7.5 mg/kg xylazine. Tropicamide (1%) was
applied to each eye prior to the test (further applications were
administered if considered necessary). The eyelids were retracted
by means of a lid speculum, and a contact lens or gold loop
electrode was placed on the surface of each eye.
[0708] A needle electrode was placed cutaneously under each eye
(reference) and on the head, posterior to the brow or at the base
of the tail (ground). Carboxymethylcellulose (1%) drops were
applied to the interior surface of the contact lens electrodes
prior to placing them on the eyes. Each ERG occasion consisted of
the following series of scotopic single flash stimuli:
[0709] 1) -30 dB single flash, a-wave amplitude and latency,
average of 5 single flashes, 10 seconds between flashes.
[0710] 2) -10 dB single flash, a- and b-wave amplitudes and
latency, average of 5 single flashes, 15 seconds between
flashes.
[0711] 3) 0 dB, average of 2 single flashes, a- and b-wave
amplitude and latency, approximately 120 seconds between flashes (a
longer time period is acceptable).
[0712] Following evaluation of the scotopic response, the animals
were adapted to background light at approximately 25 to 30 cd/m2
for a period of approximately 5 minutes (a longer time period was
acceptable), followed by an average of 20 sweeps of photopic white
flicker at 1 Hz (a- and b-wave amplitudes and latency), then 20
sweeps of photopic flicker at 29 Hz (b-wave amplitude and latency).
Waveforms were analyzed for a- and b-wave amplitudes and latency,
and oscillatory potentials (OP) 1 through 4 from the 0 dB scotopic
stimulus were filtered and analyzed for amplitude and latency.
[0713] Fluorescein angiography evaluations were performed once
pretreatment and on Days 7, 14, and 21, following
electroretinography. An isoflurane/oxygen mix was used prior to and
during the procedure as the anesthesia. The mydriatic agent, 1%
tropicamide, was used as necessary. Hydration of the eyes was
maintained by irrigation with saline solution, as needed. 0.2 mL of
10% Sodium Fluorescein Injection U.S.P. was administered via rapid
tail vein injection, followed by a 0.5 mL saline flush. Still
images of the fundus were recorded from both eyes between 10-15
minutes following the fluorescein injection. Images were taken from
the right eye first, followed by the left. A topical bland
ophthalmic ointment was administered to the eyes following the
angiographies. Images were evaluated qualitatively for vascular
integrity/diffuse leakage.
[0714] Blood Glucose Level Determination were once pre-STZ
treatment, Day-6 (the day following STZ administration) and three
times per week thereafter (all animals). Additional blood glucose
measurements may have been performed as required to monitor animal
health status. Levels were determined by glucometer using blood
drops taken from the tail vein. Values were measured in mmol/L and
converted into mg/dL by multiplying by 18 for reporting purposes.
Urine Glucose Level Determination was weekly, beginning Week-1,
following overnight collection. Animals had access to food and
water during the collection period. Urine glucose was measured by
the Clinical Laboratory department using the P800 analyzer. from
the abdominal aorta after isoflurane anesthesia. When possible, the
animals were euthanized rotating across dose groups such that
similar numbers of animals from each group, including controls were
necropsied at similar times throughout the day.
[0715] Main study animals were subjected to a complete necropsy
examination, which included evaluation of the carcass and
musculoskeletal system; all external surfaces and orifices; cranial
cavity and external surfaces of the brain; and thoracic, abdominal,
and pelvic cavities with their associated organs and tissues.
Necropsy procedures were performed by qualified personnel with
appropriate training and experience in animal anatomy and gross
pathology. A veterinary pathologist, or other suitably qualified
person, was available.
[0716] Representative samples of the tissues identified below were
collected from all animals and preserved in 10% neutral buffered
formalin, unless otherwise indicated.
Tissue Collection and Preservation
TABLE-US-00018 [0717] Microscopic Tissue Weight Collect Evaluation
Comment Animal -- X -- -- identification Eye -- X -- Bilateral;
fixed in Davidson's fixative (euthanized animals only). Gross
lesions/ -- X -- -- masses Nerve, optic -- X -- Bilateral; fixed in
Davidson's fixative (euthanized animals only) X = procedure to be
conducted; -- = not applicable.
[0718] The following parameters and end points were evaluated in
this study: mortality, clinical signs, body weights, body weight
changes, food consumption, ophthalmology, intra-ocular pressure,
electroretinography (ERG), fluorescein angiography, blood and urine
glucose determination, gross necropsy examinations.
[0719] Consistent with the diabetic retinopathy rat model, there
were hyperglycemia-related deaths, clinical signs of deteriorating
condition, and decreases in body weights, body weight gains,
increased food consumption, and severe increases blood and urine
glucose levels. Multiple ocular changes noted in the STZ-induced
groups were secondary to the nature of the hyperglycemic state,
notably the anterior cortical cataracts. There were no
XG-102-related deaths during the study. There were no
XG-102-related clinical signs or effects on body weights, body
weight gains or food consumption. Fluorescein angiography imagery
did not reveal any vascular leakage and there were no apparent
XG-102-related macroscopic findings at necropsy.
[0720] On Days 6, 13 and 20, some amplitudes of scotopic and
photopic ERG assessments for animals given .ltoreq.2 .mu.g/eye
XG-102 were mildly increased or comparable to the STZ-treated
control animals, but these responses generally remained within the
control variability. Latencies for XG-102 groups were comparable
and remained within the control and/or pretreatment variation.
There were some sporadic differences in oscillatory potential
amplitudes when comparing animals given .ltoreq.2 .mu.g/eye XG-102
with STZ-treated controls.
[0721] The following Table includes a summary of amplitudes for all
ERG stimuli by occasion (pretreatment, and Days 6, 13 and 20,
respectively). The values represent the group mean and standard
deviation (below):
TABLE-US-00019 Amplitude (.mu.V) Group Pre 6 13 20 Oscillatory
Potential #1 Scotopic Single Flash 0 dB - B-Wave Non-induced 54 50
34 41 Vehicle 9 11 12 7 XG-102 46 49 37 36 0.2 .mu.g/eye 10 12 17
18 XG-102 58 40 33 26 2 .mu.g/eye 14 11 15 7 Vehicle 53 40 14 35 21
4 11 12 Oscillatory Potential #2 Scotopic Single Flash 0 dB -
B-Wave Non-induced 180 129 107 84 Vehicle 14 32 40 8 XG-102 167 98
63 70 0.2 .mu.g/eye 34 28 25 33 XG-102 226 93 92 49 2 .mu.g/eye 49
17 31 14 Vehicle 180 98 124 58 88 27 39 12 Oscillatory Potential #3
Scotopic Single Flash 0 dB - B-Wave Non-induced 376 273 214 195
Vehicle 26 68 80 18 XG-102 326 219 165 164 0.2 .mu.g/eye 64 82 58
63 XG-102 428 239 219 137 2 .mu.g/eye 77 33 55 35 Vehicle 348 251
239 164 149 58 54 37 Oscillatory Potential #4 Scotopic Single Flash
0 dB - B-Wave Non-induced 219 162 136 142 Vehicle 26 39 24 14
XG-102 172 147 129 116 0.2 .mu.g/eye 33 64 45 38 XG-102 219 182 160
130 2 .mu.g/eye 32 27 49 50 Vehicle 162 178 143 136 52 31 45 35
Scotopic Single Flash -30 dB - B-Wave Non-induced 434 311 308 170
Vehicle 35 113 47 60 XG-102 360 269 270 240 0.2 .mu.g/eye 90 120
143 136 XG-102 417 270 292 166 2 .mu.g/eye 68 140 142 108 Vehicle
369 224 197 136 85 77 71 47 Scotopic Single Flash -10 dB - A-Wave
Non-induced -217 -152 -124 -109 Vehicle 23 39 31 30 XG-102 -191
-151 -128 -129 0.2 .mu.g/eye 39 64 59 56 XG-102 -254 -124 -152 -84
2 .mu.g/eye 48 46 75 40 Vehicle -206 -104 -111 -96 57 26 38 30
Scotopic Single Flash 0 dB - A-Wave Non-induced -355 -244 -188 -188
Vehicle 37 57 96 77 XG-102 -303 -209 -203 -198 0.2 .mu.g/eye 70 73
78 74 XG-102 -394 -205 -261 -147 2 .mu.g/eye 75 74 142 61 Vehicle
-323 -177 -208 -142 110 38 67 43 Scotopic Single Flash 0 dB -
B-Wave Non-induced 899 415 640 180 Vehicle 99 161 201 80 XG-102 739
421 442 433 0.2 .mu.g/eye 169 176 209 224 XG-102 944 383 524 278 2
.mu.g/eye 176 177 132 202 Vehicle 755 283 468 255 250 103 194 83
Photopic 1 Hz Flicker A-Wave Non-induced -3 -2 -7 -5 Vehicle 1 2 4
4 XG-102 -2 -3 -4 -2 0.2 .mu.g/eye 2 2 4 3 XG-102 -3 -3 -6 -2 2
.mu.g/eye 2 3 6 3 Vehicle -4 -2 -2 -6 2 2 2 5 Photopic 1 Hz Flicker
B-Wave Non-induced 133 72 95 40 Vehicle 15 21 36 8 XG-102 112 63 71
70 0.2 .mu.g/eye 29 25 34 32 XG-102 146 69 91 45 2 .mu.g/eye 31 28
27 28 Vehicle 100 61 100 32 14 20 39 16 Photopic 29 Hz Flicker -
B-Wave Non-induced 22 13 16 12 Vehicle 4 3 4 5 XG-102 18 9 14 10
0.2 .mu.g/eye 6 4 6 4 XG-102 27 11 17 9 2 .mu.g/eye 6 4 7 4 Vehicle
19 11 19 13 9 3 7 7
[0722] As can be retrieved from these data, there is a tendency for
XG-102 to reverse the decrease of the wave amplitude.
Example 26: Effects of XG-102 (SEQ ID No. 11) in a Diabetic
Retinopathy Prevention Study in the Streptozotocin Treated Albino
Rat (Subconjunctival)
[0723] The objective of this study was determine the ability of
XG-102 to prevent diabetic retinopathy when administered by weekly
subconjunctival injection to streptozotocin (STZ)-treated
(hyperglycemic) rats for 3 weeks.
[0724] The experimental design is shown in the following:
TABLE-US-00020 Test Item Dose STZ Level Dose Dose No. of Group No./
(mg/kg) (.mu.g/eye/ Volume Concentration Animals Identification Day
-7 week) (.mu.L) (mg/mL) Males 1/Not induced, 0 -- 50 0 8 Vehicle
2/Induced, 55 -- 50 0 10 Vehicle 3/XG-102 - low 55 2 50 0.04 8 dose
4/XG-102 - mid 55 20 50 0.4 8 dose 5/XG-102 - high 55 200 50 4 8
dose All animals from Groups 2 to 5 will receive a 55 mg/kg
intravenous (IV) dose of STZ on Day -7.
[0725] Naive Long Evans rats were used (42 male animals; 10 weeks
of age, at time of dosing; Charles River, St. Constant, QC). The
Long Evans rat was chosen as the animal model for this study as it
is an accepted species for use in the STZ-induced diabetic
retinopathy model. The total number of animals to be used in this
study is considered to be the minimum required to properly
characterize the effects of the test item and has been designed
such that it does not require an unnecessary number of animals to
accomplish its objectives. At this time, studies in laboratory
animals provide the best available basis for extrapolation to
humans. Acceptable models which do not use live animals currently
do not exist. Projected release of alternates will be Day 4.
Animals will be housed in stainless-steel cages. PMI Nutrition
International Certified Rodent Chow No. 5CR4 (14% protein) was
provided daily in amounts appropriate for the size and age of the
animals. Municipal tap water, processed through a reverse osmosis
filter and passed through UV light treatment, was freely available
to each animal. Animals were socially housed (up to 3 animals/cage)
for psychological/environmental enrichment and were provided with
items such as a hiding tube and a chewing object, except during
study procedures/activities. Only animals that are determined to be
suitable for use on study were assigned. On arrival, animals were
individually housed until randomization. Following randomization,
animals will be socialized.
[0726] Sterile vials containing 0.0412 g of inducing agent (STZ)
will be pre-weighed, sealed and transferred to the dosing room for
administration to Groups 2 to 5 animals and selected spares on
Day-7. A duplicate set of empty, appropriately labeled sterile
vials will be provided. The reconstituted STZ solution will be
filtered into these vials for dosing. The Test Item, XG-102, was
prepared using the provided correction factor. A stock solution
equal to the highest dose level was prepared in vehicle, 0.9%
Sodium Chloride for Injection, and sterile filtered through a 0.22
.mu.m polyvinylidene difluoride (PVDF) filter. The lower dose
levels were prepared by directly diluting this stock solution with
saline. Dosing aliquots were prepared and stored in a freezer set
to maintain -20.degree. C. Aliquot(s) of each dose level were
thawed at ambient temperature on each day of dosing and the
solutions maintained at room temperature for no longer than 6
hours. The vehicle, 0.9% Sodium Chloride for Injection, was
administered as received. One vial of STZ per animal (including
spares) was reconstituted within 3 minutes of injection with 1.5 mL
of Sterile Water for Injection, USP, to provide a concentration of
27.5 mg/mL. The vial was inverted or swirled to dissolve the STZ.
The reconstituted STZ solution was filtered via a 0.22 .mu.m
Millex-GV filter into empty sterile vials for dosing. STZ was
administered by intravenous injection on Day-7, within 3 minutes of
formulation via a syringe. The dose volume was 2 mL/kg and the
actual dose administration was based on the most recent practical
body weight of each animal. The animals will be restrained during
the injection. STZ-treated animals were considered diabetic if the
blood glucose level is .gtoreq.250 mg/dL. Test item or vehicle were
administered by subconjunctival injection to the left and right
eyes of each animal on Days 1, 8 and 15 and again on Day 24 (Rep
1), Day 23 (Rep 2 and 3), Day 22 (Rep 4) and Day 34 (Rep 1) Day 33
(Rep 2 and 3) and Day 32 (Rep 4). The animals were anesthetized
(isoflurane) for the dose administration, which was performed by a
board-certified veterinary ophthalmologist. Topical antibiotics
(0.3% tobramycin ointment) was applied to both eyes twice on the
day before treatment, following the injection and at least once on
the day following the injection. Prior to dosing, mydriatic drops
(1% tropicamide and/or 2.5% phenylephrine) were applied to each eye
(further applications may be performed as considered appropriate by
the veterinary ophthalmologist). During dosing, animals were
maintained under anesthesia with isoflurane/oxygen gas. The
conjunctivae were flushed with 0.9% Sodium Chloride for Injection
USP. A 29-gauge, 1/2-inch needle attached to a 0.5 cc Terumo
insulin syringe was used for each subconjunctival injection (one
syringe/group/treatment). Test items or reference item were
administered into the eyes of each animal at a dose volume of 50
.mu.L/eye. Both eyes were examined immediately following each
treatment to document any abnormalities caused by the
administration procedure. Streptozotocin is being administered IV
to induce diabetic retinopathy in the rat. The subconjunctival
route has been selected for the Test Item because this is the
intended route of administration in humans. The dose levels were
selected based on information obtained with previous proof of
concept studies as well as MTD and toxicity studies using the
subconjunctival route of administration. Morbidity/mortality checks
were performed at least twice daily (AM and PM). Cage side
observations were performed once daily. Detailed clinical
examinations were performed weekly. Quantitative food consumption
were performed weekly. Body weights were recorded twice weekly.
Ophthalmic examinations were performed once prestudy and again on
Day 37 (Rep 1), Day 36 (Rep 2 and 3) and Day 35 (Rep 4). All
animals were subjected to funduscopic (indirect ophthalmoscopy) and
biomicroscopic (slit lamp) examinations. The mydriatic used will be
1% tropicamide. Intra-ocular pressure was measured once prestudy
and on Day 37 (Rep 1), Day 36 (Rep 2 and 3) and Day 35 (Rep 4). The
pre-treatment tonometry readings were performed at the same times
as anticipated for the final measurements to reduce diurnal
variability. Intraocular pressure was measured following the
ophthalmology examinations, using a TonoVet.TM. rebound
tonometer.
[0727] Electroretinogram evaluations were performed once
pretreatment and on Days 7, 14, 21, and Day 36 (Rep 1), Day 35 (Rep
2 and 3) and Day 34 (Rep 4). Animals were dark-adapted overnight
prior to ERG recording and then anesthetized with an intramuscular
injection of 75 mg/kg ketamine and 7.5 mg/kg xylazine. Tropicamide
(1%) was applied to each eye prior to the test (further
applications may be administered if considered necessary). The
eyelids were retracted by means of a lid speculum, and a contact
lens or gold loop electrode was placed on the surface of each eye.
A needle electrode was placed cutaneously under each eye
(reference) and on the head posterior to the brow or at the base of
the tail (ground). Carboxymethylcellulose (1%) drops were applied
to the interior surface of the contact lens electrodes prior to
placing them on the eyes.
[0728] 1) -30 dB single flash, average of 5 single flashes, 10
second between flashes
[0729] 2) -10 dB single flash, average of 5 single flashes, 15
seconds between flashes.
[0730] 3) 0 dB, average of 2 single flashes, approximately 120
seconds between flashes (a longer time period is acceptable).
[0731] Following evaluation of the scotopic response, the animals
were adapted to background light at approximately 25 to 30
cd/m.sup.2 for a period of approximately 5 minutes (a longer time
period is acceptable), followed by an average of 20 sweeps of
photopic white flicker at 1 Hz, then 20 sweeps of photopic flicker
at 29 Hz. Waveforms were analyzed for a- and b-wave amplitudes and
latency and oscillatory potentials 1 through 4 from the 0 dB
scotopic stimulus will be filtered and analyzed for amplitude and
latency.
[0732] Indocyanin Green angiography evaluations were performed once
pretreatment (Day-2 or -1) and on Days 8, 15, 22, and Day 35 (Rep
1), Day 34 (Rep 2 and 3) and Day 33 (Rep 4). An isoflurane/oxygen
mix was used prior to and during the procedure as the anesthesia.
The mydriatic agent used was 1% tropicamide as necessary. Hydration
of the eyes was maintained by irrigation with saline solution, as
needed. 0.2 mL of 0.5% Indocyanin Green was administered via rapid
tail vein injection, followed by a 0.5 mL saline flush. Still
images of the fundus were recorded from both eyes between 10-15
minutes following the ICG injection. Images were taken from the
right eye first, followed by the left. A topical bland ophthalmic
ointment was administered to the eyes following the angiographies.
Images were evaluated qualitatively for vascular integrity/diffuse
leakage.
[0733] Blood glucose level were measured once pre-STZ treatment, on
Day-6 (the day following STZ administration) and again on Day-1.
Additional blood glucose measurements may be performed as required
to monitor animal health status. Levels were determined by
glucometer using blood drops taken in the tail vein. Values were
measured in mmol/L and converted into mg/dL by multiplying by 18
for reporting purposes.
[0734] Main study animals surviving until scheduled euthanasia were
euthanized by exsanguination from the abdominal aorta after
isoflurane anesthesia. When possible, the animals were euthanized
rotating across dose groups such that similar numbers of animals
from each group, including controls were necropsied at similar
times throughout the day. Representative samples of the tissues
(eye, nerve optic) were collected from all animals and preserved in
10% neutral buffered formalin, unless otherwise indicated. Eyes and
optic nerves collected bilaterally and fixed in Davidson's fixative
24 to 48 hours and then stored in 70% ethanol (euthanized animals
only).
Example 27: A Randomized, Double-Blind, Parallel Group, Controlled,
Multicentre Trial to Assess the Efficacy and Safety of a Single
Sub-Conjunctival Injection of XG-102, Compared to Dexamethasone Eye
Drops in Post-Surgery Intraocular Inflammation (Clinical Phase
II)
[0735] Despite technical advances in ocular surgery, the physical
trauma of this procedure continues to induce post-operative ocular
inflammation warranting treatment. In ocular tissue, arachidonic
acid is metabolized by cyclooxygenase (COX) to prostaglandins (PG)
which are the most important lipid-derived mediators of
inflammation. Surgical trauma causes a trigger of the arachidonic
acid cascade which in turn generates PGs by activation of COX-1 and
COX-2. Phospholipids in the cell membrane are the substrate for
phospholipase A to generate arachidonic acid from which a family of
chemically distinct PGs and leukotriens are produced. The `golden
standard` for the treatment of ocular inflammation are topical
corticosteroids and/or Non-Steroidal Anti-inflammatory Drugs
(NSAIDs). Side effects reported with (short-term) corticosteroid
use include cataract formation, increased Intra Ocular Pressure
(IOP), increased susceptibility to viral infections and retardation
of the corneal epithelial and stromal wound healing. In addition,
prolonged treatment with corticosteroids have been known to induce
systemic side effects such as glucose impairment, hypertension,
development of glaucoma, visual acuity defects, loss of visual
field, and posterior subcapsular cataract formation. The
Investigational Medicinal Product (IMP) under
investigation--XG-102--is a protease-resistant peptide that
selectively inhibits c-Jun N-terminal Kinase (JNK) activity in a
non-Adenosine Triphosphate (ATP) competitive manner. XG-102 is a 31
D-amino acids JNK inhibitor peptide with all amino acids except
glycine (which is achiral) in the D-configuration. This choice was
made to increase the resistance of the compound to proteases, which
usually degrade peptides soon after their administration. Since JNK
activation leads to the phosphorylation and activation of the
activator protein-1 (AP-1) transcription factor family and other
cellular factors implicated in autoimmune and inflammatory
diseases, compounds that inhibit the JNK pathway may have an
indicated therapeutic value. Ocular MTD (Maximum Tolerated Dose)
studies in rats and rabbits as well as ocular local tolerance in
rabbits showed that XG-102 was well-tolerated after
sub-conjunctival, intravitreal (IVT) and intravenous (iv)
administrations. Ocular MTD studies in rats and rabbits after
sub-conjunctival administration showed that the No Observed Adverse
Effect Level (NOAEL) was around 20 .mu.g in rats and 600 .mu.g in
rabbits. Ocular pharmacokinetics after single and repeated (daily
for 7 days) sub-conjunctival administration have been studied in
rabbits and showed that XG-102 was still present in choroid, bulbar
conjunctiva and iris-ciliary body 7 days after administration with
a tmax between 1 and 4 hours depending on the ocular structure,
whereas no XG-102 was detectable at any time in plasma. Given the
deleterious side effects of the current `golden standard` to treat
(post-operative) intraocular inflammation, it is clinically
justified to find other treatment alternatives which on the one
hand are efficacious in reducing the inflammation while on the
other hand, do not have the (deleterious) side effects associated
with corticosteroid use. XG-102 has shown promising results both in
the pre-clinical studies and phase I/Ib studies performed to
date.
[0736] The previous trial was an open label, single-center, dose
escalation/dose finding study which was designed to assess the
safety and tolerability of a single sub-conjunctival injection of
XG-102, administered in addition to the `usual` post-op
anti-inflammatory therapy in patients with post-surgery or
post-traumatic intraocular inflammation. The XG-102 doses which
were investigated were 45, 90, 450 and 900 .mu.g. In total, 20
patients (5 patients in each dose group) were enrolled in this
study. The conclusion of the previous study was that XG-102,
administered as a sub-conjunctival injection in patients with
recent post-surgery or trauma intraocular inflammation was safe and
well tolerated. Following the successful completion of the previous
study, it was decided to continue with the development of XG-102 in
intraocular inflammation and to perform the present study where the
objective was to evaluate the efficacy and safety, compared to
dexamethasone eye drops, of a single sub-conjunctival dose of
XG-102 administered immediately post-op in the evolution of post-op
intraocular inflammation, as assessed by chamber cell grade. This
is the first study investigating the efficacy of XG-102 when
administered as a stand-alone therapy in the evolution of
post-operative intraocular inflammation.
[0737] The objectives of the present study were to evaluate the
efficacy and safety of a single sub-conjunctival injection of
XG-102 90 or 900 .mu.g administered within maximally 3 hours after
the end of the surgical procedure compared to dexamethasone eye
drops administered 4 times/day for 21 days in post-operative
intraocular inflammation.
[0738] The primary objective of the present study was to evaluate
if a single sub-conjunctival injection of 900 .mu.g XG-102 is
non-inferior to treatment with dexamethasone eye drops administered
4 times/day for 21 days in the evolution of post-operative
intraocular inflammation. In accordance with this trial's primary
objective, the primary outcome was evaluated by the mean anterior
chamber cells grade at day 28 post-administration of the
sub-conjunctival injection of study treatment comparing XG-102 900
.mu.g with dexamethasone eye drops.
[0739] The secondary objectives were to evaluate the effect of a
single sub-conjunctival injection of either 90 .mu.g or 900 .mu.g
XG-102 compared to dexamethasone eye drops (4 times/day,
administered for 21 days) on:
[0740] Efficacy Outcome Parameters
[0741] a) Anterior chamber cells grade at day 28 (XG-102 90 .mu.g
vs dexamethasone)
[0742] b) Anterior chamber cells grade at day 7 and day 14 (XG-102
900 .mu.g vs dexamethasone)
[0743] c) Anterior chamber cells grade at day 7 and day 14 (XG-102
90 .mu.g vs dexamethasone)
[0744] d) Anterior chamber flare grade at day 7, 14 and day 28
(XG-102 900 .mu.g vs dexamethasone)
[0745] e) Anterior chamber flare grade at day 7, 14 and day 28
(XG-102 90 .mu.g vs dexamethasone)
[0746] f) Rescue medication use
[0747] g) Evolution of the intraocular inflammation over time
[0748] Safety and Tolerability Outcome Parameters:
[0749] a) Visual acuity by ETDRS method
[0750] b) Slit Lamp examination findings
[0751] c) The results of the ophthalmic fundus examination
[0752] d) Intra Ocular Pressure (IOP) measurements
[0753] e) Vital signs (blood pressure (BP), pulse rate (PR) and
rhythm)
[0754] f) The results of the hematology and chemistry laboratory
tests
[0755] g) The occurrence of Adverse Events
[0756] h) Presence (or not) of XG-102 in plasma 1 hour after the
administration of study treatment in a subset of patients
(approximately 30)
[0757] The present trial was a randomized (1:1:1), controlled,
double-blind, multicenter non-inferiority clinical trial with three
parallel groups of equal size. Randomization, which was blocked by
center, was performed using a web-based, secure, randomization
system. Eligible patients were male or female (post-menopausal, or
sterile by tubal ligation or hysterectomy), who were >18 years
of age and who had undergone one of the following ocular surgeries:
(a) anterior and posterior segment combined surgery which may
include surgery for: cataract and retinal detachment, cataract and
epimacular membrane and/or cataract and macular hole or (b)
glaucoma surgery or (c) complex posterior segment surgery or (d)
complicated intraocular surgery which may include cataract surgery
associated with diabetic retinopathy and/or complicated retinal
detachment ocular surgery. Patients were not eligible to
participate if any of the following exclusion criteria was present
at the moment of randomization:
[0758] 1. Administration of any investigational drug within 12
weeks prior to the administration of study treatment.
[0759] 2. Presence of a contraindication to prescribe dexamethasone
eye drops.
[0760] 3. Existence of a persistent fungal or bacterial eye
infection, refractory to anti-infective treatment.
[0761] 4. History of intraocular hypertension known to be provoked
by corticosteroid use.
[0762] 5. Presence of a corneal ulcer, corneal perforation or
lesion associated with an incomplete re-epithelialization.
[0763] 6. Existence of any surgical or medical condition which, in
the judgment of the Investigator, might interfere with this
study.
[0764] 7. A history of any serious adverse reaction or
hypersensitivity to protein-type drugs or to vaccines.
[0765] 8. Currently treated for seasonal allergic reactions
(example: hay fever, asthma).
[0766] 9. Females of childbearing potential.
[0767] 10. Males not willing to use an effective method of
contraception (e.g. combined contraceptive pill or barrier methods)
with non-menopausal female partners up to day 28 (i.e. the date
when the last visit is performed) in the study.
[0768] 11. Patients not willing to comply with the provisions of
this protocol.
[0769] The study protocol planned that 138 patients would be
randomized and administered the sub-conjunctival injection of study
treatment. It was also stated in the study protocol that randomized
patients for whom the sub-conjunctival injection of study treatment
was not administered would be replaced. Patients were randomly
allocated to either XG-102 90 or 900 .mu.g which was administered
as a single, sub-conjunctival injection of 250 .mu.l within
maximally 3 hours after the end of the eye surgery or to
dexamethasone eye drops, which were instilled 4 times per day for
21 days. The first study treatment eye drop was instilled within
maximally 15 minutes after the sub-conjunctival injection of study
treatment. In order to maintain the blinding, patients randomized
to the XG-102 group received eye drops containing a NaCl 0.9%
solution and patients randomized to the dexamethasone group were
administered a sub-conjunctival injection containing NaCl 0.9%.
Patients were followed for, in total, 28 (.+-.5) days after
administration of the sub-conjunctival injection of study
treatment.
[0770] They returned to the out-patient clinic to perform the
visits/investigations as required by the study protocol. The below
table shows planned visit schedule in addition to the
procedures/investigations carried out at each visit. The study
protocol planned that the data safety and monitoring board (DSMB)
would be responsible to oversee patient safety. This was to be
achieved by reviewing Serious Adverse Events (SAE) as they occurred
in addition to reviewing the cumulative patient data during the
study. Details concerning the timing of the data reviews were
detailed in the DSMB charter.
TABLE-US-00021 Screening visit: Visit 1 Visit 2 Visit 3 Visit 4
prior to eye 21 hrs (.+-.3 hrs) 7 days (.+-.1 day) 14 days (.+-.2)
after 28 days (.+-.5) after Assessment surgery after sub-conj. inj
after sub-conj. inj sub-conj. inj sub-conj. inj Written informed
consent x.sup.a Demographic data, x.sup.b ophthalmolog, MH Body
height, body weight x.sup.b Concomitant treatments x.sup.b x x x x
Seated vital signs x.sup.b x x Ophthalmic fundus x.sup.b x x x x
Intra Ocular Pressure x.sup.b x x x x Slit Lamp examination .+-.
x.sup.b x x x x Laser Flare Meter Visual acuity examination x.sup.b
x x x x (by ETDRS method) Blood sampling x.sup.b,c x x Final
Inclusion/exclusion x.sup.b criteria review Randomization X.sup.d
Preparation of study x.sup.e treatment syringe Admin. study
treatment x.sup.f Blood sampling for XG-102 x.sup.g quantification
Adverse Event reporting x.sup.h x x x x .sup.aInformed consent was
obtained prior to the surgical procedure being performed and prior
to any study related procedure being performed. .sup.bWas done
prior to ocular eye surgery being performed. .sup.cThe following
blood samples were performed prior to the surgical procedure being
performed. Chemistry blood samples to be performed were: Aspartate
Transaminase (AST), Alanine Transaminase (ALT), C-Reactive Protein
(CRP), creatine kinase (CK), glucose, creatinine and gamma-glutamyl
transferase. Hematology: hemoglobin (Hb), hematocrit (HCT) and full
white cell count. .sup.dPrior to performing the surgical procedure,
the patient eligibility was determined. Eligible patients were
randomized using the web-based system before the surgical procedure
was started. .sup.eThe study treatment vial was removed from the
freezer at least one hour prior to the sub-conjunctival injection.
The vial was placed in a secure location at ambient temperature to
defrost before preparation of the study treatment syringe. The
study treatment eye drops were retrieved from the pharmacy at the
same time as the study treatment vials. .sup.fThe administration of
study treatment (sub-conjunctival injection and eye drops) was done
within maximally 3 hours after the end of the surgical procedure.
The sub-conjunctival injection was administered first, followed by
the eye drops 5 to 15 minutes later. .sup.gWas done 1 hour after
administration of study treatment in approximately 30 patients
recruited from the Hotel-Dieu Hospital. .sup.hObservation for the
occurrence of Adverse Events started as soon as the administration
of study treatment was started.
[0771] Patients were randomized to one of the three study groups:
[0772] 1. A single sub-conjunctival injection of XG-102 90
.mu.g+placebo eye drops 4 times/day for 21 days or [0773] 2. A
single sub-conjunctival injection of XG-102 900 .mu.g+placebo eye
drops 4 times/day for 21 days or [0774] 3. A single
sub-conjunctival injection of NaCl 0.9%+dexamethasone eye drops 4
times/day for 21 days.
[0775] Randomization, which was blocked by center, was done
centrally using a web-based (i.e. e-SOCDAT.TM.) randomization
system.
[0776] XG-102 was used at doses 90 and 900 .mu.g (single
administration of 250 .mu.l). Mode of administration was a single
sub-conjunctival injection. Duration of treatment was one single
administration (sub-conjunctival injection).
[0777] The Reference product Dexamethasone (Dexafree.RTM.) was used
at a dose of 1 mg/ml. Mode of administration was eye drop (4
times/day, 21 days). Duration of treatment was 21 days-4
times/day.
[0778] The Placebo NaCl was used at a dose of 0.9%. Mode of
administration was a single sub-conjunctival injection (250 .mu.L)
or eye drop (4 times/day, 21 days). Duration of treatment was one
single administration (sub-conjunctival injection) and for the eye
drops, 21 days-4 times/day.
[0779] Based on preclinical pharmacology and toxicology studies in
addition to the safety and preliminary efficacy data obtained from
the previous study, two doses -90 and 900 .mu.g XG-102--were
selected for this trial. In the previous study, the safety profile
of the 90 and 900 .mu.g doses were similar. In addition, the
reduction of the intraocular inflammation, in combination with
corticosteroid eye drops, behaved in the same manner in both dose
groups. Taking into account the precautionary measures taken for
this study (role of the DSMB, and possibility to introduce open
label anti-inflammatory treatment in the case of persistent
inflammation), the XG-102 doses selected for this study were on the
one hand, considered not to compromise patient safety while on the
other hand, were sufficiently high to provide meaningful data for
the objectives of the study. The sub-conjunctival route of
administration is one of the intended routes of administration for
patients with the diagnosis under investigation as both safety and
efficacy has been shown in animals and in humans using this route
of administration. The dexamethasone dose (i.e. 1 mg/ml/0.4 ml eye
drops) in addition to the frequency (i.e. 4 drops per day) and
duration (i.e. 21 days) for use chosen for this study is the
standard dose/duration of use for dexamethasone eye drops as used
in clinical practice for post-operative ocular inflammation.
[0780] The study protocol stipulated that the sub-conjunctival
injection of study treatment was to be administered within
maximally 3 hours at the end of the eye surgery and that this was
to be followed within maximally 15 minutes by the instillation of
the first study treatment eye drop. The administration of the study
treatments at the end of the ocular surgery followed the standard
routine for the administration of anti-inflammatory treatments
following the eye surgery procedures which were part of the study
inclusion.
[0781] Neither the Investigator, the patient, the operational team
at the CC (Coordinating Center) nor the Sponsor personnel (other
than pharmacovigilance staff) had access to the randomization plan.
The study treatment vials containing the XG-102 solution or placebo
(i.e. NaCl 0.9% solution) were identical in appearance and
consistency. The eye drop solutions in single dose containers
containing either dexamethasone solution or NaCl 0.9% were
identical in appearance and consistency. The packaging and labeling
of study treatment was performed according to GMP (Good
Manufacturing Practice) and GCP (Good Clinical Practice). In
addition, the content of the labels affixed on the study treatment
packs was in accordance with local regulations for clinical trials.
For each patient two identically numbered study treatment packs
were supplied. One study treatment `pack` contained 1 vial of
XG-102 solution (90 or 900 .mu.g) or 1 vial of placebo (NaCl
0.9%)--depending on the treatment group to which the patient was
randomized--and the second `pack` contained the eye drop solution
in single dose containers containing either dexamethasone or
placebo (NaCl 0.9%) with sufficient supplies to enable treatment
for 4 times/day for 21 days. Once allocated to a patient, a study
treatment pack number was not allocated to another patient. The
patient's study identification number (i.e. patient identification
number) was written on the label by hand by the person who handed
out the study treatment. The size and shape of the outer study
treatment boxes were identical for the XG-102 and placebo
solutions. In an emergency situation where knowledge of a patient's
study treatment allocation would have been necessary to determine
the further medical management of the patient concerned, or if
knowledge of a patient's treatment allocation was required for
regulatory reporting purposes, the blinded Investigator or the
Sponsor delegated pharmacovigilance officer, respectively had the
user access rights to the study treatment code for the patient
concerned via the secure, web-based trial-specific treatment
allocation system within e-SOCDAT.TM.. If the treatment code was
accessed for any one patient, all information (i.e. the name of the
person who accessed the treatment code, the reason, date and time
and patient for whom the code was accessed) concerning study
treatment code access, would be tracked and stored in the web-based
system if the study treatment code was accessed.
[0782] The primary objective was evaluated by the mean anterior
chamber cells grade at day 28 post-administration of the
sub-conjunctival administration of study treatment. The criteria
for evaluation of the primary objective was
[0783] a. Anterior chamber cells grade at day 28 (XG-102 900 .mu.g
vs dexamethasone).
[0784] The criteria for evaluation of the secondary objectives
were
[0785] a. Anterior chamber cells grade at day 28 (XG-102 90 .mu.g
vs dexamethasone
[0786] b. Anterior chamber cells grade at day 7 and day 14 (XG-102
900 .mu.g vs dexamethasone)
[0787] c. Anterior chamber cells grade at day 7 and day 14 (XG-102
90 .mu.g vs dexamethasone)
[0788] d. Anterior chamber flare grade at day 7, 14 and day 28
(XG-102 900 .mu.g vs dexamethasone)
[0789] e. Anterior chamber flare grade at day 7, 14 and day 28
(XG-102 90 .mu.g vs dexamethasone)
[0790] f. Rescue medication use
[0791] g. Evolution of the intraocular inflammation over time as
assessed by Cleared ocular inflammation.
[0792] The ophthalmology examinations were performed at baseline
(i.e. either on the day of surgery, but before the surgery was
performed). Thereafter, patients were seen at 21 (.+-.3) hours
after the sub-conjunctival injection was administered, and then at
7 (.+-.1), 14 (.+-.2) and 28 (.+-.5) days. In order to reduce
operator variability, the sites were instructed that, where
possible, the same operator should perform all ophthalmology
examinations for the same patient throughout the trial. The
ophthalmology measurements were performed in accordance with the
study-specific instructions. The latter were reviewed and discussed
with the site teams during the initiation visit and during each
monitoring visit. For the determination of the cell/flare count and
cell/flare grade, the SUN Working Group's consensus was used by the
sites using the SUN Working group definitions ("Standardization of
Uveitis Nomenclature for Reporting Clinical Data. Results of the
First International Workshop.," American Journal of Ophthalmology,
vol 140, no. 3, pp. 509-516, 2005).
[0793] The criteria for evaluation of safety were:
[0794] a. Visual acuity by ETDRS method
[0795] b. Slit Lamp examination findings
[0796] c. The results of the ophthalmic fundus examination
[0797] d. Intra Ocular Pressure (IOP) measurements
[0798] e. Vital signs (blood pressure (BP), pulse rate (PR) and
rhythm)
[0799] f. The results of the hematology and chemistry laboratory
tests
[0800] g. The occurrence of Adverse Events
[0801] h. Presence (or not) of XG-102 in plasma 1 hour after the
administration of study treatment in a subset of patients
(approximately 30).
[0802] The definitions for an adverse event were:
[0803] An Adverse Event (AE) is defined as `any untoward medical
occurrence in a patient administered a medicinal product and which
does not necessarily have a causal relationship with this
treatment`. An AE is therefore any unfavorable and unintended sign,
symptom or disease temporally associated with the use of a
medicinal product, whether or not considered related to the
medicinal product.
[0804] An AE was considered to be serious if the event: [0805]
resulted in death [0806] was life-threatening [0807] required
in-subject hospitalization or prolongation of existing
hospitalization [0808] resulted in persistent or significant
disability/incapacity [0809] resulted in a congenital anomaly/birth
defect in an offspring conceived during the treatment period [0810]
was medically significant and jeopardizes the patient or requires
intervention to prevent one of the above outcomes
[0811] The term "life-threatening" in the definition of "serious"
referred to an event in which the subject was at risk of death at
the time of the event; it did not refer to an event which
hypothetically might have caused death, were it more severe. A
Suspected Unexpected Serious Adverse Reaction (SUSAR) was defined
as a suspected adverse reaction related to the treatment that is
both unexpected (i.e. not consistent with the expected outcomes of
the study treatment being administered) and serious.
[0812] The quantification (plasma) of XG-102 in plasma was
evaluated in a subset of 32 patients located in one site. A venous
blood sample (2 ml) was obtained using a Li-Heparin tube 60 minutes
after sub-conjunctival administration of study treatment. The exact
time when the sample was performed was entered in the space
provided on the e-CRF. The blood sample was centrifuged for 10
minutes at 2,500 RPM at room temperature. After centrifugation,
using a pipette, the plasma was transferred to two 1.5 ml
cryotubes. The cryotubes were then placed in a freezer at
-80.degree. C. and were then subsequently sent in dry ice with a
temperature data logger to the central laboratory responsible for
the analysis. Upon receipt at the central laboratory, the samples
were stored at -80.degree. C. until analyzed.
[0813] Statistical methods: The primary objective was a non
in-inferiority comparison between XG-102 900 .mu.g and
dexamethasone eye drops on anterior chamber cell grade at day 28
following the sub-conjunctival injection of study treatment. The
primary outcome was analyzed for the Per-Protocol (PP) population
and repeated for sensitivity reasons on the Full Analysis Set
(FAS). Non-inferiority of XG-102 900 .mu.g to dexamethasone could
be declared if the upper bound of the 95% Cl around the estimated
difference lay below 0.5 anterior chamber cell grade. The first
secondary end-point-anterior chamber cell grade at day 28 comparing
XG-102 90 .mu.g and dexamethasone was analyzed in the same manner
as for the primary outcome. All other secondary outcomes were
evaluated by superiority testing on the FAS using a two-sided alpha
value of 0.005. The safety analyses were performed on the FAS group
by treatment received.
[0814] The disposition of patients included in the present study is
shown in FIG. 59. In total, 157 patients provided informed consent
and 151 of these were randomized. Of the 151 randomized patients, 6
were not administered the sub-conjunctival injection of study
treatment. As per the requirements in the study protocol, these
randomized patients were replaced. In total, 145 patients were
administered the sub-conjunctival injection of study treatment
(i.e. XG-102 or placebo) and 144 patients completed the study as
planned by the study protocol. In total, 1 patient withdrew from
follow-up. The following Table displays the completeness of
follow-up for the three study groups:
TABLE-US-00022 90 .mu.g XG-102 900 .mu.g XG-102 Dexamethasone (N =
47) (N = 48) (N = 50) # patients (%) # patients (%) # patients (%)
Randomized 50 50 51 Randomized but not 3 2 1 administered study tx
Randomized and 47 (100.0%) 48 (100.0%) 50 (100.0%) administered
study tx Premature withdrawal 10 (21.3%) 8 (16.7%) 3 (6.0%) of
study tx eye drops Premature withdrawal 0 (0.0%) 1 (2.1%) 0 (0.0%)
from follow-up Visit 4 performed as 47 (100.0%) 47 (97.9%) 50
(100.0%) planned by the protocol Lost to follow-up 0 (0.0%) 1
(2.1%) 0 (0.0%) Data are number of patients (%). N = Number of
patients in each group, # = number, .mu.g = microgram, % =
percentage, tx = treatment.
[0815] The Full Analysis Set (FAS) comprised all randomized
patients for whom the sub-conjunctival injection of study treatment
was started/administered. The FAS set was analyzed according to the
intention-to-treat principle, i.e. patients were evaluated in the
treatment group to which they were randomized irrespective of the
treatment received. In addition, data was removed from the FAS
analysis sets for visits which were performed outside the allowed
time windows.
[0816] The PP analysis set was a subset of the FAS. Patients were
excluded from the PP analysis data set in case because of either
major violations after randomization and/or introduction of open
label anti-inflammatory treatment during follow-up. In addition,
data was removed from the PP analysis sets for visits which were
performed outside the allowed time windows.
[0817] The safety set included all randomized patients for whom the
sub-conjunctival injection of study treatment was
started/administered. Patients were analyzed as treated, i.e.
according to the treatment which they received. The safety set was
the primary analysis set for the safety analysis.
[0818] The baseline characteristics and comorbidities were balanced
between the three treatment groups both for FAS and PP populations.
The table below shows some of the main baseline co-morbidities by
treatment group for the PP analysis population. The percentage of
patients with retinal detachment was higher in patients allocated
to the XG-102 90 .mu.g (52%) compared to the XG-102 900 .mu.g (41%)
and the dexamethasone groups (40%) while the percentage of patients
with diabetes was higher in patients randomized to XG-102 900 .mu.g
group (33%) compared to XG-102 90 .mu.g group (22%) and
dexamethasone group (26%).
TABLE-US-00023 90 .mu.g XG-102 900 .mu.g XG-102 Dexamethasone (N =
46) (N = 46) (N = 50) # patients (%) # patients (%) # patients (%)
Retinal detachment 24 (52.2%) 19 (41.3%) 20 (40.0%) Glaucoma 6
(13.0%) 6 (13.0%) 6 (12.0%) Diabetic retinopathy 5 (10.9%) 6
(13.0%) 4 (8.0%) Hypertension 16 (34.8%) 25 (54.3%) 24 (48.0%)
Diabetes 10 (21.7%) 15 (32.6%) 13 (26.0%) Hypercholesterolemia 17
(37.0%) 20 (43.5%) 21 (42.0%) Data are number of patients (%). N =
Number of patients in each group, # = number, .mu.g = microgram, %
= percentage.
[0819] The following table shows, by treatment group, the
indication for ocular surgery at baseline in addition to the type
of surgery performed for the PP analysis population. The percentage
of patients who underwent complex posterior segment surgery was
higher in patients allocated to the XG-102 90 .mu.g (50%) compared
to those allocated to the XG-102 900 .mu.g (46%) and the
dexamethasone groups (42%). The percentage of patients in each
treatment group for whom gas (SF6 or C2F6) was instilled during the
surgery performed at baseline was 43% (XG-102 90 .mu.g), 37%
(XG-102 900 .mu.g) and 38% (dexamethasone) respectively.
TABLE-US-00024 90 .mu.g XG-102 900 .mu.g XG-102 Dexamethasone (N =
46) (N = 46) (N = 50) # patients (%) # patients (%) # patients (%)
Type of ocular surgery Anterior and posterior segment 18 (39.1%) 22
(47.8%) 26 (52.0%) combined surgery Glaucoma surgery 5 (10.9%) 3
(6.5%) 3 (6.0%) Complex posterior segment surgery 23 (50.0%) 21
(45.7%) 21 (42.0%) Eye concerned Left 17 (37.0%) 23 (50.0%) 25
(50.0%) Right 29 (63.0%) 23 (50.0%) 25 (50.0%) Indication of ocular
surgery Cataract 19 (28.8%) 22 (31.4%) 25 (31.6%) Epimacular
membrane 8 (12.1%) 8 (11.4%) 10 (12.7%) Epiretinal membrane 4
(6.1%) 6 (8.6%) 10 (12.7%) Foveoschisis 0 (0.0%) 0 (0.0%) 1 (1.3%)
Intravitreous hemorrhage 5 (7.6%) 4 (5.7%) 3 (3.8%) Macular hole 2
(3.0%) 6 (8.6%) 2 (2.5%) Neovascular glaucoma 1 (1.5%) 0 (0.0%) 0
(0.0%) Relief of intraocular pressure 5 (7.6%) 3 (4.3%) 3 (3.8%)
Retinal detachment 22 (33.3%) 19 (27.1%) 20 (25.3%) Subluxation of
intraocular lens 0 (0.0%) 1 (1.4%) 0 (0.0%) Subluxation of lens 0
(0.0%) 0 (0.0%) 1 (1.3%) Vitreomacular traction 0 (0.0%) 1 (1.4%) 4
(5.1%) Type of gas 20 (43.5%) 17 (37.0%) 19 (38.0%) SF6 11 (55.0%)
11 (64.7%) 12 (63.2%) C2F6 9 (45.0%) 6 (35.3%) 7 (36.8%) Data are
number of patients (%). N = Number of patients in each group, # =
number, .mu.g = microgram, % = percentage, SD = Standard deviation.
Nr. available = Number of patients for whom data are available
[0820] Anterior chamber cell grade at day 28--XG-102 900 .mu.g vs
dexamethasone: The primary endpoint was analyzed as the mean
difference in the anterior chamber cells grade at day 28, comparing
the XG-102 900 .mu.g dose with the dexamethasone group, using an
adjusted repeated measures model. Only data collected for the day
7, 14 and 28 visits were used in the repeated model. The primary
analysis was performed on the PP analysis data set and a
sensitivity analysis was performed on the FAS data set. For the
first secondary outcome,--i.e. Anterior chamber cells grade at day
28 (XG-102 90 .mu.g vs dexamethasone)--non-inferiority was
determined in the same manner as for the primary endpoint, using
the same non-inferiority margin of 0.5 anterior chamber cell grade.
The mean anterior chamber cell grade up to 28 days after the
administration of the sub-conjunctival injection of study treatment
for the PP analysis population is shown in FIG. 60 for the three
treatment groups--i.e. XG-102 90 .mu.g, XG-102 900 .mu.g and the
dexamethasone--while the statistical model results are shown in the
following table:
TABLE-US-00025 Model adjusted Estimated Pvalue mean Dose group
difference (non- Pvalue Dose group [95% CI] comparison [95% CI]
inferiority) (superiority) Visit 2 (7 days +/- 2 days after
administration of study tx) XG-102 90 .mu.g 1.05 [0.84-1.26] XG-102
90 .mu.g versus 0.142 0.327 Dexamethasone [-0.142-0.425] XG-102 900
.mu.g 0.96 [0.76-1.16] XG-102 900 .mu.g versus 0.056 0.694
Dexamethasone [-0.222-0.333] Dexamethasone 0.91 [0.72-1.10] XG-102
900 .mu.g versus -0.086 0.561 XG-102 90 .mu.g [-0.377-0.205] Visit
3 (14 days +/- 3 days after administration of study tx) XG-102 90
.mu.g 0.80 [0.58-1.01] XG-102 90 .mu.g versus 0.009 0.948
Dexamethasone [-0.278-0.296] XG-102 900 .mu.g 0.77 [0.56-0.98]
XG-102 900 .mu.g versus -0.017 0.906 Dexamethasone [-0.300-0.266]
Dexamethasone 0.79 [0.60-0.98] XG-102 900 .mu.g versus -0.026 0.862
XG-102 90 .mu.g [-0.323-0.271] Visit 4 (28 days +/- 8 days after
administration of study tx) XG-102 90 .mu.g 0.58 [0.36-0.81] XG-102
90 .mu.g versus 0.086 0.003 0.573 Dexamethasone [-0.214-0.385]
XG-102 900 .mu.g 0.44 [0.23-0.66] XG-102 900 .mu.g versus -0.054
<0.001* 0.720 Dexamethasone [-0.350-0.242] Dexamethasone 0.50
[0.30-0.70] XG-102 900 .mu.g versus -0.140 0.381 XG-102 90 .mu.g
[-0.453-0.174] CI = Confidence Interval, .mu.g = microgram, % =
percentage, tx = treatment. *Primary comparison`
[0821] The results of the primary outcome in addition to the first
secondary outcome are shown in FIG. 61 for both the PP and FAS data
sets. XG-102 900 .mu.g was non-inferior to dexamethasone eye drops
in the evolution of post-operative intraocular inflammation as
assessed by anterior chamber cell grade at day 28 (difference of
-0.054 anterior cell grade, 95% Confidence Interval (CI)
-0.350-0.242, p<0.001). The same analysis was repeated on the
FAS and XG-102 900 .mu.g was found to be non-inferior to
dexamethasone eye drops (difference -0.032 cell grade, 95% Cl
-0.301-0.238, p<0.001). Given that the upper boundary crossed
zero for the FAS and PP analysis sets, XG-102 900 .mu.g was not
superior to dexamethasone eye drops (p=0.818 for the FAS and
p=0.720 for the PP analysis set) for anterior chamber cell grade at
day 28.
[0822] Anterior chamber cell grade at day 28--XG-102 90 .mu.g vs
dexamethasone: Concerning the secondary endpoint comparing XG-102
90 .mu.g with dexamethasone eye drops, XG-102 90 .mu.g was
non-inferior to dexamethasone in the evolution of post-operative
intraocular inflammation (difference 0.086 anterior cell grade, 95%
Cl -0.214-0.385, p=0.003). The same analysis was repeated on the
FAS and XG-102 90 .mu.g was found to be non-inferior to
dexamethasone eye drops (difference of 0.053 anterior cell grade
95% Cl -0.215-0.321 p<0.001).
[0823] Anterior chamber cell grade at day 7 and 14 for XG-102 90
.mu.g vs dexamethasone and XG-102 900 .mu.g vs dexamethasone:
[0824] The statistical analyses for the anterior chamber cell grade
at day 7 and 14 for XG-102 90 .mu.g vs dexamethasone and XG-102 900
.mu.g vs dexamethasone were performed on the FAS data set. There
were no statistically significant differences in anterior chamber
cell grade between XG-102 90 .mu.g and dexamethasone and between
XG-102 900 .mu.g and dexamethasone at either day 7 or day 14.
[0825] Anterior chamber flare grade at day 7, 14 and day 28 for
XG-102 90 .mu.g vs dexamethasone and XG-102 900 .mu.g vs
dexamethasone:
[0826] The anterior chamber flare grade (for the FAS) obtained up
to day 28 is shown in FIG. 62 and the model results is shown in the
table below. There was no statistically significant difference in
the anterior chamber flare grade between XG-102 90 .mu.g and
dexamethasone and between XG-102 900 .mu.g and dexamethasone at
either day 7 or day 14 or at day 28.
TABLE-US-00026 Model adjusted mean Estimated difference Dose group
[95% CI] Dose group comparison [95% CI] Pvalue Visit 2 (7 days +/-
2 days after administration of study tx) XG-102 90 ug 0.93
[0.73-1.14] XG-102 90 ug versus Dexamethasone 0.133 [-0.154-0.420]
0.363 XG-102 900 ug 0.80 [0.60-1.00] XG-102 900 ug versus
Dexamethasone -0.003 [-0.284-0.278] 0.983 Dexamethasone 0.80
[0.60-1.00] XG-102 900 ug versus XG-102 90 ug -0.136 [-0.424-0.152]
0.353 Visit 3 (14 days +/- 3 days after administration of study tx)
XG-102 90 ug 0.72 [0.52-0.92] XG-102 90 ug versus Dexamethasone
0.142 [-0.140-0.424] 0.322 XG-102 900 ug 0.80 [0.59-1.00] XG-102
900 ug versus Dexamethasone 0.220 [-0.061-0.502] 0.125
Dexamethasone 0.58 [0.38-0.77] XG-102 900 ug versus XG-102 90 ug
0.078 [-0.210-0.366] 0.595 Visit 4 (28 days +/- 8 days after
administration of study tx) XG-102 90 ug 0.48 [0.28-0.69] XG-102 90
ug versus Dexamethasone 0.027 [-0.255-0.309] 0.851 XG-102 900 ug
0.44 [0.23-0.64] XG-102 900 ug versus Dexamethasone -0.017
[-0.301-0.267] 0.906 Dexamethasone 0.46 [0.26-0.65] XG-102 900 ug
versus XG-102 90 ug -0.044 [-0.333-0.245] 0.764 CI = Confidence
Interval, .mu.g = microgram, % = percentage, tx = treatment.
[0827] Cleared Ocular Inflammation:
[0828] The evaluation of ocular inflammation over time was assessed
by cleared ocular inflammation. The latter was defined as the
proportion of subjects that had a summed ocular inflammation score
of grade 0 defined as anterior cell grade=0 and anterior chamber
flare grade=0. This outcome was evaluated at day 7, 14 and day 28
comparing XG-102 900 .mu.g with dexamethasone and XG-102 90 .mu.g
with dexamethasone. The summary statistic results for the FAS and
PP populations is shown in the table below. Concerning the analysis
performed on the FAS, compared to the usual care group, for
patients allocated to the XG-102 900 .mu.g group the odds of having
cleared inflammation at day 7 post-surgery was 0.76 (95% Cl
0.25-2.28), at day 14 post-surgery, 1.25 (95% Cl 0.47-3.32) and at
day 28 post-surgery, 1.13 (95% Cl 0.49-2.60). Concerning patients
allocated to the XG-102 90 .mu.g group, compared to the usual care
group, the odds of having cleared inflammation at day 7
post-surgery was 0.52 (95% Cl 0.15-1.83), at day 14 post-surgery,
0.85 (95% Cl 0.30-2.40) and at day 28 post-surgery, 1.24 (95% Cl
0.54-2.87). Concerning the analysis performed on the PP analysis
set, compared to the usual care group, for patients allocated to
the XG-102 900 .mu.g group the odds of having cleared inflammation
at day 7 post-surgery was 0.84 (95% Cl 0.28-2.46), at day 14
post-surgery, 1.12 (95% Cl 0.41-3.05) and at day 28 post-surgery,
1.26 (95% Cl 0.52-3.04). Concerning patients allocated to the
XG-102 90 .mu.g group, compared to the usual care group, the odds
of having cleared inflammation at day 7 post-surgery was 0.56 (95%
Cl 0.16-1.97), at day 14 post-surgery, 0.97 (95% Cl 0.34-2.77) and
at day 28 post-surgery, 1.45 (95% Cl 0.58-3.61).
TABLE-US-00027 Full analysis population Per-protocol analysis
population 90 .mu.g XG- 900 .mu.g XG- 90 .mu.g XG- 900 .mu.g XG-
102 102 102 102 (N = 47) (N = 48) Dexamethasone (N = 46) (N = 46)
Dexamethasone # patients # patients (N = 50) # patients # patients
(N = 50) (%) (%) # patients (%) (%) (%) # patients (%) Visit 1 (21
hours after administration of study tx) Nr. available (%) 47
(100.0%) 48 (100.0%) 49 (98.0%) 46 (100.0%) 46 (100.0%) 49 (98.0%)
Yes 2 (4.3%) 2 (4.2%) 4 (8.2%) 2 (4.3%) 2 (4.3%) 4 (8.2%) Visit 2
(7 days +/- 2 days after administration of study tx) Nr. available
(%) 42 (89.4%) 47 (97.9%) 48 (96.0%) 39 (84.8%) 44 (95.7%) 48
(96.0%) Yes 5 (11.9%) 7 (14.9%) 9 (18.8%) 5 (12.8%) 7 (15.9%) 9
(18.8%) Visit 3 (14 days +/- 3 days after administration of study
tx) Nr. available (%) 45 (95.7%) 43 (89.6%) 49 (98.0%) 37 (80.4%)
40 (87.0%) 47 (94.0%) Yes 8 (17.8%) 10 (23.3%) 10 (20.4%) 8 (21.6%)
9 (22.5%) 10 (21.3%) Visit 4 (28 days +/- 8 days after
administration of study tx) Nr. available (%) 45 (95.7%) 43 (89.6%)
48 (96.0%) 33 (71.7%) 34 (73.9%) 43 (86.0%) Yes 18 (40.0%) 17
(39.5%) 17 (35.4%) 14 (42.4%) 15 (44.1%) 15 (34.9%) Data are number
of patients (%). N = Number of patients in each group, # = number,
.mu.g = microgram, % = percentage. Tx = treatment. **Cleared ocular
inflammation is defined as 0 cell (i.e. cell grade as 0) and no
flare (i.e. flare grade as 0)
[0829] Laser Flare Meter (LFM):
[0830] The LFM measurements which were obtained at the defined time
points throughout the study are depicted as the LFM measurements
over time and up to day 28 for the FAS in FIG. 63.
[0831] Rescue medication was defined in the study protocol as any
open-label anti-inflammatory ocular treatment which was prescribed
for patients during follow-up because of persistent eye
inflammation as judged by the Investigator. The study protocol
stipulated that the study treatment eye drops were to be stopped at
the introduction of open-label anti-inflammatory ocular treatment.
The percentage of patients for whom rescue medication was
introduced in the XG-102 90 .mu.g group was statistically different
when compared to the dexamethasone group (21.3% vs 4.0% for the
XG-102 90 .mu.g and dexamethasone groups respectively (p=0.013))
while the difference between XG-102 900 .mu.g and dexamethasone
(14.6% and 4.0% respectively for the two groups) was not
statistically significant (p=0.88)
[0832] Pharmacokinetics in Plasma:
[0833] Blood sampling for quantification of XG-102 was taken 60
minutes after the sub-conjunctival administration of XG-102 in a
subset of 32 patients. The analytical report of quantification of
XG-102 in plasma shows that XG-102 was not detected in the plasma
samples for any patient--see the following table:
TABLE-US-00028 90 .mu.g XG- 102 900 .mu.g XG-102 Dexamethasone (N =
11) (N = 9) (N = 12) # patients (%) # patients (%) # patients (%) #
patients with sample 11 (100.0%) 9 (100.0%) 12 (100.0%) <LLOQ*
11 (100.0%) 9 (100.0%) 12 (100.0%) Data are number of patients (%).
N = Number of patients for whom an XG-102 quantification sample was
obtained, # = number, .mu.g = microgram, % = percentage. LLOQ =
Less than the Limit of Quantification *Values which were below the
LLOQ (i.e. <10 ng/mL) were considered as `not detectable`
[0834] In summary, XG-102 900 .mu.g was non-inferior to
dexamethasone eye drops in the evolution of post-operative
intraocular inflammation (difference of -0.054 anterior cell grade,
95% Cl--0.350-0.242, p<0.001). The same analysis was repeated on
the FAS and XG-102 900 .mu.g was found to be non-inferior to
dexamethasone eye drops (difference -0.032 cell grade, 95% Cl
-0.301-0.238, p<0.001). Given that the upper boundary crossed
zero for the FAS and PP analysis sets, XG-102 900 .mu.g was not
superior to dexamethasone eye drops (p=0.818 for the FAS and
p=0.720 for the PP analysis set) for the anterior chamber cell
grade at day 28.
[0835] Concerning the secondary endpoint comparing XG-102 90 .mu.g
with dexamethasone eye drops, XG-102 90 .mu.g was non-inferior to
dexamethasone eye in the evolution of post-operative intraocular
inflammation (difference 0.086 anterior cell grade, 95% Cl
-0.214-0.385, p=0.003). The same analysis was repeated on the FAS
and XG-102 90 .mu.g was found to be non-inferior to dexamethasone
eye drops (difference of 0.053 anterior cell grade 95% Cl
-0.215-0.321 p<0.001).
[0836] There were no statistically significant differences in
anterior chamber cell grade between XG-102 90 .mu.g and
dexamethasone and between XG-102 900 .mu.g and dexamethasone at
either day 7 or day 14. There was no statistically significant
difference in the anterior chamber flare grade between XG-102 90
.mu.g and dexamethasone and between XG-102 900 .mu.g and
dexamethasone at either day 7 or day 14 or at day 28.
[0837] The evaluation of ocular inflammation over time was assessed
by cleared ocular inflammation. The latter was defined as the
proportion of subjects that had a summed ocular inflammation score
of grade 0 defined as anterior cell grade=0 and anterior chamber
flare grade=0. This outcome was evaluated at day 7, 14 and day 28
comparing XG-102 900 .mu.g with dexamethasone and XG-102 90 .mu.g
with dexamethasone. Concerning the analysis performed on the FAS,
compared to the usual care group, for patients allocated to the
XG-102 900 .mu.g group the odds of having cleared inflammation at
day 7 post-surgery was 0.76 (95% Cl 0.25-2.28), at day 14
post-surgery, 1.25 (95% Cl 0.47-3.32) and at day 28 post-surgery,
1.13 (95% Cl 0.49-2.60). Concerning patients allocated to the
XG-102 90 .mu.g group, compared to the usual care group, the odds
of having cleared inflammation at day 7 post-surgery was 0.52 (95%
Cl 0.15-1.83), at day 14 post-surgery, 0.85 (95% Cl 0.30-2.40) and
at day 28 post-surgery, 1.24 (95% Cl 0.54-2.87).
[0838] Safety Evaluation
[0839] Extent of Exposure:
[0840] The present study was a double-blind study. All patients who
were randomized and for whom the sub-conjunctival injection was
started are included in the safety analysis by dose group. Only
treatment emergent AEs have been analyzed, i.e. AEs that occurred
after the start of the sub-conjunctival injection of study
treatment. If the study treatment eye drops were stopped
prematurely (i.e. before day 21), the patients concerned continued
follow-up until day 28, in accordance with the study protocol. The
sub-conjunctival injection of study treatment was administered for
145 patients in total of which 47 patients were administered XG-102
90 .mu.g, 48 patients were administered XG-102 900 Ng and 50
patients allocated to the dexamethasone group were administered to
NaCL 0.9%. For all patients in whom the sub-conjunctival injection
of study treatment was started, the total amount (i.e. 250 .mu.L)
of study treatment was administered.
[0841] The exposure by patient for the study treatment eye drops is
shown in the table below. Concerning the study treatment eye drops,
the overall compliance with the instillation of the study treatment
eye drops as required by the study protocol was >90% in the
three study groups. Patients allocated to the XG-102 treatment
groups had a slightly higher compliance with the instillation of
the study treatment eye drops (95% and 94% for the XG-102 90 .mu.g
and XG-102 900 .mu.g groups respectively) compared to patients
allocated to the dexamethasone group where the compliance was 91%.
Fifty patients received dexamethasone eye drops for an average of
20 days (6-21 days, min-max) with a maximal cumulated dose of 81
drops (81.times.0.05 mg=4.05 mg).
TABLE-US-00029 90 .mu.g XG-102 900 .mu.g XG-102 Dexamethasone (N =
47) (N = 48) (N = 50) mean mean mean (min-max) (min-max) (min-max)
Days under treatment 18 (1-21) 19 (1-21) 20 (6-21) eye drops
Compliance with study 95.3% 93.8% 90.6% treatment eye drops*
(75.3%-100%) (33.3%-100%) (33.3%-100%) footnote: For patients who
stopped the study treatment eye drops prematurely, compliance was
calculated as used/planned *100 where planned = 4 * (days from
start and up to withdrawal). For patients who used the study
treatment eye drops as planned by the protocol, compliance was
calculated as ((81 - unused eye drops bottles)/81) * 100
[0842] Adverse Events
[0843] Summary of Adverse Events by Dose Group:
[0844] The overview of reported adverse events (serious and
non-serious) is displayed in FIG. 64 by dose group. There was not a
statistically significant difference between the XG-102 90 .mu.g
and dexamethasone groups and between the XG-102 900 .mu.g and
dexamethasone groups with respect to the number of patients for
whom an AE was reported. For patients allocated to XG-102 90 .mu.g,
a total of 78 AEs were reported for 31/47 (66%) patients allocated
to this group and for patients allocated to XG-102 900 .mu.g, a
total of 69 AEs were reported for 32/48 (67%) patients. For
patients allocated to the dexamethasone group, a total of 55 AEs
were reported for 29/50 (58%) patients. The percentage of patients
who experienced an AE within 24 hours after administration of study
treatment was similar between the three study treatment groups
(i.e. 34%, 27% and 30% for the XG-102 90 .mu.g, the XG-102 900
.mu.g and dexamethasone groups' respectively).
[0845] The distribution of the reported AEs by severity and dose
group is shown in table below. The majority (approximately 70%) of
reported AEs were considered by the Investigator as being `mild`
for the three dose groups.
TABLE-US-00030 Severity Dose group Mild Moderate Severe XG-102 90
.mu.g 55 (70.5%) 6 (7.7%) 17 (21.7%) XG-102 900 .mu.g 49 (71.0%) 12
(17.4%) 8 (11.6%) Dexamethasone 42 (76.4%) 9 (16.4%) 4 (7.3%) Data
are number of events (% or reported events)
[0846] The summary overview of AEs which led to an interruption of
the study treatment eye drops is shown by dose group in the
following table:
TABLE-US-00031 Adverse events which led to an Dose group
interruption of study treatment XG-102 90 .mu.g 11 (14.1%) XG-102
900 .mu.g 8 (11.6%) Dexamethasone 3 (5.5%) Data are number of
events (% of reported events)
[0847] For patients allocated to the XG-102 90 .mu.g dose group, 11
events (14% of all reported AEs in this dose group) resulted in the
premature withdrawal of study treatment while in the XG-102 900
.mu.g and dexamethasone dose groups, the study treatment eye drops
were interrupted for 8 events (12% of all reported AEs in this dose
group) and 3 events (6% of all reported AEs in this dose group)
respectively.
[0848] Investigators assessed (in a blinded manner) the
relationship of each reported AE to any of the study treatments. An
event was considered to be related to study treatment if the
Investigator ticked either `possible` or `probable` as the reply to
this question. In addition, the Investigator had to specify to
which of the study treatments (i.e. XG-102 or dexamethasone) the
event was considered related to-see the table below. AEs were
considered by the Investigators (blinded assessment) to be possibly
or probably related to study medication for 18 events reported for
patients in the XG-102 90 .mu.g, for 13 events reported for
patients in the XG-102 900 .mu.g, and for 15 events reported for
patients in dexamethasone group (see table below). None of the
reported SAEs were considered by the Investigator to be either
possibly or probably related to either of the study treatments.
TABLE-US-00032 Relationship to study treatment 90 .mu.g XG-102 900
.mu.g XG-102 Dexamethasone as assessed by (N = 47) (N = 48) (N =
50) the Investigator Total # events Total # events Total # events
Possibly or Probably 18 13 15 related AEs considered by the
Investigator to be related to: XG-102 16 (20.5%) 12 (17.4%) 13
(23.64%) Dexamethasone 2 (2.6%) 1 (1.5%) 2 (3.6%) Data are number
of events. N = Number of patients in each group, # = number, .mu.g
= microgram, % = percentage.
[0849] Display of Adverse Events:
[0850] A reported event was considered to be related to study
treatment if the Investigator had ticked either `Possible` or
`Probable` as the reply to the question `Related to study
treatment` on the e-CRF. The summary of the AEs (sorted by MedDRA
SOC and PT term) which were reported for at least 2% of patients
randomized to either of the three study groups may be found in FIG.
65.
[0851] Analysis of Adverse Events
[0852] Overall, there was not a statistically significant
difference between either of the XG-102 dose groups and the
dexamethasone dose group with respect to the number of patients for
whom an AE was reported. For patients allocated to XG-102 90 .mu.g,
a total of 78 AEs were reported for 31/47 (66%) patients allocated
to this group and for patients allocated to XG-102 900 .mu.g, a
total of 69 AEs were reported for 32/48 (67%). For patients
allocated to the dexamethasone group, a total of 55 AEs were
reported for 29/50 (58%). The percentage of patients who
experienced an AE within 24 hours after administration of study
treatment was similar between the three study treatment groups
(i.e. 34%, 27% and 30% for the XG-102 90 .mu.g, the XG-102 900
.mu.g and dexamethasone groups' respectively).
[0853] The most frequently reported AEs were in the SOC `EYE
DISORDERS. Within this SOC, 49 events were reported for 26 (55%)
patients allocated to XG-102 90 .mu.g, 43 events were reported for
24 (50%) patients allocated to XG-102 900 .mu.g and 30 events were
reported for 16 (32%) of patients allocated to dexamethasone. There
was a statistically significant difference between the XG-102 90
.mu.g and dexamethasone group with respect to the number of
patients for whom an event was reported in this SOC (p=0.025).
Events suggestive of inflammation (such as `eye inflammation`,
`Corneal oedema`, `Eyelid oedema`) were more frequently reported
for patients allocated to XG-102 90 .mu.g compared to patients
allocated to either the XG-102 900 .mu.g or dexamethasone dose
groups. Eye pain was more frequently reported for patients
allocated to the XG-102 900 .mu.g group and when compared to the
dexamethasone group, the difference in the number of patients for
whom this event was reported was statistically significant
(p=0.029). Within the SOC `investigations`, `Intraocular pressure
increased` was reported more frequently for patients allocated to
XG-102 90 .mu.g (23%) when compared to 10% and 14% for the XG-102
900 .mu.g and dexamethasone groups respectively. The difference in
number of patients for whom this event was reported (between XG-102
90 .mu.g and dexamethasone) was not statistically significant. The
study treatment eye drops were interrupted because of an AE for 11
patients allocated to XG-102 90 .mu.g, for 8 patients allocated to
XG-102 900 .mu.g and for 3 patients allocated to dexamethasone.
FIG. 65 displays a summary of the AEs (sorted by MedDRA SOC and
Preferred Term (PT)) which occurred for at least 2% of patients,
irrespective of the randomized group.
[0854] Serious Adverse Events
[0855] The serious adverse events concerned are listed in FIG. 66.
In total, 9 SAEs were reported for 9 patients--i.e. for 4 patients
randomized to the XG-102 90 .mu.g dose group, for 3 patients
randomized to the XG-102 900 .mu.g dose group and for 2 patients
randomized to the dexamethasone dose group. In total, one SAE (for
a patient randomized to the XG-102 90 .mu.g dose group) was
reported within the first 24 hours after administration of the
sub-conjunctival injection of study treatment. None of the reported
SAEs were considered by the Investigator as being related to study
treatment. The `reason for seriousness` for all reported SAEs was
`hospitalization`. The overview of the reported SAEs is shown in
FIG. 66.
[0856] Clinical Laboratory Evaluation
[0857] The hematology and chemistry assays which were performed for
the study are shown in the following table. All laboratory tests
were performed locally.
TABLE-US-00033 Hematology: Hemoglobin, hematocrit, White blood cell
count (WBC), neutrophils, basophils, eosinophils, monocytes and
lymphocytes Chemistry: Creatinine, Aspartate Transaminase (AST),
Alanine Transaminase (ALT), gamma-glutamyltransferase (gamma-GT),
glucose, CK, CRP
[0858] Safety Conclusions
[0859] Overall, XG-102 90 .mu.g and XG-102 900 .mu.g was well
tolerated in patients who underwent complex ocular surgery. The
study treatment eye drops were stopped prematurely for 11 patients
randomized to XG-102 90 .mu.g, for 8 patients randomized to XG-102
900 .mu.g and for 3 patients randomized to dexamethasone. The
reason for the premature withdrawal of study treatment was
primarily because of persistent eye inflammation which in the
opinion of the Investigator necessitated intensification of
anti-inflammatory treatment. For the patients concerned, treatment
with open-label anti-inflammatory ocular treatment was
initiated.
[0860] No fatal events were reported in this study. In total, 9
SAEs were reported for 9 patients and none of these events were
considered as being related to the study treatment.
[0861] Concerning the overall number of reported AEs, there are not
a statistically significant difference between either of the XG-102
dose groups and the dexamethasone group with respect to the number
of patients for whom an AE was reported. For patients allocated to
XG-102 90 .mu.g, a total of 78 AEs were reported for 31/47 (66%)
patients allocated to this group and for patients allocated to
XG-102 900 .mu.g, a total of 69 AEs were reported for 32/48 (67%)
patients. For patients allocated to the dexamethasone group, a
total of 55 AEs were reported for 29/50 (58%) patients. The
percentage of patients who experienced an AE within 24 hours after
administration of study treatment was similar between the three
study treatment groups (i.e. 34%, 27% and 30% for the XG-102 90
.mu.g, the XG-102 900 .mu.g and dexamethasone groups,
respectively). The number of patients who experienced an AE
suggestive of eye inflammation was higher in patients allocated to
the XG-102 90 .mu.g group compared to the XG-102 900 .mu.g and
dexamethasone groups which suggests that XG-102 90 .mu.g may be
less efficacious in the treatment of eye inflammation secondary to
complex ocular surgery. The number of patients who experienced an
AE suggestive of eye pain was higher in patients allocated to the
XG-102 900 .mu.g group compared to the XG-102 90 .mu.g and
dexamethasone groups. For two patients in the XG-102 90 .mu.g
group, eye pain was reported less than 24 hours after the injection
of the sub-conjunctival injection of study treatment--for one of
these patients, analgesic treatment had not been prescribed
post-operatively. For one of these patients, eye pain was again
reported as an AE 35 days later which was at the same time when the
patient was reported as having an elevated IOP.
[0862] For three patients in the XG-102 900 .mu.g group, eye pain
was reported less than 24 hours after the injection of the
sub-conjunctival injection of study treatment and for one of these
patients, eye pain was again reported as an AE five days later. For
four patients in the same dose group, eye pain was reported >24
hours after the sub-conjunctival injection of study treatment
concomitantly. For three of these patients, eye pain was reported
concomitantly with other AEs. Eye pain was reported for one patient
in the dexamethasone group >24 hours after the sub-conjunctival
injection of study treatment concomitantly. This event was reported
concomitantly with another AE. Given that complex surgery was
performed, eye pain could also be related to the presence of
stitches following the surgery.
[0863] Summary
[0864] Compliance: For all patients in whom the sub-conjunctival
injection of study treatment was started, the total amount (i.e.
250 .mu.L) was administered. In the three study treatment groups,
the overall compliance with the study treatment eye drops was
>90%.
[0865] Safety: There was not a statistically significant difference
between either of the XG-102 dose groups and the dexamethasone
group with respect to the number of patients for whom an adverse
event was reported. For patients allocated to XG-102 90 .mu.g, a
total of 78 adverse events were reported for 31/47 (66%) patients
allocated to this group and for patients allocated to XG-102 900
.mu.g, a total of 69 adverse events were reported for 32/48 (67%)
patients. For patients allocated to the dexamethasone group, a
total of 55 adverse events were reported for 29/50 (58%) patients.
The percentage of patients who experienced an adverse event within
24 hours after administration of study treatment was similar
between the three study treatment groups (i.e. 34%, 27% and 30% for
the XG-102 90 .mu.g, the XG-102 900 .mu.g and dexamethasone groups'
respectively). More patients allocated to the XG-102 90 .mu.g
group, compared to the XG-102 900 .mu.g and dexamethasone groups,
experienced an adverse event suggestive of eye inflammation which
may suggest that XG-102 90 .mu.g may be less efficacious (compared
to the 900 pig and dexamethasone dose groups) in the treatment of
eye inflammation secondary to complex ocular surgery. The number of
patients who experienced an adverse event suggestive of eye pain
was higher in patients allocated to the XG-102 900 .mu.g group
compared to the XG-102 90 .mu.g and dexamethasone groups. The eye
pain may be related to the presence of stitches following the
surgery. `Intraocular pressure increased` was reported more
frequently for patients allocated to XG-102 90 .mu.g (23%) when
compared to 10% and 14% for the XG-102 900 .mu.g and dexamethasone
groups respectively. The difference in number of patients for whom
this event was reported (between XG-102 90 .mu.g and dexamethasone
was not statistically significant).
[0866] The majority (approximately 70%) of all reported adverse
events (AE) were considered by the Investigator as being mild. In
total, AEs were considered by the Investigators (blinded
assessment) to be possibly or probably related to study medication
for 18 events reported for patients in the XG-102 90 .mu.g, for 13
events reported for patients in the XG-102 900 .mu.g, and for 15
events reported for patients in dexamethasone group. No fatal
events were reported in this study. In total, 9 SAEs were reported
for 9 patients and none of these events were considered as being
related to the study treatment.
[0867] The quantification of XG-102 was performed in a sub-set of
32 patients. A blood sample was obtained 1 hour after the
sub-conjunctival injection of study treatment. For all samples
obtained (and irrespective of the assigned dose group) the XG-102
concentration was analyzed as being below the Lower Limit of
Quantification (LLOQ) of <10 ng/ml.
[0868] According to our definitions of non-inferiority, both XG-102
900 .mu.g and XG-102 90 .mu.g administered as a single
sub-conjunctival injection was non-inferior to treatment with
dexamethasone eye drops instilled 4 times/day for 21 days in the
treatment of post-operative intraocular inflammation as assessed by
anterior chamber cell grade, in patients who underwent complex
ocular surgery.
[0869] Overall, XG-102 90 .mu.g and XG-102 900 .mu.g was well
tolerated. None of the reported adverse events were suggestive of
an intolerable or irreversible side effect of XG-102. The increased
number of events suggestive of eye inflammation reported in the
XG-102 90 .mu.g suggests that this dose is less effective in the
treatment of post-operative intraocular inflammation in patients
following complex intraocular surgery. This is also probably
enforced by the percentage of patients for whom rescue medication
was introduced due to persistent eye inflammation in the XG-102 90
.mu.g group. The plasma quantification of XG-102 which was assessed
1 hour after administration of the sub conjunctival injection of
study treatment demonstrated that there was no systemic passage of
XG-102.
Example 28: Effects of XG-102 on In Vivo Hepatocarcinoma in p-38
(Mapk14) Deficient Mice
[0870] Mapk14, which is also known as p-38, is a well-known
negative regulator of cell proliferation and tumorigenesis. In this
study, Mapk14.sup.f/f and Mapk14.sup..DELTA.Ii* mice as well as
Mapk14.sup.f/f Jun.sup.f/f and Mapk14.sup..DELTA.Ii*
Jun.sup..DELTA.Ii* mice have been used. "Mapk14.sup..DELTA.Ii*",
and "Mapk14.sup..DELTA.Ii*" Jun.sup..DELTA.Ii*" respectively, means
herein polylC-treated Mx-cre/Mapk14.sup.f/f mice, and Mx-cre
deleted Mapk14.sup.f/f Jun.sup.f/f mice respectively, thus
resulting in Mapk14 "deletion", and Jun deletion respectively, by
the Mx-cre process, i.e. "Mapk14.sup.-/-" mice, and "Mapk14.sup.-/-
Jun.sup.-/-" mice respectively.
[0871] XG-102 has been administered intraperitoneal twice weekly at
a dose of 20 mg/kg to study its effects on the diethylnitrosamine
(DEN)-induced hyperproliferation of hepatocytes and liver tumor
cells (cf. Hui L. et al, p38a suppresses normal and cancer cell
proliferation by antagonizing the JNK-c-Jun pathway. Nature
Genetics, 2007; 39: 741-749). PBS has been used as control.
Specifically, the Mapk14.sup.f/f and Mapk14.sup..DELTA.Ii* mice
were injected with either PBS or XG-102 (20 mg per kg body weight)
before DEN treatment. The proliferation of hepatocytes in the mice
was then analyzed by Ki67 staining 48 h after DEN treatment and
quantified.
[0872] FIG. 67 shows in the left panel the proliferation of
hepatocytes (quantification of Ki67-positive cells) in XG-102 (in
the figure referred to as "D-JNKI1") or PBS treated Mapk14.sup.f/f
and Mapk14.sup..DELTA.Ii* mice. In PBS conditions (control),
Mapk14.sup.-/- cells (Mapk14.sup..DELTA.Ii*) are proliferating more
intensively than Mapk14.sup.+/+ cells (Mapk14.sup.f/f), since the
negative regulation of Mapk14 (p38) on cell proliferation and
tumorigenesis is not present. Administration of XG-102 reverts this
"non-activity" of Mapk14 (in Mapk14.sup.-/- cells) by the activity
of XG-102 (DJNKI1).
[0873] In the right panel of FIG. 67 the proliferation of
hepatocytes (quantification of Ki67-positive cells) in XG-102 (in
the figure referred to as "D-JNKI1") treated Mapk14.sup.f/f
Jun.sup.f/f (meaning Mapk14.sup.+/+ Jun.sup.+/+) and
Mapk14.sup..DELTA.Ii* Jun.sup..DELTA.Ii* mice (meaning
Mapk14.sup.-/- Jun.sup.-/-) is shown. The results are equivalent
and mimic those of Mapk14.sup.f/f in PBS condition and
Mapk14.sup..DELTA.Ii* in XG-102 (DJNKI1) condition. Thus, XG-102
activity is "equivalent" to deleting the Jun gene in the cell
line.
[0874] Taken together, these results are confirming that XG-102 has
an activity on the growth of cancer cell lines (reverting the
overgrowth induced by Mapk14 deletion) and this is probably
mediated by Jun.
Example 29: Effects of XG-102 on In Vivo Human Liver Cancer Cells
(Implanted)
[0875] To study the effect of XG-102 on in vivo human liver cancer
cells, 3.times.10.sup.6 Huh7 human liver cancer cells were injected
subcutaneously to both flank area of nude mice at 4 weeks of age.
Nude mice treated with XG-102 intraperitoneally twice a week at 5
mg/kg after Huh7 injection. Tumor volumes were measured twice a
week. Mice were killed 4 week after xenograft.
[0876] As shown in FIG. 68, XG-102 administered intraperitoneally
twice weekly after subcutaneous injection of human hepatocellular
carnimoma in nude mice markedly reduced tumor growth at a dose of 5
mg/kg.
Example 30: Antitumor Activity of 1 mg/kg XG-102 in Swiss Nude Mice
Bearing Orthotopic HEP G2 Human Liver Carcinoma
[0877] The objective of this study was to determine the antitumor
activity of 1 mg/kg XG-102 in the model of SWISS Nude mice bearing
the orthotopic Hep G2 human hepatocarcinoma tumor.
[0878] 20 healthy female SWISS Nude mice were obtained from Charles
River (L'Arbresles, France).
[0879] Animal experiments were performed according to the European
ethical guidelines of animal experimentation and the English
guidelines for welfare of animals in experimental neoplasia. The
animals were maintained in rooms under controlled conditions of
temperature (23.+-.2.degree. C.), humidity (45.+-.5%), photoperiod
(12 h light/12 h dark) and air exchange. Animals were maintained in
SPF conditions and room temperature and humidity was continuously
monitored. The air handling system was programmed for 14 air
changes per hour, with no recirculation. Fresh outside air pass
through a series of filters, before being diffused evenly into each
room. A high pressure (20.+-.4 Pa) was maintained in the
experimentation room to prevent contamination or the spread of
pathogens within a mouse colony. All personnel working under SPF
conditions followed specific guidelines regarding hygiene and
clothing when they entered the animal husbandry area. Animals were
housed in polycarbonate cages (UAR, Epinay sur Orge, France) that
are equipped to provide food and water. The standard area cages
used were 800 cm2 with a maximum of 10 mice per cage according to
internal standard operating procedures. Bedding for animals was
sterile wood shavings (SERLAB, Cergy-Pontoise, France), replaced
once a week. Animal food was purchased from SERLAB (Cergy-Pontoise,
France). The type of sterile controlled granules was DIETEX. The
food was provided ad libitum, being placed in the metal lid on top
of the cage. Water was also provided ad libitum from water bottles
equipped with rubber stoppers and sipper tubes. Water bottles was
cleaned, filled with water, sterilized by filtration and replaced
twice a week.
[0880] For XG-102 administration a stock solution was prepared at
10 mM (corresponding to 38.22 mg/ml) in sterile water (WFI,
Aguettant). Aliquots were prepared for each treatment day and
stored at approximately -80.degree. C. Dilutions with WFI of this
stock solution to 0.2 mg/ml was performed on each treatment day and
stored at 2-4.degree. C. for maximum 24 hours. The stability of the
stock solution is more than 100 days at approximately -80.degree.
C.; the stability of the diluted formulations for animal dosing is
24 hours at 2-4.degree. C. Diluted formulations were maintained on
ice until use and unused diluted material was discarded. The
treatment dose of XG-102 was injected at 1 mg/kg/inj. Injections
were performed at days D10, D14, D18, D22, D41, D45, D49 and D53
([Q4D.times.4].times.2). XG-102 substances were injected
intravenously (IV) at 5 ml/kg via the caudal vein of mice. The
injection volumes were adapted according to the most recent
individual body weight of mice.
[0881] The tumor cell line and culture media were purchased and
provided by Oncodesign:
TABLE-US-00034 Cell line Type Specie Origin Reference Hep G2 Human
hepatocarcinoma human ATCC* 4 *American Type Culture Collection,
Manassas, Virginia, USA.
[0882] The Hep G2 cell line was established from the tumor tissue
of a 15-year old Argentine boy with a hepatocellular carcinoma in
1975 (ADEN D. P. et al., Nature, 282: 615-616, 1979). Tumor cells
grew as adherent monolayers at 37.degree. C. in a humidified
atmosphere (5% CO2, 95% air). The culture medium was RPMI 1640
containing 2 mM L-glutamine (Ref BE12-702F, Lonza, Verviers,
Belgium) and supplemented with 10% FBS (Ref DE14-801E, Lonza). For
experimental use, the cells were detached from the culture flask by
a 5-minute treatment with trypsin-versene (Ref 02-007E, Cambrex),
diluted in Hanks' medium without calcium or magnesium (Ref
BE10-543F, Cambrex) and neutralized by addition of complete culture
medium. Cells were counted in a hemocytometer and their viability
was assessed by 0.25% trypan blue exclusion. Mycoplasma detection
was performed using the MycoAlert.RTM. Mycoplasma Detection Kit
(Ref LT07--318, Lonza) in accordance with the manufacturer
instructions. The MycoAlert.RTM. Assay is a selective biochemical
test that exploits the activity of mycoplasmal enzymes. The viable
mycoplasma are lysed and the enzymes react with the MycoAlert.RTM.
substrate catalyzing the conversion of ADP to ATP. By measuring the
level of ATP in a sample both before and after the addition of the
MycoAlert.RTM. substrate a ratio can be obtained which is
indicative of the presence or absence of mycoplasma. The mycoplasma
test was assayed in duplicate from the culture supernatants of the
cell lines and compared to negative and positive controls
(MycoAlert.RTM. Assay Control Set Ref LT07-518, Lonza) (Internal
Standard Operating Procedure No TEC-007/002, data not shown but
archived).
[0883] Experimental Design:
[0884] Twenty four hours before tumor induction, 20 female SWISS
Nude mice were irradiated with a .gamma.-source (2.5 Gy, Co60,
INRA, Dijon, France). At D0, Hep G2 tumors were induced
orthotopically on 20 female SWISS Nude. Under anesthesia, the
animal abdomen was opened through a median incision under aseptic
conditions. Ten millions (10.sup.7) Hep G2 tumor cells suspended in
50 .mu.l of RPMI 1640 culture medium were injected in the
subcapsular area of the liver. The abdominal cavity was
subsequently closed in 2 layers with 5-0 sutures.
[0885] At D10, mice were randomized before treatment start
according to their body weight to form 2 groups of 10 mice. The
body weight of each group was not statistically different from the
others (analysis of variance). Mice from group 1 received one IV
injection of vehicle at 5 ml/kg/inj. at D10, D14, D18, D22, D41,
D45, D49 and D53 ([Q4D.times.4].times.2) and mice from group 2
received one IV injection of XG-102 at 1 mg/kg/inj. at D10, D14,
D18, D22, D41, D45, D49 and D53 ([Q4D.times.4].times.2):
TABLE-US-00035 Group No. Treatment Dose Route Treatment 1 8 vehicle
-- IV [Q4Dx4]x2 2 7 XG-102 1 IV [Q4Dx4]x2
[0886] Surviving mice were sacrificed at D185.
[0887] Mice were monitored every day throughout the study for
behaviour and survival. The body weight was monitored twice a week
for all mice throughout the study. Isoflurane.RTM. Forene
(Centravet, Bondoufle, France) was used to anaesthetize the animals
before cell injection, IV treatments and sacrifice. During the
course of the experiment, animals were killed under anaesthesia
with Isoflurane.RTM. by cervical dislocation if any of the
following occurred: [0888] Signs of suffering (cachexia, weakening,
difficulty to move or to eat), [0889] Compound toxicity (hunching,
convulsions), [0890] 20% weight loss for 3 consecutive days or 25%
body weight loss on any day.
[0891] An autopsy was performed in each case. When mice looked
moribund, they were sacrificed and necropsied. Livers were
collected and weighed.
[0892] For the body weight analysis body weight curves of mice were
drawn. Mean body weight change (MBWC): Average weight change of
treated animals in grams (weight at day X minus weight at D10) was
calculated.
[0893] Efficacy parameters were expressed as a percent (T/C %). T
will be the median survival times of animals treated with drugs and
C is the median survival times of control animals treated with
vehicle. Survival systems indicated a degree of success when T/C
percents exceed 125. T/C % was expressed as follows: T/C
%=[T/C].times.100. Survival curves of mice were drawn. Mean
survival time was calculated for each group of treatment as the
mean of the days of death. Median survival time was calculated for
each group of treatment as the median of the days of death. The
log-Rank (Kaplan-Meier) test was used to compare the survival
curves. Statistical analysis of the body weight and MBWC was
performed using the Bonferroni/Dunn test (ANOVA comparison) using
StatView.RTM. software (Abacus Concept, Berkeley, USA). A p value
<0.05 is considered significant. All groups were compared with
themselves.
[0894] In FIG. 69 the mean body weight and mean body weight change
curves of mice bearing orthotopically injected HEP G2 tumor are
shown. Mice were IV treated with XG-102 at 1 mg/kg/inj following
the Q4D.times.4 treatment schedule repeated two times, at D10 and
D41. As shown in FIG. 69, no apparent differences occurred for the
body weight, indicating that XG-102 was well-tolerated.
Accordingly, in FIG. 70 the respective statistical data are
presented.
[0895] FIG. 71 shows the mice long survival curves, whereby
proportion of surviving mice per group until sacrifice day (D185)
is depicted. Mice were treated with XG-102 at the indicated doses
following the Q4D.times.4 treatment schedule repeated two times, at
D10 and D41. These data clearly show a prolonged survival for mice
treated with XG-102. Accordingly, the statistical data are
presented below (survival analysis of mice xenografted with HepG2
tumor and treated with XG-102):
TABLE-US-00036 Treatment (D10 Median survival Mean survival time
&D41, Q4Dx4) time .+-. SD (day) (day) T/C (%) Vehicle 102 .+-.
8 102 -- XG-102 1 mg/kg 111 .+-. 14 123 120 Group Chi df p
significance XG-102 1 mg/kg 5.1550 1 0.0232 S
[0896] Mice survival time was expressed as median survival time as
T/C (%) values (the ratio between the median of the days of death
of treated group and the tumor bearing untreated control group).
Statistical analysis was performed with the Log-Rank test, taking
vehicle treated group as reference.
[0897] Taken together, these data indicate that administration of
XG-102 prolongs the survival time of mice xenografted with HepG2
tumor.
Example 31: Antitumor Activity of XG-102 (Dose/Response) in Swiss
Nude Mice Bearing Orthotopic HEP G2 Human Liver Carcinoma
[0898] The objective of this study was to determine the antitumor
activity of XG-102 (dose/response) in the model of SWISS Nude mice
bearing the orthotopic Hep G2 human hepatocarcinoma tumor.
[0899] 32 healthy female SWISS Nude mice were obtained from Charles
River (L'Arbresles, France). Animal experiments were performed
according to the European ethical guidelines of animal
experimentation and the English guidelines for welfare of animals
in experimental neoplasia. The animals were maintained in rooms
under controlled conditions of temperature (23.+-.2.degree. C.),
humidity (45.+-.5%), photoperiod (12 h light/12 h dark) and air
exchange. Animals were maintained in SPF conditions and room
temperature and humidity was continuously monitored. The air
handling system was programmed for 14 air changes per hour, with no
recirculation. Fresh outside air pass through a series of filters,
before being diffused evenly into each room. A high pressure
(20.+-.4 Pa) was maintained in the experimentation room to prevent
contamination or the spread of pathogens within a mouse colony. All
personnel working under SPF conditions followed specific guidelines
regarding hygiene and clothing when they entered the animal
husbandry area. Animals were housed in polycarbonate cages (UAR,
Epinay sur Orge, France) that are equipped to provide food and
water. The standard area cages used were 800 cm2 with a maximum of
10 mice per cage according to internal standard operating
procedures. Bedding for animals was sterile wood shavings (SERLAB,
Cergy-Pontoise, France), replaced once a week. Animal food was
purchased from SERLAB (Cergy-Pontoise, France). The type of sterile
controlled granules was DIETEX. The food was provided ad libitum,
being placed in the metal lid on top of the cage. Water was also
provided ad libitum from water bottles equipped with rubber
stoppers and sipper tubes. Water bottles was cleaned, filled with
water, sterilized by filtration and replaced twice a week.
[0900] For XG-102 administration XG-102 was prepared at the
concentration of 1 mg/ml with sterile water (WFI, Aguettant,
France). Lt was then diluted at the concentrations of 0.2 and 0.02
mg/ml with sterile water. All these steps were performed within one
hour prior to injection to mice. XG-102 was injected at 0.1, 1 and
5 mg/kg/inj. Four injections were performed, each separated by four
days (Q4D.times.4). XG-102 substances were injected intravenously
(IV) at 5 ml/kg via the caudal vein of mice. The injection volumes
were adapted according to the most recent individual body weight of
mice.
[0901] The tumor cell line and culture media were purchased and
provided by Oncodesign:
TABLE-US-00037 Cell line Type Specie Origin Reference Hep G2 Human
hepatocarcinoma human ATCC* 4 *American Type Culture Collection,
Manassas, Virginia, USA.
[0902] The Hep G2 cell line was established from the tumor tissue
of a 15-year old Argentine boy with a hepatocellular carcinoma in
1975 (ADEN D. P. et al., Nature, 282: 615-616, 1979). Tumor cells
grew as adherent monolayers at 37.degree. C. in a humidified
atmosphere (5% CO2, 95% air). The culture medium was RPMI 1640
containing 2 mM L-glutamine (Ref BE12-702F, Lonza, Verviers,
Belgium) and supplemented with 10% FBS (Ref DE14-801E, Lonza). For
experimental use, the cells were detached from the culture flask by
a 5-minute treatment with trypsin-versene (Ref 02-007E, Cambrex),
diluted in Hanks' medium without calcium or magnesium (Ref
BE10-543F, Cambrex) and neutralized by addition of complete culture
medium. Cells were counted in a hemocytometer and their viability
was assessed by 0.25% trypan blue exclusion. Mycoplasma detection
was performed using the MycoAlert.RTM. Mycoplasma Detection Kit
(Ref LT07--318, Lonza) in accordance with the manufacturer
instructions. The MycoAlert.RTM. Assay is a selective biochemical
test that exploits the activity of mycoplasmal enzymes. The viable
mycoplasma are lysed and the enzymes react with the MycoAlert.RTM.
substrate catalyzing the conversion of ADP to ATP. By measuring the
level of ATP in a sample both before and after the addition of the
MycoAlert.RTM. substrate a ratio can be obtained which is
indicative of the presence or absence of mycoplasma. The mycoplasma
test was assayed in duplicate from the culture supernatants of the
cell lines and compared to negative and positive controls
(MycoAlert.RTM. Assay Control Set Ref LT07-518, Lonza) (Internal
Standard Operating Procedure No TEC-007/002).
[0903] Experimental Design:
[0904] Twenty four hours before tumor induction, 32 female SWISS
Nude mice were irradiated with a .gamma.-source (2.5 Gy, Co.sup.60,
INRA, Dijon, France). At D0, Hep G2 tumors were induced
orthotopically on 32 female SWISS Nude. Under anesthesia, the
animal abdomen was opened through a median incision under aseptic
conditions. Ten millions (10.sup.7) Hep G2 tumor cells suspended in
50 .mu.l of RPMI 1640 culture medium were injected in the
subcapsular area of the liver. The abdominal cavity was
subsequently closed in 2 layers with 5-0 sutures.
[0905] At D10, mice were randomized before treatment start
according to their body weight to form 4 groups of 8 mice. The body
weight of each group was not statistically different from the
others (analysis of variance). Mice from group 1 received one IV
injection of vehicle at 5 ml/kg/inj. once every four days repeated
four times (Q4D.times.4), mice from group 2 received one IV
injection of XG-102 at 0.1 mg/kg/inj. once every four days repeated
four times (Q4D.times.4), mice from group 3 received one IV
injection of XG-102 at 1 mg/kg/inj. once every four days repeated
four times (Q4D.times.4), and mice from group 4 received one IV
injection of XG-102 at 5 mg/kg/inj. once every four days repeated
four times (Q4D.times.4):
TABLE-US-00038 Group No. Treatment Dose Route Treatment 1 8 vehicle
-- IV Q4Dx4 2 8 XG-102 0.1 IV Q4Dx4 3 8 XG-102 1 IV Q4Dx4 4 8
XG-102 5 IV Q4Dx4
[0906] Surviving mice were sacrificed at D171.
[0907] Mice were monitored every day throughout the study for
behaviour and survival. The body weight was monitored twice a week
for all mice throughout the study. Isoflurane.RTM. Forene
(Centravet, Bondoufle, France) was used to anaesthetize the animals
before cell injection, IV treatments and sacrifice. During the
course of the experiment, animals were killed under anaesthesia
with Isoflurane.RTM. by cervical dislocation if any of the
following occurred: [0908] Signs of suffering (cachexia, weakening,
difficulty to move or to eat), [0909] Compound toxicity (hunching,
convulsions), [0910] 20% weight loss for 3 consecutive days or 25%
body weight loss on any day.
[0911] An autopsy was performed in each case.
[0912] At D67, 3 mice randomly selected per group during
randomization were sacrificed for observation of macroscopic
development. The remaining mice in each group were kept for
survival monitoring. Final sacrifice was performed at D171. Primary
tumors and livers were collected and weighed from sacrificed
animals. Each liver was fixed in 10% neutral buffered fonnalin.
Forty eight (48) hours after collection, they were embedded in
paraffin (Histosec.RTM.) and used for anapathological analysis. For
the estimation of metastatic invasion in mouse liver by
histological analysis, paraffin-embedded sections (5 .mu.m) were
deparaffinized in xylene and rehydrated by serial incubations in
100%, 95%, and 70% ethanol. All sections were stained with
haematoxylin and eosin (HE) (Ref. S3309, Dakocytomation, Trappes,
France) for histological analyses. The coverslip was mounted with
aqueous mountant (Aquatex, Ref 1.08562, Merck) and sections were
viewed under a light microscope (DMRB Leica). Histological sections
were analyzed by a pathologist expert to determine the metastatic
invasion in liver.
[0913] For the body weight analysis body weight curves of mice were
drawn. Mean body weight change (MBWC): Average weight change of
treated animals in grams (weight at day X minus weight at D10) was
calculated.
[0914] Efficacy parameters were expressed as a percent (T/C %). T
will be the median survival times of animals treated with drugs and
C is the median survival times of control animals treated with
vehicle. Survival systems indicated a degree of success when T/C
percents exceed 125. T/C % was expressed as follows: T/C
%=[T/C].times.100. Survival curves of mice were drawn. Mean
survival time was calculated for each group of treatment as the
mean of the days of death. Median survival time was calculated for
each group of treatment as the median of the days of death. The
log-Rank (Kaplan-Meier) test was used to compare the survival
curves. Statistical analysis of the body weight and MBWC was
performed using the Bonferroni/Dunn test (ANOVA comparison) using
StatView.RTM. software (Abacus Concept, Berkeley, USA). A p value
<0.05 is considered significant. All groups were compared with
themselves.
[0915] FIG. 72 shows the statistical data regarding the mean body
weight and mean body weight change curves of mice bearing
orthotopically injected HEP G2 tumor. Mice were IV treated with
XG-102 following the Q4D.times.4 treatment schedule repeated two
times, at D10 and D41. As shown in FIG. 72, no apparent differences
occurred for the body weight, indicating that XG-102 was
well-tolerated.
[0916] FIG. 73 shows the mice long survival curves, whereby
proportion of surviving mice per group until sacrifice day (D171)
is depicted. Mice sacrificed at D67 for autopsy were excluded from
calculation. Mice were treated with XG-102 at the indicated doses
following the Q4D.times.4 treatment schedule repeated two timed, at
D10 and D41. These data clearly show a prolonged survival for mice
treated with XG-102 in a dose-dependent manner. Accordingly, the
statistical data are presented below (survival analysis of mice
xenografted with HepG2 tumor and treated with XG-102):
TABLE-US-00039 Treatment (D10 Median survival &D41, Q4Dx4) time
(day) T/C (%) Statistical analysis Vehicle 86 -- -- XG-102 0.1
mg/kg 105 123 NS XG-102 1 mg/kg 138 161 NS XG-102 5 mg/kg 118 137
NS
[0917] Mice sacrificed as D67 for autopsy were excluded from
calculation. Mice survival time was expressed as median survival
time as T/C (%) values (the ratio between the median of the days of
death of treated group and the tumor bearing untreated control
group). A T/C % value >125% is indicative of anti-tumor
effectiveness.
[0918] The following table shows the tumor development of HepG2
cancer cells into liver. Detection of tumor masses on liver was
performed by microscopic observation after HE staining on mice
sacrificed at D171:
TABLE-US-00040 Group Animal ID Observation Vehicle 933 Tumor on
liver XG-102 0.1 mg/kg 8665 Tumor (1.3 cm) on liver 2925 No tumor
detected XG-102 1 mg/kg 8631 No tumor detected 8641 No tumor
detected 2929 Tumor (1.9 cm) on liver XG-102 5 mg/kg 2931 No tumor
detected 2765 No tumor detected 2767 No tumor detected
[0919] In FIG. 74 the tumor invasion observed by microscopic
evaluation of mice sacrificed at D67 or between D67 and final
sacrifice are shown as histogram representations. The level of
tumor take was classified in 4 different categories specified in
the figure legend.
Example 32: Antitumor Activity of XG-102 in Balb/c Nude Mice
Bearing Subcutaneous PC-3 Human Prostate Tumors
[0920] The objective of this study was to determine the antitumor
activity of XG-102 (dose/response) in the model of Balb/c Nude mice
bearing the subcutaneous PC-3 human prostate tumors.
[0921] 15 healthy male Balb/c Nude mice were obtained from Charles
River (L'Arbresles, France). Animal experiments were performed
according to the European ethical guidelines of animal
experimentation and the English guidelines for welfare of animals
in experimental neoplasia. The animals were maintained in rooms
under controlled conditions of temperature (23.+-.2.degree. C.),
humidity (45.+-.5%), photoperiod (12 h light/12 h dark) and air
exchange. Animals were maintained in SPF conditions and room
temperature and humidity was continuously monitored. The air
handling system was programmed for 14 air changes per hour, with no
recirculation. Fresh outside air pass through a series of filters,
before being diffused evenly into each room. A high pressure
(20.+-.4 Pa) was maintained in the experimentation room to prevent
contamination or the spread of pathogens within a mouse colony. All
personnel working under SPF conditions followed specific guidelines
regarding hygiene and clothing when they entered the animal
husbandry area. Animals were housed in polycarbonate cages (UAR,
Epinay sur Orge, France) that are equipped to provide food and
water. The standard area cages used were 800 cm2 with a maximum of
10 mice per cage according to internal standard operating
procedures. Bedding for animals was sterile wood shavings (SERLAB,
Cergy-Pontoise, France), replaced once a week. Animal food was
purchased from SERLAB (Cergy-Pontoise, France). The type of sterile
controlled granules was DIETEX. The food was provided ad libitum,
being placed in the metal lid on top of the cage. Water was also
provided ad libitum from water bottles equipped with rubber
stoppers and sipper tubes. Water bottles was cleaned, filled with
water, sterilized by filtration and replaced twice a week.
[0922] For XG-102 administration XG-102 was prepared at the
concentration of 0.2 mg/ml with sterile water (WFI, Aguettant,
France). It was then diluted to the concentration of 0.02 mg/ml
with sterile water. All these steps were performed within one hour
prior to injection to mice. XG-102 was injected at 0.1 and 1
mg/kg/inj. Four injections were performed, each separated by four
days (Q4D.times.4). XG-102 substances were injected intravenously
(IV) at 5 ml/kg via the caudal vein of mice. In case of necrosis of
the tail during the injection period, the intraperitoneal (IP)
route was used. The injection volumes were adapted according to the
most recent individual body weight of mice.
[0923] The tumor cell line and culture media were purchased and
provided by Oncodesign:
TABLE-US-00041 Cell line Origin Source Reference PC-3 Human
prostatic ATCC* BISSERY M. C. et al., Bull. adenocarcinoma Cancer
1991, 78: 587-602. *American Type Culture Collection, Manassas,
Virginia, USA.
[0924] The PC-3 was initiated from a bone metastasis of a grade IV
prostatic adenocarcinoma from a 62-year old male Caucasian (VOLENEC
F. J. et al., J Surg Oncol 1980; 13(1):39-44). Tumor cells grew as
adherent monolayers at 37.degree. C. in a humidified atmosphere (5%
CO2, 95% air). The culture medium was RPMI 1640 containing 2 mM
L-glutamine (Ref BE12-702F, Lonza, Verviers, Belgium) and
supplemented with 10% FBS (Ref DE14-801E, Lonza). For experimental
use, the cells were detached from the culture flask by a 5-minute
treatment with trypsin-versene (Ref 02-007E, Cambrex), diluted in
Hanks' medium without calcium or magnesium (Ref BE10-543F, Cambrex)
and neutralized by addition of complete culture medium. Cells were
counted in a hemocytometer and their viability was assessed by
0.25% trypan blue exclusion. Mycoplasma detection was performed
using the MycoAlert.RTM. Mycoplasma Detection Kit (Ref LT07--318,
Lonza) in accordance with the manufacturer instructions. The
MycoAlert.RTM. Assay is a selective biochemical test that exploits
the activity of mycoplasmal enzymes. The viable mycoplasma are
lysed and the enzymes react with the MycoAlert.RTM. substrate
catalyzing the conversion of ADP to ATP. By measuring the level of
ATP in a sample both before and after the addition of the
MycoAlert.RTM. substrate a ratio can be obtained which is
indicative of the presence or absence of mycoplasma. The mycoplasma
test was assayed in duplicate from the culture supernatants of the
cell lines and compared to negative and positive controls
(MycoAlert.RTM. Assay Control Set Ref LT07-518, Lonza) (Internal
Standard Operating Procedure No TEC-007/002).
[0925] Experimental Design:
[0926] Forty-eight hours before tumor induction, 15 male Balb/c
Nude mice were irradiated with a .gamma.-source (2.5 Gy, Co.sup.60,
INRA, Dijon, France). At D0, twenty millions (2.times.10.sup.7)
PC-3 cells suspended in 200 .mu.l of RPMI medium were
subcutaneously injected in the right flank of the 60 male Balb/c
Nude mice.
[0927] When the mean tumor volume reached 80.+-.38 mm.sup.3, mice
were randomized before treatment start according to their tumor
volume to form 3 groups of 5 mice. The tumor volume of each group
was not statistically different from the others (analysis of
variance).
[0928] The treatment schedule of the test substance was as follows:
Mice from group 1 received one IV injection of vehicle at 5
ml/kg/inj. once every four days repeated four times (Q4D.times.4),
Mice from group 2 received one IV injection of XG-102 at 0.1
mg/kg/inj. once every four days repeated four times (Q4D.times.4),
and Mice from group 3 received one IV injection of XG-102 at 1
mg/kg/inj. once every four days repeated four times
(Q4D.times.4):
TABLE-US-00042 Group No. Treatment Dose Route Treatment 1 5 vehicle
-- IV Q4Dx4 2 5 XG-102 0.1 IV Q4Dx4 3 5 XG-102 1 IV Q4Dx4
[0929] Mice were sacrificed when tumors reached a maximum volume of
2000 mm.sup.3.
[0930] Mice were monitored every day throughout the study for
behaviour and survival. The body weight and tumor volume was
monitored twice a week for all mice throughout the study.
Isoflurane.RTM. Forene (Centravet, Bondoufle, France) was used to
anaesthetize the animals before cell injection, IV treatments and
sacrifice. During the course of the experiment, animals were killed
under anaesthesia with Isoflurane.RTM. by cervical dislocation if
any of the following occurred: [0931] Signs of suffering (cachexia,
weakening, difficulty to move or to eat), [0932] Compound toxicity
(hunching, convulsions), [0933] 20% weight loss for 3 consecutive
days or 25% body weight loss on any day, [0934] Tumor volume of
more than 2000 mm.sup.3.
[0935] An autopsy was performed in each case.
[0936] For the body weight analysis body weight curves of mice were
drawn. Curves were stopped when more than 40% of dead mice were
recorded in at least one group. Mean body weight change (MBWC):
Average weight change of treated animals in grams (weight at day X
minus weight at D33) was calculated.
[0937] The tumor volume was calculated with the following formula
where length corresponds to the largest tumor diameter and width to
the smallest tumor diameter: TV=(length.times.width.sup.2)/2. Tumor
growth curves were drawn using the mean tumor volumes (MTV)+/-SD.
Curves were stopped when more than 40% of mice were dead.
Individual tumor volume curves were also drawn. Relative tumor
volume curve using the relative tumor volumes (RTV) at different
time points calculated as shown below were drawn. Curves were
stopped when more than 40% of mice were dead. The RTV was
calculated following the formula:
RTV=(Tumor volume at DX)/(Tumor volume at D33).times.100
[0938] Tumor doubling time (DT) defined as the period required to
reach a MTV of 200% during the exponential tumor growth phase was
calculated using Vivo Manager.RTM. software. Time to reach V was
calculated. Volume V was defined as a target volume deduced from
experimental data and chosen in the exponential phase of tumor
growth. Volume V was chosen as close as possible for all mice of
each group, the time to reach this Volume V was deduced from
experimental data. Tumor growth inhibition (T/C %) defined as the
ratio of the median tumor volumes of treated groups versus vehicle
treated group was calculated. The effective criteria for the T/C %
ratio according to NCl standards, is .about.42% (BISSERY M. C. et
al., Bull. Cancer 1991, 78: 587-602). All statistical analyses were
performed using Vivo Manager.RTM. software. Statistical analysis of
the toxicity and the efficiency of the treatment (BWC, MBWC, TV,
RTV, TTRV and DT) was performed using the Bonferroni/Dunn test
(ANOVA comparison). All groups were compared with each other.
[0939] In FIG. 75 shows the mean tumor volume of PC-3 tumor bearing
mice during the antitumor activity experiment. At D33, 3 groups of
5 animals were treated with vehicle and XG-102 (0.1 and 1
mg/kg/inj, Q4D.times.4). These data indicate a reduction of tumor
volume over time for XG-102 treatment in a dose-dependent manner,
whereby the effects were more prominent for 1 mg/kg XG-102.
Example 33: Effects of XG-102 on Tumor Growth in SCID Mice Bearing
Orthotopic HCT 116 Human Colon Tumors
[0940] The objective of this study was to determine the effect of
XG-102 on the growth of HCT 116 human colon tumor orthotopically
xenografted in SCID mice.
[0941] 80 healthy female SCID mice were obtained from Charles River
(L'Arbresles, France). Animal experiments were performed according
to the European ethical guidelines of animal experimentation and
the English guidelines for welfare of animals in experimental
neoplasia. The animals were maintained in rooms under controlled
conditions of temperature (23.+-.2.degree. C.), humidity
(45.+-.5%), photoperiod (12 h light/12 h dark) and air exchange.
Animals were maintained in SPF conditions and room temperature and
humidity was continuously monitored. The air handling system was
programmed for 14 air changes per hour, with no recirculation.
Fresh outside air pass through a series of filters, before being
diffused evenly into each room. A high pressure (20.+-.4 Pa) was
maintained in the experimentation room to prevent contamination or
the spread of pathogens within a mouse colony. All personnel
working under SPF conditions followed specific guidelines regarding
hygiene and clothing when they entered the animal husbandry area.
Animals were housed in polycarbonate cages (UAR, Epinay sur Orge,
France) that are equipped to provide food and water. The standard
area cages used were 800 cm2 with a maximum of 10 mice per cage
according to internal standard operating procedures. Bedding for
animals was sterile corn cob bedding (LAB COB 12, SERLAB,
CergyMPontoise, France), replaced once a week. Animal food was
purchased from DIETEX. The type of sterile controlled granules was
DIETEX. The food was provided ad libitum, being placed in the metal
lid on top of the cage. Water was also provided ad libitum from
water bottles equipped with rubber stoppers and sipper tubes. Water
bottles was cleaned, filled with water, sterilized by filtration
and replaced twice a week.
[0942] For XG-102 administration the required amount of XG-102 was
dissolved in the vehicle. The formulation was prepared according to
the procedure detailed below. Concentrations were calculated and
expressed taking into account test item purity and peptide content
(multiplier coefficient was 74.6%). After thawing of XG-102, a
stock solution was prepared at 10 mM (corresponding to 38.22 mg/ml)
in sterile water (Wfi, Batch 500 111 00 J, Aguettant, France) and
allowed to equilibrate to room temperature for 20 minutes minimum.
Aliquots were prepared for each treatment day and stored at
approximately -80.degree. C. Dilutions of this stock solution to
the required concentrations were performed on each treatment day
and stored at 2-4.degree. C. for maximum 24 hours. The period of
stability of the stock solution is more than 100 days at
approximately -80.degree. C. The period of stability of the diluted
formulations for animal dosing is 24 hours at 2-4.degree. C.
Diluted solutions were maintained on ice until use. Unused material
was discarded. XG-102 was injected once daily at 0.1 and 1
mg/kg/inj. for a total of fourteen consecutive administrations
(Q1D.times.14). The routes of substance administrations were:
injected subcutaneously (SC) at 5 ml/kg/inj., administered per os
(PO) to mice by oral gavage via a canula at 5 ml/kg/adm. The
injection and administration volumes were adapted, according to the
daily individual body weight of mice.
[0943] The tumor cell line and culture media were purchased and
provided by Oncodesign:
TABLE-US-00043 Cell line Origin Source Reference HTC116 Human colon
ATCC* BRATTAIN M. G. et al., adenocarcinoma Cancer Res. 1981, 41:
1751M1756. *American Type Culture Collection, Manassas, Virginia,
USA.
[0944] The HCT 116 variant cell line was isolated from a primary
cell culture of a single colonic carcinoma of a male patient
(BRATTAIN M. G. et al., Cancer Res. 1981, 41:I751 M1756).
[0945] Tumor cells grew as adherent monolayers at 37.degree. C. in
a humidified atmosphere (5% CO2, 95% air). The culture medium was
RPMI 1640 containing 2 mM L-glutamine (Ref BE12-702F, Lonza,
Verviers, Belgium) and supplemented with 10% FBS (Ref DE14-801E,
Lonza). For experimental use, the cells were detached from the
culture flask by a 5-minute treatment with trypsin-versene (Ref
02-007E, Cambrex), diluted in Hanks' medium without calcium or
magnesium (Ref BE10-543F, Cambrex) and neutralized by addition of
complete culture medium. Cells were counted in a hemocytometer and
their viability was assessed by 0.25% trypan blue exclusion.
Mycoplasma detection was performed using the MycoAlert.RTM.
Mycoplasma Detection Kit (Ref LT07--318, Lonza) in accordance with
the manufacturer instructions. The MycoAlert.RTM. Assay is a
selective biochemical test that exploits the activity of
mycoplasmal enzymes. The viable mycoplasma are lysed and the
enzymes react with the MycoAlert.RTM. substrate catalyzing the
conversion of ADP to ATP. By measuring the level of ATP in a sample
both before and after the addition of the MycoAlert.RTM. substrate
a ratio can be obtained which is indicative of the presence or
absence of mycoplasma. The mycoplasma test was assayed in duplicate
from the culture supernatants of the cell lines and compared to
negative and positive controls (MycoAlert.RTM. Assay Control Set
Ref LT07-518, Lonza) (Internal Standard Operating Procedure No
TEC-007/002).
[0946] Experimental Design:
[0947] Twenty four to Forty-eight hours before tumor induction, 5
SCID mice were irradiated with a .gamma.-source (1.8 Gy, Co.sup.6,
INRA, Dijon, France). Ten millions (10.sup.7) HCT 116 cells
suspended in 200 .mu.l of RPMI medium were subcutaneously injected
in the right flank of the 5 female SCJD mice. When tumors reached
1000-2000 mm.sup.3, mice were sacrificed. Tumors were surgically
excised from the animal to obtain fresh tumor fragments (20-30 mg)
to be orthotopically implanted on the caecum of 75 mice at D0.
[0948] Twenty four to forty-eight hours before tumor implantation,
75 SCID mice were irradiated with a .gamma.-source (1.8 Gy,
Co.sup.60, INRA, Dijon, France). The surgery was performed in the
afternoon, with a minimum delay of two hours after the 7th XG-102
treatment. The abdomen from anaesthetized animal was opened through
a median incision under aseptic conditions. The caecum was
exteriorized and a small lesion was performed on caecum wall. The
tumor fragment was placed on lesion and fixed with 6/0 sutures. The
abdominal cavity was subsequently closed in 2 layers with 4/0
sutures.
[0949] At D-7, mice were randomized according to their body weight
before treatment start to form 5 groups of 15 mice. The body weight
of each group was not statistically different from the others
(analysis of variance). The treatment began at D-7 according to
following treatment schedule: [0950] Mice from group 1 received one
PO administration of XG-102 vehicle at 5 ml/kg/inj. once daily for
a total of fourteen consecutive administrations (Q 1D.times.14),
[0951] Mice from group 2 received one PO administration of XG-102
at 0.1 mg/kg/inj. once daily for a total of fourteen consecutive
administrations (QI D.times.14), [0952] Mice from group 3 received
one PO administration of XG-102 at 1 mg/kg/inj. Once daily for a
total of fourteen consecutive administrations (Q1D.times.14),
[0953] Mice from group 4 received one SC injection of XG-102 at 0.1
mg//kg/inj. once daily for a total of fourteen successive
administrations (Q1D.times.14), [0954] Mice from group 5 received
one SC injection of XG-102 at 1 mg//kg/inj. once daily for a total
of fourteen consecutive administrations (Q1D.times.14):
TABLE-US-00044 [0954] Treatment Group No. Treatment Route Dose
(mg/kg/inj.) Schedule 1 15 vehicle po -- Q1Dx14 2 15 XG-102 po 0.1
Q1Dx14 3 15 XG-102 po 1 Q1Dx14 4 15 XG-102 sc 0.1 Q1Dx14 5 15
XG-102 sc 1 Q1Dx14
[0955] Mice were monitored every day throughout the study for
behaviour and survival. The body weight and tumor volume was
monitored twice a week for all mice throughout the study.
Isoflurane.RTM. Forene (Centravet, Bondoufle, France) was used to
anaesthetize the animals before cell injection, surgery (orthotopic
tumor implantation) and sacrifice.
[0956] During SC tumor amplification, the tumor volume was
monitored twice a week for all mice throughout the study.
[0957] Mice were sacrificed at D26. The liver and tumors were
collected and weighed for all animals. Invasion of liver by tumor
nodules was evaluated macroscopically. Livers and tumors were fixed
in 10% neutral buffered formalin. Forty eight (48) hours after
collection, they were embedded in paraffin (Histosec.RTM.) and used
for histology analysis. Two slides were issued from two different
parts into the core of each tumor. Each slide was identified by the
mouse identification number. One slide was issued per liver,
localized at its center. It was identified by the mouse
identification number. For determination of proliferating index by
Ki67 marker, paraffin-embedded sections (5 .mu.m) were
deparaffinized in xylene (Ref. 11699027, Labonord, Templemars,
France) and rehydrated by serial incubations in 100%, 95%, and 70%
ethanol (Ref. 13099500, Labonord). Endogenous peroxidase was
inhibited by incubating tissues in hydrogen peroxide containing
solution for 10 min at room temperature before addition of the
first antibody. A biotin blocking system was used to reduce
background. Sections were treated for 20 min with 3% bovine serum
albumin (BSA) in PBS (1.times.) completed with 3% goat serum at
room temperature to inhibit crossreactivity before addition of the
first antibody. Tissue sections were incubated for 1 hour at room
temperature with the mouse anti-human Ki-67 clone MIB-1 monoclonal
antibody (Ref M7240, Dako cytomation; 1:100 dilution, 80 .mu.g/ml).
A non-relevant biotinylated mouse IgGI antibody (Ref X0931, Dako
cytomation, 1:120 dilution, 100 .mu.g/ml) was used as a negative
control slide ensuring the specificity of reaction. The sections
were further incubated with the secondary goat anti-mouse antibody
(Ref. 89904, Sigma) coupled to biotin. Then, tissue sections were
incubated for 30 min at room temperature with the
avidin-biotin-peroxidase conjugate (Ref PK-6100, Vector
Laboratories, 1:50 dilution). DAB peroxydase substrate (Ref
SK-4100, Vector Laboratories) was used as a chromogen to visualize
the reaction. Sections were counterstained with Mayer's
haematoxylin for histological study. After each incubation,
sections were washed two times with 1.times.PBS. The coverslip was
mounted with aqueous mountant and sections were visualized under a
light microscope (DMRB Leica).
[0958] For detection of metastasis in mouse liver by histological
analysis, paraffin-embedded sections (5 .mu.m) were deparaffinized
in xylene and rehydrated by serial incubations in 100%, 95%, and
70% ethanoJ. All sections were stained with haematoxylin and eosin
(HE) (Ref. 83309, Dakocytomation, Trappes, France) for histological
analyses. The coverslip was mounted with aqueous mountant (Aquatex,
Ref 1.08562, Merck) and sections were viewed under a light
microscope (DMRB Leica). Histological sections were analyzed by an
experienced pathologist to determine the metastatic invasion in
liver.
[0959] For the body weight analysis body weight curves of mice were
drawn. Curves were stopped when more than 40% of dead mice were
recorded in at least one group. Mean body weight change (MBWC):
Average weight change of treated animals in grams (weight at day X
minus weight at D-7) was calculated.
[0960] Tumor weights were calculated. Tumor growth inhibition (T/C
%) was defined as the ratio of the median tumor weight of treated
groups versus vehicle treated group. The effective criteria for the
T/C % ratio according to NCl standards is .ltoreq.42%. For
semi-quantification of proliferating index (Ki-67 staining), the
numeric images of stained tumor sections were blindly analyzed and
classified as no staining (level 0 corresponding to none stained
area), low staining (level 1 corresponding to less than 10% of
stained area), moderate staining (level 2 corresponding to 10 to
30% of stained area) and strong staining (level 3 corresponding to
more than 30% of stained area). Representative pictures were taken.
For detection of metastasis in the liver mean liver weights were
measured, and the number of metastasis per liver was estimated on
entire liver macroscopically and on section by histological
analysis. Results were reported in a table. Representative pictures
were taken. All statistical analyses were performed using Vivo
Manager.RTM. software, Statistical analysis of the toxicity and the
efficiency of the treatment (MBWC, TV, Volume V and time to reach
V, DT) were performed using the Bonferroni/Dunn test (ANOVA
comparison). All groups were compared with each other.
[0961] Ten millions (10.sup.7) HCT 116 cells were SC injected in 5
irradiated female SCID mice. No mycoplasma was detected in cells
and their viability was 99% before injection. Thirty-nine days
after, when mean tumor volume was 864.+-.426 mm3, mice were
sacrificed. Their tumor was isolated and cut into pieces of
approximately 20.about.30 mg. These pieces were implanted at D0
onto the ceacum of 75 treated animals. From D0 to 09, surgery
complications due to tumor implantation induced death of 33% of
mice in vehicle treated group. In the treated groups, percentages
of death were 40%, 34%, 47% and 40%, with no dose related effect.
The fact that treatments with XG-102 did not significantly modify
lethality compared to vehicle treated group suggest that treatments
were tolerated by animals. Moreover, between the day of treatment
start (D-7) and two days before surgery (D-2), the six daily
treatments did not induce any significant body weight loss,
indicating again that XG-102 was well tolerated. At D-2, MBWC was
distributed between +5.2.+-.4.6% for vehicle treated group to
+7.1.+-.4.6% in the group PO treated at 0.1 mg/kg/adm. In addition,
after surgery, no MBWC difference was observed between the group
treated with vehicle and those treated with XG-102 at different
doses, even if a significant decrease caused by surgery was
observed for all groups, when comparing MBWC before and after
surgery.
[0962] The mean liver weights in mice sacrificed at D26 were
distributed between 0.82.+-.0.17 g in vehicle treated group and
0.91.+-.0.17 g in the group PO treated at 0.1 mg/kg. They were not
significantly different. In the vehicle treated group, 20% did not
develop any metastasis in liver. As shown in FIG. 76, this control
group was the one where the number of mice developing more than 1
metastasis in liver was the highest (40%). In the treated groups,
this score was distributed between 12.5% for the group PO treated
at 1 mg/kg to 25% for the groups PO treated at 0.1 mg/kg or SC
treated at 1 mg/kg. Remarkably, the group PO treated at 1 mg/kg had
the highest number of mice with no liver metastasis, suggesting
that XG-102 might decrease metastatic power of HCT 116 orthotopic
tumor.
Example 34: Evaluation of Efficacy of XG-102 in Reducing the
Photoreceptors Light Damage in Rat (AMD Model)
[0963] The aim of this study was to investigate the dose effect of
XG-102 on light-induced photoreceptor cell death.
[0964] 50 male Rat (Sprague-Dawley (albinos rat); approximately 8
weeks; 200-250 g (on ordering)) have been used. Rats are most
commonly used in this experimental model. Animals were examined
before study, and particular attention was paid to the eyes.
Animals were held in observation for 2 weeks following their
arrival. Animals were observed daily for signs of illness. Only
healthy animals with no ocular abnormalities were accepted for use
in experiments. Animals were housed individually in standard cages
(420.times.270.times.190 mm).sup.ii. All animals were housed under
identical environmental conditions. The temperature was held at
22.+-.2.degree. C. and the relative humidity at 55.+-.10%. Rooms
were continuously ventilated (15 times per hour). A cycle of 12
hours light (200-300 lx) and 12 hours darkness was automatically
controlled. These parameters were continuously controlled and
recorded. Throughout the study, animals had free access to food and
water. They were fed a standard dry pellet diet. Tap water was
available ad libitum from plastic bottles.
[0965] Study Design:
[0966] Forty-eight (48) rats were randomly divided into six (6)
groups of eight (8) animals each.
[0967] Test item (XG-102: 30 mg/ml, 3 mg/ml, and 0.3 mg/ml) and
vehicle (0.9% NaCl) were administered by intravitreal injection in
right eyes the day before induction. The reference
(Phenyl-N-test-Butylnitrone (PBN) (50 mg/kg)) and vehicle were
intraperitoneally injected 30 min before induction then, 3 times
during 12 hours of light exposition, then once after induction.
Animals were placed in constant light (7000 lux) for 24 h.
Electroretinograms (ERG) were recorded before light treatment and
on days 9, 16 and 23 after induction. Eyes were then taken for
histology and outer nuclear layer (ONL) thickness assessment. The
table below summarizes the allocation of animals in treatment
groups:
TABLE-US-00045 Route of Group administration Time of No. Treatment
Dose (volume) administration Animals Identification 1 XG-102 30 000
.mu.g/mL i.v.t. (5 .mu.l) Day before 13, 38, 9, 35, 2, 23, 25, 36
150 .mu.g/eye induction (D0) 2 3 000 .mu.g/mL 18, 28, 5, 27, 16,
12, 30, 1 15 .mu.g/eye 3 300 .mu.g/mL 3, 11, 8, 17, 31, 7, 22, 15
1.5 .mu.g/eye 4 Vehicle -- 6, 29, 24, 21, 40, 32, 14, 37 5 PBN 50
mg/kg i.p. 30 min before induction 4, 39, 19, 33, 10, 26, 34, 20 6
Vehicle -- (2.5 ml/kg) then 2 h, 4 h, 6 h 41, 42, 43, 44, 45, 46,
47, 48 (5 times) (during light exposure - D 1) and 24 h (at the
cessation of exposure - D 2) after induction
[0968] Forty-eight (48) animals out of fifty (50) were used in this
study. Only animals with no visible sign of ocular defect were
selected. Then, the randomization in the treatment groups was done
by a random function in Excel.RTM. software.
[0969] Route and Method of Administration
[0970] For the intravitreal injection animals were anesthetized by
intramuscular injection of a mixture of xylazine/ketamine. Test
item (5 .mu.l) and vehicle (5 .mu.l) were injected in the right
eye. The injection was performed under an operating microscope in
the supratemporal area at pars plana using a 33G-needle mounted on
a 50 .mu.l Hamilton. The filled syringe was mounted into the
UltraMicroPump III to achieve accurate injection in microliter
range. Reference and vehicle were injected intraperitoneally at a
dose volume of 2.5 ml/kg using a 30G-needle mounted on a 1
ml-syringe.
[0971] Light Exposure: The rats that had been dark-adapted
overnight were exposed for 24 hours to a continuous white
fluorescent light (7000 lx) in clear plastic cages. Each cage
contained one rat. After exposure, the rats returned to rearing
cyclic light conditions.
[0972] The body weight of all animals was recorded before the start
of the study then at the end of the study. Each day, the general
behavior and the aspect of all animals were observed. ERG was
recorded before induction and 7, 14 and 21 days after cessation of
exposure (Days 9, 16 and 23) on right eyes of dark-adapted and
anesthezied animals. The latency times (for a- and b-wave) and the
a-wave and b-wave amplitudes were measured for each ERG; the
latency times were expressed as millisecond and the a-wave and
b-wave as a percentage of the baseline value obtained before light
exposure. 15 min before measurement 10 .mu.l Mydriaticum.RTM. (0.5%
tropicamide) were instilled for pupillary dilatation.
[0973] ERG Parameters: [0974] Color: white maximum. [0975] Maximum
intensity: 2.6 cd.s/m.sup.2 (0 dB); Duration 0.24 ms; number of
flash: 1. [0976] Filter: 50 Hz. [0977] Impedence Threshold: 90
k.OMEGA..
[0978] Measurement of the ONL Thickness: After ERG testing, animal
was euthanized by an overdose of pentobarbital and the right eyes
were enucleated, fixed and embedded in paraffin. Sections (5 .mu.m
thick) were performed along the vertical meridian and stained with
Trichrome-Masson. The vertical meridian included the optic nerve.
ONL Thickness was done every 500 .mu.m (seven points) between 500
and 3500 .mu.m from the optic nerve in the inferior retina using a
standard microscope (Leica).
[0979] Results were expressed in the form of individual and
summarized data tables using Microsoft Excel.RTM. Software. Group
mean values and standard deviation were calculated. A statistical
Mann and Whitney test was used to evaluate the differences between
pair-wise groups. For comparison between time-point into each
vehicle groups, a Friedman test was used.
[0980] Results
[0981] General behavior and appearance were normal in all
animals.
[0982] The animal body weights all were within a normal range at
baseline: 379.+-.13 g (mean.+-.SD; n=48). On sacrifice day (Day 23)
no visible differences between test articles, and vehicle were
observed. The mean body weights, recorded for each group just
before the start of the study (baseline) and on the day of
euthanasia were within a normal range with a body weight gain about
31.+-.5% (mean.+-.SD; n=48).
[0983] Electroretinograms
[0984] To investigate the protective effect on photoreceptors,
test, vehicle and reference items were evaluated in light-induced
photodegeneration model. The functional status of retina was
evaluated by electroretinography. Electroretinography waves'
amplitudes were normalized to baseline values and expressed as a
percent of the baseline. FIG. 77 illustrates the time course of
recovery for the different groups.
[0985] Phenyl-N-tert-Butylnitrone, a synthetic anti-oxidant that
has been shown to protect albino rat from light-induced
photoreceptor death was used as reference in the assay. Three doses
of XG-102 were tested: 1.5 .mu.g/eye (0.3 mg/ml, Low dose), 15
.mu.g/eye (3 mg/ml, Mid dose) and 150 .mu.g/eye (30 mg/ml, High
dose). The mean values of the a and b-waves for amplitude (in %;
mean.+-.SD) are summarized in the following tables:
TABLE-US-00046 Time after the beginning of exposure Day 9 Day 16
Day 23 A-wave Mean .+-. SD Mean .+-. SD Mean .+-. SD Vehicle (IVT)
5.7 .+-. 4.9 13.2 .+-. 9.7 22.5 .+-. 9.6 XG-102 (ivt, 1.5
39.5.sctn. .+-. 19.0 47.5.sctn. .+-. 16.5 48.3.sctn. .+-. 15.2
.mu.g/eye) XG-102 (ivt, 15 35.7.sctn. .+-. 22.0 51.1.sctn. .+-.
18.9 41.8 .+-. 23.6 .mu.g/eye) XG-102 (ivt, 150 23.6 .+-. 25.3 24.1
.+-. 28.1 23.7 .+-. 27.2 .mu.g/eye) Vehicle (ip) 9.0 .+-. 19.2 13.6
.+-. 24.0 18.6 .+-. 22.0 PBN (ip, 50 70.4.sctn. .+-. 16.2
79.6.sctn. .+-. 9.4 76.2.sctn. .+-. 13.1 mg/kg) .sctn.p < 0.05
by Mann and Whitney test, x vs. vehicle.
TABLE-US-00047 Time after the beginning of exposure Day 9 Day 16
Day 23 B-wave Mean .+-. SD Mean .+-. SD Mean .+-. SD Vehicle (IVT)
15.3 .+-. 11.4 24.8 .+-. 15.9 30.6 .+-. 13.8 XG-102 (ivt, 1.5
55.3.sctn. .+-. 23.8 61.7.sctn. .+-. 19.7 73.5.sctn. .+-. 22.0
.mu.g/eye) XG-102 (ivt, 15 60.6.sctn. .+-. 32.8 62.3.sctn. .+-.
18.5 56.5 .+-. 29.8 .mu.g/eye) XG-102 (ivt, 150 31.9 .+-. 42.6 38.9
.+-. 51.5 37.1 .+-. 54.4 .mu.g/eye) Vehicle (ip) 15.6 .+-. 29.3
17.3 .+-. 30.6 24.9 .+-. 33.9 PBN (ip, 50 100.sctn. .+-. 18.6
103.6.sctn. .+-. 12.1 102.4.sctn. .+-. 11.0 mg/kg) .sctn.p <
0.05 by Mann and Whitney test x vs. vehicle.
[0986] As also shown in FIG. 77, the mean a-wave amplitude in the
induced groups that was injected with vehicle by intraperitoneal or
intravitreal injection showed reduction at Days 9, 16 and 23
compared with control values on baseline. The a-wave was reduced to
less than 50% of control values on Day 9 (p<0.01), Days 16
(p<0.01) and 23 (p<0.05). The b-wave was significantly
reduced to less than 50% of control values on Day 9 (p<0.01) and
Day 16 (p<0.01). On Day 23, the reduction was not statistically
significant. In the group treated with PBN and exposed to the
damaging light, the retinal function was preserved to a large
extent. The recovery of the a-wave was significantly improved
compared with vehicle at Day 9 (p<0.01), at Day 16 (p<0.01)
and Day 23 (p<0.01) and was 70.4%, 79.6% and 76.2%,
respectively. Similarly, the recovery of the b-wave was
significantly greater (p<0.01) than the vehicle, 100%, 103.6%
and 102.4% at days 9, 16 and 23, respectively.
[0987] Rats treated with different doses of intravitreous XG-102 up
to 15 .mu.g/eye and exposed to the damaging light, showed a
preservation of the retinal function to a large extend compared
with vehicle at Days 9, 16 and 23. The recovery of the a-wave was
47.5% (p<0.01) and 51.1% (p<0.01) at Day 16 and 48.3%
(p<0.01) and 41.8% (p<0.05) at Day 23 for the low and mid
dose, respectively. Similarly, the recovery of the b-wave was
greater than the vehicle and was 55.3% and 60.6% at Day 9, 61.7%
and 62.3% at Day 16, 73.5% and 56.5% at Day 23, for the low and mid
dose respectively. On the other hand, high-dose (150 .mu.g/eye
group) XG-102 showed no effect in preventing light damage. The
recovery of the a-wave was 23.6%, 24.1% and 23.7% versus 5.7%,
13.2% and 22.5% for the vehicle group at Days 9, 16 and 23,
respectively. Similarly, the recovery of the b-wave was 31.9%,
38.9% and 37.1% versus 15%, 24.8% and 30.6% for the vehicle group
at Days 9, 16 and 23, respectively.
[0988] ONL Thickness
[0989] To assess the ability of treatment to preserve photoreceptor
structure, the thickness of the ONL was evaluated 21 days after
cessation of exposure (Day 23). The mean values are summarized in
the following table:
TABLE-US-00048 ONL thickness Loss ONL thickness (% comparison with
Treatment (.mu.m) control non-induced eye) Non-induced eyes
(internal 40.6 .+-. 4.6 -- data) Vehicle (IVT) 13.94 .+-. 3.35 66%
XG-102 (ivt, 1.5 .mu.g/eye) 24.89 .+-. 4.01.sctn. 39% XG-102 (ivt,
15 .mu.g/eye) 24.42 .+-. 5.99.sctn. 40% XG-102 (ivt, 150 .mu.g/eye)
18.95 .+-. 9.17 53% Vehicle (ip) 12.56 .+-. 8.15 69% PBN (ip, 50
mg/kg) 34.05 .+-. 4.00.sctn. 16% .sctn.p < 0.05 by Mann and
Whitney test, x vs. vehicle (ivt, ip).
[0990] A decrease in ONL thickness was observed in the eyes of
vehicle-treated rats. A 66% to 69% loss of mean ONL thickness was
observed in vehicle-treated eyes after exposure compared with
untreated eyes. Administration of PBN showed a significant
protection compared with vehicle groups (ivt and ip, p<0.001).
When the rats were treated with PBN, the ONL was preserved. Only a
small decrease (16%) was observed compared with untreated eyes in
normal condition (40.6.+-.4.6 .mu.m, internal data). The decrease
in ONL thickness was inhibited in the XG-102-treated rats with the
low and mid doses (p<0.01 compared with vehicle). No protection
was observed with high dose XG-102. A 40% loss of the mean ONL
thickness was observed in low and mid doses XG-102-treated
eyes.
[0991] Thus, under these experimental conditions, it can be stated
that: [0992] In vehicle treated groups (2 routes of administration:
ivt, ip) a bright light exposure induced a decrease of retinal
function and a loss of photoreceptor. 23 days after exposure, the
recovery of the a-wave was 18.6% (ip) and 22.5% (ivt); 69% (ip) and
66% (ivt) loss of mean ONL thickness was observed. [0993] Systemic
administration (i.p.) of PBN protects significantly the retina from
light damage. The PBN-treated group maintained 76.2% of a-wave and
only a small loss (16%) of mean ONL thickness was observed. [0994]
Intravitreal injection of 1.5 and 15 .mu.g/eye XG-102 protects
significantly the retina from light damage. The XG-102 treated
group maintained 48.3% and 41.8% of a-wave and a 40% loss of mean
ONL thickness was observed.
[0995] Taken together, according to the statistical analyses,
intravitreal injection of XG-102 (1.5 and 15 .mu.g/eye) was
efficient to protect retinal function. Under these experimental
conditions, the results indicate that XG-102 by IVT at doses 1.5
.mu.g and 15 .mu.g/eye protects the structure and function of the
retina from acute light-induced damage.
Example 35: Efficacy and Safety of XG-102 in Reduction of
Post-Cataract Surgery Intraocular Inflammation (Clinical Phase
III)
[0996] A multicenter, randomized, double-masked,
vehicle-controlled, parallel group phase III study served to assess
the efficacy and safety of a single sub-conjunctival injection of
XG-102 for the reduction of post-cataract surgery intraocular
inflammation. The purpose of this study is to evaluate the clinical
efficacy and safety of XG-102 (900 .mu.g) compared to vehicle (NaCl
0.9%) in the treatment of subjects with inflammation and pain
following uncomplicated cataract surgery.
[0997] The study focuses on inflammation and pain following eye
surgery, in particular unilateral cataract extraction via
phacoemulsification and posterior chamber intraocular lens (PCIOL)
implantation in the study eye. Treatment by a single
sub-conjunctival injection of 900 .mu.g of XG-102 is compared
versus placebo (vehicle: NaCl 0.9%) sub-conjunctival injection.
Visits #3, 4, 5, 6 and 7 are planned at days 2, 8, 15, 22 and 85
respectively.
[0998] In particular the absence of anterior chamber cells for the
900 .mu.g XG-102 sub-conjunctival injection compared to vehicle,
preferably at visit 5 at day 15, and the absence of pain for the
900 .mu.g XG-102 compared to vehicle, preferably at visit 3 at day
2, serve as primary outcome measures. Secondary outcome measures
are in particular absence of anterior chamber cells, preferably at
visits 3, 4 and 6 (Days 2, 8 and 22 respectively), absence of pain,
preferably at visits 4, 5 and 6 (Days 8, 15 and 22 respectively),
absence of flare, preferably at visits 3, 4, 5 and 6 (Days 2, 8, 15
and 22 respectively), absence of anterior chamber cells and flare,
preferably at visits 3, 4, 5 and 6 (Days 2, 8, 15 and 22
respectively), and use of rescue medication on or prior each visit
and overall. Other pre-specified outcome measures include in
particular pin-hole visual acuity, preferably at visits 3, 4, 5, 6
and 7 (Days 2, 8, 15, 22 and 85 respectively), slit-lamp
biomicroscopy, preferably at visits 3, 4, 5, 6 and 7 (Days 2, 8,
15, 22 and 85 respectively), dilated indirect ophthalmoscopy,
preferably at visit 6 (Day 22), intraocular pressure (IOP),
preferably at visits 3, 4, 5 and 6 (Days 2, 8, 15 and 22
respectively), specular microscopy, preferably at visit 7 (Day 85),
and adverse event (AE) monitoring, preferably at visits 3, 4, 5, 6
and 7 (Days 2, 8, 15, 22 and 85 respectively).
Example 36: Effects of XG-102 (SEQ ID No. 11) on Renal
Ischemia/Reperfusion Lesions
[0999] Renal Ischemia/Reperfusion (Renal I/R) injury is a commonly
employed model of acute kidney injury (AKI), also known as acute
renal failure. In addition to the clinical relevance of studies
that examine renal I/R injury to acute kidney injury, experimental
renal I/R injury is also an important model that is used to assess
the conditions that occur in patients receiving a kidney
transplant. Depending upon the donor, transplanted kidneys are not
perfused with blood for a variable amount of time prior to
transplantation. Because AKI has such serious effects in patients,
and all transplanted kidneys experience renal I/R injury to some
extent, the clinical relevance and translational importance of this
type of research to human health is extremely high. The aim of this
study is thus to investigate the influence of the JNK inhibitor
XG-102 (SEQ ID NO: 11) on experimental renal ischemia/reperfusion
in rats.
[1000] Twenty-six (26) male Wistar rats (age 5-6 weeks) were used
in this study (divided into 2 groups of 10 rats and 1 group of 6
rats). Rats were housed in standard cages and had free access to
food and tap water. Each day, the general behavior and the
appearance of all animals were observed. The health of the animals
was monitored (moribund animals, abnormal important loss of weight,
major intolerance of the substance, etc. . . . ). No rats were
removed.
[1001] Renal ischemia was induced by clamping both renal pedicles
with atraumatic clamp. A single dose of 2 mg/kg XG-102 (in 0.9%
NaCl as vehicle) or vehicle, respectively, was administered by IV
injection in the tail vein on Day 0, one hour after clamping period
(after reperfusion) both renal pedicles with atraumatic clamp. The
administration volume was 2 ml/kg. Heparin (5000 UI/kg) was
administered intraperitoneally 1 hour before clamping (in all
groups).
[1002] The table below summarizes the random allocation:
TABLE-US-00049 Treatment Dose volume/ Renal Number Group (1 hour
after Route of Ischemia of No clamping) administration
Concentration time (min) animals 1 NaCl 0.9% 2 mL/kg, IV 0 6 2 NaCl
0.9% 2 mL/kg, IV 0 40 10 3 XG-102 (2 mg/kg) 2 mL/kg, IV 1 mg/mL 40
10
[1003] For sample collection, rats were housed individually in
metabolic cages (Techniplast, France). Urine was collected at 72
hours. Blood samples were obtained from tail vein before and at 24
hours after reperfusion. After animal sacrifice, both kidneys were
collected.
[1004] For evaluation of proteinuria and albuminuria appropriate
kits from Advia Chemistry 1650 (Bayer Healthcare AG, Leverkusen,
Germany) were used.
[1005] For evaluation of renal function, blood was collected from
the tail vein at 24 hours after reperfusion. Serum creatinine
(pmol/mL) and urea concentrations (mmol/mL) were measured using
appropriate kits (Bayer Healthcare AG, Leverkusen, Germany).
[1006] Evaluation of histological lesions was performed at 24 and
72 hours after reperfusion.
[1007] For light microscopy, kidneys were be incubated for 16 hours
in Dubosq-Brazil, dehydrated, embedded in paraffin, cut into
sections and stained with hematoxylin and eosin (H&E) or with
periodic acid-Schiff (PAS).
[1008] For immunohistochemistry, kidney samples were fixed for 16
hours in Dubosq Brazil, and subsequently dehydrated and embedded in
paraffin. Antigen retrieval was performed by immersing the slides
in boiling 0.01 M citrate buffer in a 500 W microwave oven for 15
min. The endogenous peroxidase activity was blocked with 0.3%
H.sub.2O.sub.2 in methanol for 30 min. Slides were incubated with
the blocking reagents consisting of the Avidin-biotin solution for
30 min and the normal blocking serum for 20 min. For
immunodetection, the slides were incubated overnight with an
antibody, then with a biotinylated secondary antibody. An
avidinbiotinylated horseradish peroxidase complex (Vectastain ABC
Reagent, Vector Laboratories; Burlingame, Calif.) and
3,3'-diaminobenzidine (Sigma Biochemicals; St Louis, Mo.) as a
chromogen were applied for visualization of the immunoreaction.
Slides were counterstained with hematoxylin. Omission of the
primary antibody was considered as a negative control.
[1009] Immunofluorescence labeling was carried out on 4 mm thick
cryostat sections of kidney tissue fixed in acetone for 10 min,
air-dried for 30 min at room temperature, then incubated in PBS for
3 min and blocked in 1% BSA in PBS. The sections were incubated
with the indicated antibodies for 1 hour at room temperature,
washed in PBS and incubated with Red Texas-conjugated secondary
antibodies. Sections will be examined by fluorescence microscopy
(Zeiss).
[1010] Moreover, expression of several markers specific of podocyte
damage, inflammation and renal fibrosis (RelA, TGF .beta.,
TNF.alpha., Masson trichrome) were evaluated by
immunohistochemistry and immunofluorescence. Quantitative
transcription profile of TNF.alpha., IL6, CXCL 1 (KC), CXCL2
(MIP-2) and MCP1 in kidneys were determined.
[1011] Results:
[1012] Results are shown in FIG. 78. Serum creatinine (FIG. 78A)
and urea (FIG. 78B) were increased in vehicle-treated ischemic rats
(G2) 24 h following ischemia, as compared to vehicle-treated
controls rats without ischemia (G1). On the other hand,
XG-102-treated-ischemic rats (G3) exhibited lower serum creatinine,
relatively to untreated ischemic rats (G2). These results suggest
that XG102 may prevent the ischemia-induced renal failure.
Example 37: Antitumour Activity of XG-102 (SEQ ID No. 11) Against
Human Liver Tumour Cell Lines
[1013] The aim of this study is to determine the cytotoxic activity
of XG-102 (SEQ ID No. 11) against human hepatocarcinoma and human
hepatoma cell lines using MTS assay.
[1014] The human hepatocarcinoma cell line HepG2 (origin: American
Type Culture Collection, Manassas, Va., USA; the HepG2 cell line
was established from the tumor tissue of a 15-year old Argentine
boy with a hepatocellular carcinoma in 1975, there is no evidence
of a Hepatitis B virus genome in this cell line) and the human
hepatoma cell line PLC/PRF/5 (origin: American Type Culture
Collection, Manassas, Va., USA; the PLC/PRF/5 cell line secrete
hepatitis virus B surface antigen (HBsAg)) are used. Tumor cells
are grown as monolayer at 37.degree. C. in a humidified atmosphere
(5% CO.sub.2, 95% air). The culture medium is EMEM (ref: BE12-611F,
Lonza) supplemented with 10% fetal bovine serum (ref: 3302, Pan),
0.1 mM NEAA (ref: BE13-114E, Lonza) and 1 mM NaPyr (ref: BE13-115E,
Lonza). The cells are adherent to plastic flasks. For experimental
use, tumor cells are detached from the culture flask by a 5-minute
treatment with trypsin-versene (ref: BE02-007E, Lonza), in Hanks'
medium without calcium or magnesium (ref: BE10-543F, Lonza) and
neutralized by addition of complete culture medium. The cells are
counted in a hemocytometer and their viability is assessed by 0.25%
trypan blue exclusion assay.
[1015] Tumor cells are plated at the optimal seeding density in
flat-bottom microtitration 96-well plates (ref 167008, Nunc,
Dutscher, Brumath, France) and incubated in 190 .mu.L drug-free
culture medium at +37.degree. C. in a humidified atmosphere
containing 5% CO.sub.2 for 24 hours before treatment.
[1016] Dilutions of XG-102 (SEQ ID No. 11) as well as distribution
to plates containing cells are performed manually. At treatment
start 10 .mu.L of XG-102 (SEQ ID No. 11) dilutions are added to
wells at the following final concentrations (for both cell lines):
0, 3.8.times.10.sup.3, 1.5.times.10.sup.2, 6.1.times.10.sup.-2,
0.24, 0.98, 3.9, 15.6, 63, 250 and 1000 .mu.M. Then cells are
incubated for 72 hours in 200 .mu.L final volume of culture medium
containing XG-102 at +37.degree. C. in a humidified atmosphere
containing 5% CO.sub.2. At the end of treatments, the cytotoxic
activity is evaluated by a MTS assay.
[1017] The in vitro cytotoxic activity of the XG-102 is revealed by
a MTS assay using tetrazolium compound (MTS,
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and an electron coupling
reagent named PMS (phenazine methosulfate). Like MTT, MTS is
bioreduced by cells into a formazan product that is directly
soluble in culture medium without processing, unlike MTT. At the
end of cell treatment, 40 .mu.L of a 0.22 .mu.m freshly filtered
combined solution of MTS (20 mL at 2 mg/mL, ref: GII 11, Promega,
Charbonnieres, France) and PMS (1 mL at 0.92 mg/mL, ref: P9625,
Sigma) in Dulbecco's Phosphate Buffered Saline (DPBS, ref: 17-513F,
Cambrex), are added in each well. Absorbance (Optical Density, OD)
is measured at 490 nm in each well using a VICTOR3.TM. 1420
multilabeled counter (Wallac, PerkinElmer, Courtaboeuf, France).
Individual OD values of MTS assays are provided. Dose response for
index of cytotoxicity (IC) is expressed as follows:
IC=(OD.sub.drug-exposed/OD.sub.vehicle-exposed wall).times.100
whereby IC.sub.50 refers to the drug concentration to obtain a 50%
inhibition of cell proliferation. IC.sub.50 represent drug
concentration required to obtain 50% of cellular cytotoxicity.
Dose-response curves are plotted using XLFit5 (IDBS, United
Kingdom) and provided. The IC.sub.50 determination values are
calculated using the XLFit5 software from semi-log curves. Each
individual IC.sub.50 determination values are provided as well as
mean.+-.SD IC.sub.50 values.
[1018] FIG. 107 shows the results of the determination of the
cytotoxic activity of XG-102 against HepG2 and PLC/PRF/5 tumour
cell lines using MTS assay.
Example 38: Effects of XG-102 (SEQ ID No. 11) in a Rat Model of
Experimental Autoimmune Uveitis (Posterior Uveitis)
[1019] In the United States, there are approximately 70,000 cases
of uveitis per year, and autoimmune uveitis is responsible for
approximately 10% of severe vision loss (Caspi et al., 2012).
Experimental autoimmune uveitis (EAU) is an organ specific
autoimmune disease that targets the neural retina, i.e. it is a
model of posterior uveitis. This autoimmune response is induced
when animals are immunized with retinal antigens, e.g.
Interphotoreceptor retinoid-binding protein (IRBP). In this study,
animals are immunized with IRBP. After a period of 9-14 days,
animals develop uveitis in the eye. At the end of the study,
animals are sacrificed and eyes submitted for histology.
[1020] Sixty-four (64) male Lewis rats (8 weeks, Charles River) are
randomly assigned to test groups. Groups 1 to 6 are immunized with
an emulsion of interphotoreceptor binding protein (IRBP) in
Complete Freund's Adjuvant (CFA).
[1021] Group Assignment:
TABLE-US-00050 Dose Concentration Number of (.mu.g/.mu.L or Group
animals Route Dose mg/mL) Dose Volume 1/Vehicle 10 Sub-conj. 0
.mu.g/eye 0 5 .mu.L/Eye 2/XG-102 10 Sub-conj. 20 .mu.g/eye 4 5
.mu.L/Eye 3/Vehicle 10 Intravitreal 0 .mu.g/eye 0 5 .mu.L/Eye
4/XG-102 10 Intravitreal 2 .mu.g/eye 0.4 5 .mu.L/Eye 5/FTY-720 10
Oral 0.3 mg/kg 0.03 10 mL/kg 6/No Treatment 10 n/a n/a n/a n/a
7/Naive 4 n/a n/a n/a n/a
[1022] FTY 720 is used as positive control (group 5). Animals
(group 5) are given 0.3 mg/kg/day FTY 720 in 10% PEG and sterile
water (once daily from day-3 to day 13; route: oral gavage). The
total volume per day is no more than 10 m/kg/day. Rats are weighed
every Monday, Wednesday, and Friday, and the volume to be
administered is determined by the group's average weight.
[1023] XG-102 is given at a single dose at day-1 either at 20
.mu.g/eye subconjunctivally (group 2) or at 2 .mu.g/eye
intravitreally (group 4). To this end, animals are sedated with an
intraperitoneal (IP) injection of a mixture of ketamine and
xylazine (k/x) at a concentration of 33.3 mg/kg ketamine and 6.7
mg/kg xylazine. Once fully sedated (as confirmed by lack of toe
pinch reflex), each eye is given a drop of Proparacaine. Under a
dissection microscope, 5 .mu.L of XG-102 (as described above) are
carefully administered into the vitreous or sub-conjunctiva of each
eye. Lubrication (such as Puralube.RTM.) is added to the eye to
prevent corneal ulcer formation. The animal is then placed on a
warm heating pad and monitored until fully awake.
[1024] On day 0, groups 1 to 6 are immunized by a single
subcutaneous administration of IRBP/CFA. To this end, an emulsion
of IRBP in CFA is made at the day of injection. Animals are lightly
anesthetized with isoflurane and receive 50 .mu.g IRBP in 200 .mu.L
of CFA.
[1025] All animals are daily checked for general health/mortality
and morbidity. Prior to any dose (or Day-3 for the untreated but
immunized and naive groups) and prior to euthanasia on Day 14,
fundus exams are performed. To this end, animals are sedated with
k/x (the same amount as specified above). Once sedated, a drop of
GONAK is placed on each eye and is gently placed on a platform. The
eye is positioned to make gentle contact with a special lens for
fundus imaging. Images are taken with the Micron Ill. Animals
receiving IVT injections have baseline fundus exams just prior to
injection when they are already sedated. All other animals not
receiving IVT injections are sedated on Day-3. For clinical
evaluation, on day 13 animals are observed under a dissection
microscope and scored on a scale of 0-4 based on their clinical
disease. After sacrifice on day 14 and upon verification of death,
both eyes of each animal are carefully removed via forceps, being
sure to keep as much of the optic nerve intact as possible. Eyes
are placed in Davidson's fixative for 24 hours. Eyes are
transferred to 70% ethanol for histology. Each eye is stained with
hematoxylin and eosin for histological analysis.
[1026] The experimental design is summarized below:
TABLE-US-00051 Day Days Day Day Procedure Day -3 Day -2 Day -1 0
1-12 13 14 Baseline X Fundus - Groups 5-7 Baseline X Fundus -
Groups 1-4 Oral dose X X X X X X FTY720 - Group 5 Sub-conj or X IVT
of XG-102 or vehicle groups 1-4 Immunization X group 1-6 Clinical X
Evaluations/ Photographs Final Fundus - X All groups Euthanasia X
and Tissue Collection
Example 39: Effects of XG-102 (SEQ ID No. 11) in a Rat Model of
Diabetic Retinopathy
[1027] The objective of this study is to determine the
dose-dependent effect of XG-102 on loss of visual acuity, ocular
clinical signs and cytokine profiling after repeated
sub-conjunctival administration in a rat model of streptozotocin
(STZ)-induced diabetes.
[1028] To this end, 30 rats (female, Brown Norway, 6-8 weeks at
time of STZ-treatment are assigned to the following 5 groups (6
animals per group):
TABLE-US-00052 Group STZ Treatment Assessment 1 - Vehicle Weekly
recording of body weight and NaCl 0.9% blood glucose levels (Weeks
1-16); 2 + Vehicle Weekly Draize scoring of chemosis, NaCl 0.9%
hyperernia, and discharge (Weeks 1-16); 3 + XG-102 Quantification
of contrast threshold at (2 .mu.g/eye/ Days 43, 57, 71, 85, 99, and
113; 2-week) Quantification of spatial frequency 4 + XG-102
threshold at Days 43, 57, 71, 85, 99, (20 .mu.g/eye/ and 113;
2-week) Quantification of scotopic a-wave, 5 + XG-102 scotopic
b-wave, and photopic b-wave at (200 .mu.g/eye/ Day 114; 2-week)
Multiplex cytokine quantification of retinas using Bio-Rad rat
23-plex kit
[1029] The "treatment" (vehicle or XG-102) is for each group
bilateral sub-conjunctival administration (vehicle or XG-102,
respectively) on Days 22, 36, 50, 64, 78, 92, and 106.
[1030] The experimental design is the following:
[1031] Day 1: IP injection of streptozotocin (Groups 2-5)
[1032] Day 4: Blood glucose quantification
[1033] Day 22: Bilateral sub-conjunctival injection of vehicle or
XG-102 (Groups 2-5)
[1034] Day 36: Bilateral sub-conjunctival injection of vehicle or
XG-102 (Groups 2-5)
[1035] Day 43: OKT* assessment of contrast sensitivity and spatial
frequency threshold
[1036] Day 50: Bilateral sub-conjunctival injection of vehicle or
XG-102 (Groups 2-5)
[1037] Day 57: OKT assessment of contrast sensitivity and spatial
frequency threshold
[1038] Day 64: Bilateral sub-conjunctival injection of vehicle or
XG-102 (Groups 2-5)
[1039] Day 71: OKT assessment of contrast sensitivity and spatial
frequency threshold
[1040] Day 78: Bilateral sub-conjunctival injection of vehicle or
XG-102 (Groups 2-5)
[1041] Day 85: OKT assessment of contrast sensitivity and spatial
frequency threshold
[1042] Day 92: Bilateral sub-conjunctival injection of vehicle or
XG-102 (Groups 2-5)
[1043] Day 99: OKT assessment of contrast sensitivity and spatial
frequency threshold
[1044] Day 106: Bilateral sub-conjunctival injection of vehicle or
XG-102 (Groups 2-5)
[1045] Day 113: OKT assessment of contrast sensitivity and spatial
frequency threshold
[1046] Day 114: Scotopic and Photopic ERG analyses
[1047] Day 114: Enucleation of retinas for multiplex cytokine
analyses
[1048] *OKT: optokinetic tracking
[1049] For streptomycin administration, rats of the same age are
weighed the day prior to injections and are fasted overnight, and
cages are marked with yellow cards in animal facility. The weights
are averaged, and a single dose is calculated for all rats based
upon the average weight. No more than ten animals are injected with
a single preparation of STZ due to quick decrease of STZ activity
in solution, and the procedure is repeated for each batch of 10
animals. STZ powder is dissolved in 10 mM sodium citrate, pH 4.5,
immediately before injection and rats receive 50 mg/kg STZ
intraperitoneally in a volume of 1 mL using a 22 gauge syringe with
care to avoid stomach and any vital organs.
[1050] For subconjunctival administration, animals are anesthetized
with ketamine/xylazine (Ketamine and xylazine are mixed using a
U-100 syringe utilizing 20 units of ketamine (100 mg/mL) and 100
units of xylazine (20 mg/mL) and the anesthesia mixture is applied
via IP injection at 1 mL/kg (body weight)) and pupils are dilated
with topical administration of Cyclogel and/or Tropicamide.
Following sedation and dilation, a total volume of 50 .mu.L per eye
is injected into conjunctiva using a 31-gauge needle attached to an
insulin syringe.
[1051] For Draize scoring of hyperemia, chemosis, and discharge,
animals are restrained by hand and scores for chemosis, hyperemia,
and discharge are recorded by a masked observer using the "EyeCRO
ocular scoring system".
[1052] All optokinetic tracking experiments are performed using an
Optomotry designed for rodent use (Cerebra) Mechanics Inc.). In
this non-invasive assessment, rats are placed onto a platform
surrounded by 4 LCD screens which resides within a light-protected
box. Visual stimuli are then presented to the rats via the LCD
screens and a masked observer visualizes and scores optokinetic
tracking reflexes from a digital camcorder which is mounted on the
top of the box. For measurements of spatial frequency threshold,
the rats are tested at a range of spatial frequencies from 0.034 to
0.664 cycles/degree. The Optomotry device employs a proprietary
algorithm to accept the input from the masked observer and
automatically adjust the testing stimuli based upon whether the
animal exhibited the correct or incorrect tracking reflex. All
measurements of contrast threshold are performed at a spatial
frequency threshold of 0.064 cycles/degree.
[1053] For electroretinography (ERG), after a minimum of 12 h dark
adaptation, animals are anesthetized by intraperitoneal injection
of 85 mg/kg ketamine and 14 mg/kg xylazine. Animal preparation is
performed under a dim red light (<50 lux). ERG analyses are
performed using an Espion system from Diagnosys. For the assessment
of scotopic response, a stimulus intensity of 40 (S) cd.s/m2 is
presented to the dark-adapted dilated eyes. The amplitude of the
scotopic a-wave is then measured from the prestimulus baseline to
the a-wave trough. The amplitude of the b-wave is then measured
from the trough of the a-wave to the crest of the b-wave. To
evaluate photopic response, animals are light adapted for 10 min
then presented a strobe flash to the dilated eyes with an intensity
of 10 (S) cd.s/m2. A total of 25 repeated flashes and measurements
are averaged to produce the final waveform. The amplitude of the
photopic b-wave is then measured from the trough of the a-wave to
the crest of the b-wave.
[1054] For multiplex cytokine analysis, at study termination, the
retinas will be individually isolated and immediately snap-frozen
in liquid N.sub.2. The Bio-Rad "Bio-plex Pro Rat Cytokine 23-plex
assay (Cat# L80-01V11 SS) is used according to manufacturer's
specifications to quantify the protein expression of EPO, G-CSF,
GM-CSF, GRO/KC, IFN-.gamma., IL-1.alpha., IL-1.beta., IL-2, IL-4,
IL-5, IL-6, IL-7, IL-10, IL-12, p70, IL-13, IL-17A, IL-18, M-CSF,
MCP-1, MIP-3.alpha., RANTES, TNF-.alpha., and VEGF in each retina
isolated in the study.
[1055] Results:
[1056] To determine the ocular tolerability of bilateral
sub-conjunctival administration of XG-102, a comprehensive
ophthalmic examination of signs of ocular irritation indicated by
chemosis, hyperemia, or discharge was performed once a week for the
duration of the study. The rats were scored on a scale of 0
(normal) to 4 (severe) for each indicator. There was no observed
ocular irritation at any time point in any of the animals
administered either vehicle or XG-102.
[1057] Optokinetic tracking was used to measure the maximum spatial
frequency distinguished by STZ-diabetic Brown Norway rats at 2-week
intervals beginning on Day 43 (6 weeks post-STZ). There is no loss
in visual acuity in the non-diabetic group administered vehicle. At
Day 43, there was no difference in visual acuity across groups.
Visual acuity decreases in STZ-diabetic rats treated with vehicle
at Day 57. All STZ-diabetic rats show a decline in visual acuity
between Day 57 and Day 99 (FIG. 89 A-C). However, treatment with
either 20 .mu.g/eye, or 200 .mu.g/eye XG-102, significantly delays
the progressive decline in visual acuity at each of these time
points (FIG. 89 A-C). At Day 113, all groups administered XG-102
display higher visual acuity scores relative to the vehicle treated
STZ-diabetic group, with the groups receiving either 2 .mu.g/eye or
200 .mu.g/eye having significantly higher spatial frequency
thresholds (FIG. 89D).
[1058] Optokinetic tracking was used to measure the threshold at
which STZ-diabetic rats were able to distinguish contrast in
visually presented stimuli at 2-week intervals beginning on Day 43.
There is no loss in contrast sensitivity in the non-diabetic group
administered vehicle. At Day 43 and Day 57, the STZ-diabetic groups
administered either vehicle, or 2 .mu.g/eye XG-102 had decreased
contrast threshold values relative to all other groups. All
STZ-diabetic groups show a decline in contrast threshold values
over the course of the study, but the decline is significantly
delayed in the group treated with 200 .mu.g/eye (FIG. 90 A, B). At
Day 99, all XG-102 treated groups have significantly higher
contrast threshold values relative to the vehicle group (FIG. 90 C,
and the values remain higher through Day 113 (FIG. 90 D).
[1059] At Day 114, eyes were enucleated, and retinal tissue was
collected and processed for Multiplex cytokine analysis of 23
unique cytokines. STZ-induced diabetes raised retinal levels in
vehicle treated animals for 13 of the 23 cytokines observed (FIG.
91). Seven of the 13 elevated cytokines were reduced in
STZ-diabetic animals treated with 2 .mu.g/eye XG-102 (FIG. 91). All
cytokines were BLQ in the retinal tissue collected from the groups
of rats receiving either 20 .mu.g/eye, or 200 .mu.g/eye XG-102
(FIG. 91). The total protein concentration was equal for all
samples used to detect cytokines, and the standard curves for each
individual cytokine had high r-squared values. Therefore, there is
no evidence for protein degradation or deficiencies in the assay
itself. The cytokines that were upregulated in vehicle treated
diabetic animals, and downregulated by treatment with 2 .mu.g/eye
XG-102 were: IL-3, IL-13, IL-17, RANTES, GM-CSF, MCSF, and IL-7.
Each of these cytokines is linked to inflammation-associated
diabetic retinopathy disease progression.
[1060] Taken together, bilateral sub-conjunctival delivery of
XG-102 was well tolerated by Brown Norway rats as there was no
indication of chemosis, hyperemia, or discharge at any point
throughout the study. Visual acuity and contrast sensitivity
display a progressive decline in STZ-diabetic rats in all treatment
groups. The vision loss is greatest in rats treated with vehicle
alone. All treatment doses of XG-102 conferred improvements in
vision relative to vehicle alone. Treatment with either 2 .mu.g/eye
or 200 .mu.g/eye XG-102 significantly rescues visual acuity at Day
113 post-STZ administration; and treatment with 2 .mu.g/eye
SDD-1002 significantly rescues contrast sensitivity at Day 113
post-STZ administration relative to vehicle treated STZ-diabetic
rats. STZ-induced diabetes resulted in higher retinal cytokine
levels in 18 of the 23 cytokines analyzed in this study. 10 of the
18 elevated cytokine levels were decreased by treatment with 2
.mu.g/eye XG-102. All cytokine levels were below the limit of
quantification (BLQ) in the retinal tissue of STZ-diabetic rats
receiving the two highest doses of XG-102 (20 and 200
.mu.g/eye).
[1061] The results of this study indicate that sub-conjunctival
delivery of XG-102 is well tolerated by rats and does not cause
adverse effects. XG-102 is effective in improving loss of both
visual acuity and contrast sensitivity over 16 weeks in STZ-induced
diabetic retinopathy in rats.
Example 40: Evaluation of XG-102 (SEQ ID NO: 11) in Langerhans
Islet Isolation and Transplantation
[1062] This study is based on the previous studies on islet
isolation and transplantation (cf. Examples 17 and 22) and aims at
determining the effects of XG-102 on islet viability.
[1063] In the first part of this study, the model described in
Example 22 was used, i.e. ischemia for a period of 30 min and
XG-102 was applied at 100 .mu.M.
[1064] As shown in FIG. 79 concerning the impact of ischemia and
XG-102 on islet viability, it was again observed that XG-102
decreases apoptosis and necrosis. These results show that XG-102
has a beneficial effect on islet viability.
[1065] Because islet isolation is a long process, in which
differents pathways could be activated to impact islet function and
viability, in the second part of this study another model than
ischemia was used to investigate the effects of the JNK inhibitor
XG-102 on islet viability.
[1066] Therefore, hypoxia was used as a model for islet
isolation/transplantation, since it is known to induce JNK
phosphorylation. In these experiments, 18 h after isolation, islets
were pre-treated or not with XG-102 100 .mu.M for 1 h and then
submitted to hypoxia for 4 h, whereby XG-102 was still present (or
not in control groups) during the 4 hour hypoxia ("H4").
[1067] As shown in the western blot shown in FIG. 80. hypoxia
("H4") induces JNK and JUN phosphorylation as compared to islets
maintained in normoxia conditions ("N4"), as expected.
Surprisingly, however, the JNK inhibitor XG-102 did not inhibit
phosphorylation of JNK and JUN induced by hypoxia (cf. FIG. 80
"H4+XG102").
[1068] Regarding the viability, hypoxia increased apoptosis and
necrosis, as shown in FIG. 81 (H4 vs. N4). However when islets were
treated with XG-102, apoptosis and necrosis were decreased either
in normoxia and hypoxia conditions. In conclusion XG102 had also a
beneficial effect on islet viability in this hypoxia model.
Example 41: Effects of XG-102 on Puromycine Aminonucleoside
(PAN)-Induced Nephropathy--Frequency of Administration
[1069] The aim of this study was to determine the frequency of
administration of XG-102 in a model of glomerulonephritis, namely
in chronic puromycine aminonucleoside-induced nephropathy in rats.
This study is thus based on the study described in Example 20 and
the dose of 4 mg/kg XG-102 was chosen based on the results of the
study described in Example 20.
[1070] This study thus includes the following 8 groups of 15 rats
each, whereby "SDD-1002" refers to XG-102:
TABLE-US-00053 Number Number Day of i.v. of of PAN Treatment
adminis- animals/ Day(s) of sacri- Group (i.p.) (i.v.) trations
group administration fice 1 no vehicle 2 12 21 and 35 49 2 yes
vehicle 2 12 21 and 35 49 3 yes SDD-1002 4 12 21, 28, 35 and 49 42
4 yes SDD-1002 2 12 21 and 35 49 5 yes SDD-1002 1 12 21 49 6 no
vehicle 1 12 21 77 7 yes vehicle 1 12 21 77 8 yes SDD-1002 1 12 21
77
[1071] The dose of XG-102 is 4 mg/kg for a single administration in
all groups (group 3, 4, 5, and 8). The groups thus vary in the
number of i.v. administrations as specified above.
[1072] Male Wistar rats are treated with two repeated
intraperitoneal injections (i.p.) of PAN (Sigma Aldricht, France)
at day 0 (130 mg/kg of body weight) and at day 14 (60 mg/kg of body
weight) in saline (0.9% NaCl). Control rats (groups 1 and 6)
receive an equal amount of saline i.p at day 0 and at day 14.
[1073] XG-102 or its vehicle (0.9% NaCl) are administered into the
tail vein (i.v.) at different time points as listed above. XG-102
or vehicle administration will start at day 21 after the first PAN
injection at day 0. XG-102 will be administered at the dose of 4
mg/kg.
[1074] The temporal schedule is summarized as follows:
[1075] Day 0 and day 14: PAN or its vehicle (saline) injection for
induction of nephropathy.
[1076] From day 21 to day 42: XG-102 or its vehicle administration
by i.v. route as described above.
[1077] Day 21: Blood sample collection in conscious animals for
creatinine and urea quantification (n=12 chosen by randomization in
vehicle, n=6, and PAN-treated, n=6, animals).
[1078] Day 49 or day 77: Blood sample collection, sacrifice of
animals and sample collections (kidneys).
[1079] The study design is shown schematically in FIG. 82.
[1080] Blood samples are collected in conscious animals at day 21
after the first PAN injection at day 0. For blood and kidney
sampling at days 49 and 77 animals are anesthetized by injection of
pentobarbital (60 mg/kg; Ceva Sante Animale; Libourne, France).
Blood samples are collected and transferred into tubes coated with
EDTA 3K (4.degree. C.), then centrifuged (10 minutes, 3000 rpm,
4.degree. C.) for plasma collection. Plasma is stored at
-20.degree. C. until use for creatinine and urea assays.
[1081] Kidneys are removed, cleaned from all connective tissue and
capsule and weighted on an electronic microbalance (Mettler,
Toledo). Kidneys are transferred in Formalin solution 10% (Sigma
Aldrich, France) for 48 h and then transferred in ethanol 70% for
further histological preparation and imaging by Histalim
(Montpellier, France). At the end of the protocol, animals are
sacrificed by cervical dislocation.
[1082] For biomarker quantification, e.g. plasma creatinine and
urea, will be quantified using an ABX Pentra 400 Clinical Chemistry
analyzer (HORIBA) by the Phenotypage platform of Genotoul (Rangueil
Hospital, Toulouse, France).
[1083] Histological preparation and imaging are performed by
Histalim (Montpellier, France). Kidney sections of paraffin
embedded tissue are stained by Hematoxylin/eosin, PAS-methenamine
silver and Sirius Red for histological evaluation of morphological
alterations, glomerular damage evaluation and interstitial fibrosis
quantification, respectively. Results are expressed by
semi-quantitative scoring following to expert histopathologist
evaluation.
[1084] Fibrosis is expressed as percentage of Red Sirius stained
area on total kidney section surface. All the slides are
digitalized at .times.20 with the Nanozoomer 2.0HT from Hamamatsu
(Japan).
[1085] Histological examination of glomerulosclerosis Glomerular
changes were evaluated on H&E, PAS and PAS-M stained sections
using a semi quantitative scoring system as adapted from Nakajima
et al. (2010). Briefly, the degree of glomerular injury was
assessed in 25 glomeruli per kidney section (2 sections per animal)
for a total of 50 glomeruli per animal. Degree of injury in
individual glomeruli was graded using a scale from 0 to 4, based on
the percentage of glomerular involvement.
[1086] Score 0: normal,
[1087] Score 1: lesions in up to 25% of the glomerulus,
[1088] Score 2: lesions between 26-50% of the glomerulus,
[1089] Score 3: lesions between 51-75% of the glomerulus, and
[1090] Score 4: lesions between 76-100% of the glomerulus
[1091] The incidence of glomerular damage was expressed as
percentage (%) of injured glomeruli (from score 1 to 4) of the
total number of evaluated glomeruli (50/animal).
[1092] Scores were determined blinded by a histopathologist at
Histalim.
[1093] Expression and Analysis of Results
[1094] For each group results were expressed as mean
values.+-.s.e.m.
[1095] Statistical test used: [1096] Comparison of all groups using
two-way ANOVA for body weight results. [1097] Comparisons between
Group 1 or 6 (Saline/vehicle) and Group 2 or 7 (PAN/vehicle) were
performed using unpaired Student t-test. [1098] Comparison between
Group 2 (PAN/vehicle) and Groups from 3 to 5 (PAN/XG-102) were
performed using a one-way ANOVA followed by Bonferroni's or
Newman-Keuls post-test. [1099] Comparisons between Group 7
(PAN/vehicle) and Group 8 (PAN/XG-102) were performed using
unpaired Student t-test. [1100] For statistical analysis of
histological scores, when all data were identical or equal to zero
one value was modified (for example: 0 to 0.0001) to allow the
statistical test to be performed.
[1101] A P<0.05 value was accepted as statistical
significance.
[1102] Results: Glomerular Injury Score and Incidence
[1103] Glomerular injury was evaluated after collection at day 49
(Groups 1-5) and at day 77 (Groups 6-8).
[1104] Glomerular injury score (FIG. 83) represents an evaluation
of severity of glomerular damage and sclerosis. Quantification of
glomerular damage incidence expressed as percentage of injured
glomeruli (FIG. 84) is an index of the frequency of the lesions and
indirectly of the remaining functional nephrons.
[1105] Day 49 (groups 1-5):
[1106] In naive control rats (Group 1: Saline/vehicle; FIG. 85
A-C), more than 90% of glomeruli were of normal appearance
histologically while a small percentage of the glomeruli showed
slight segmental evidence of glomerulosclerosis which was mainly
characterized by a minimal increase in mesangial matrix and focal
hypercellularity. There was low inter-individual variability in the
extent of glomerular changes. The glomerular injury score (GIS) in
Group 1 (saline/vehicle) was 0.09.+-.0.01 (FIG. 83).
[1107] In comparison, animals receiving puromycin alone (Group 2)
showed histological changes in more than 90% of glomeruli (FIG. 84)
with a GIS of 1.50.+-.0.06 (FIG. 83). Changes (FIG. 85 D-F)
included a mild to moderate increase in mesangial matrix
accompanied by a variable hypercellularity of the glomerular tuft.
The number of mesangial cells appeared often slightly increased.
The presence of large and pale cells was also noted. These pale
cells are likely enlarged podocytes with the presence of occasional
macrophages. A small percentage of glomeruli showed a greater
degree of glomerular injury with a thickening of the Bowman's
capsule and hypertrophy/hyperplasia of parietal epithelial cells in
addition to changes in the glomerular tuft. It some cases,
glomerular changes were mainly associated with increased PAS
positive material in the glomerular tuft and with a slight increase
in cellularity. More than 80% of glomeruli were graded with a score
of 2 or 3, and some Grade 4 glomeruli were observed. These Grade 4
glomeruli were characterized by an almost global sclerosis and a
significant decrease in cellularity. They were representative of
terminal glomerulosclerosis.
[1108] Glomeruli in Group 3 (PAN/XG-102, 4 i.v.) were less affected
in percentage (76.9%, FIG. 84) and severity in comparison to Group
2 (PAN/vehicle) animals. The Group 3 (PAN/XG-102) GIS was
0.94.+-.0.05 (FIG. 83) and significantly different compared to
Group 2 (P<0001). The glomerular changes were associated with
segmental hypercellularity of the mesangial cells often accompanied
by a slight increase in mesangial matrix deposition (FIG. 85 G-I)
as described for Group 2 (PAN/vehicle) animals. There were also
certain glomeruli showing a slight increase number of large and
pale podocytes, as observed mainly in group 4 and 5, but not as
much in Group 2 (PAN/vehicle). The percentage of affected glomeruli
was significantly lower than that observed in Group 2 (FIG. 7,
P<0.001). A clear difference in the percentage of Grade 1 and
Grade 2 glomeruli was noted between the groups: Group 3 animals
showed an average of 61% of glomeruli with a Grade 1 in comparison
to 37% for Group 2, and an average of 15% of glomeruli with Grade
2, whereas the average was 46% in Group 2.
[1109] In Group 4 (PAN/XG-102, 2 i.v.; FIG. 85 J-L), the glomerular
changes were a mixture of segmental membranoproliferative to more
diffuse proliferative glomerulosclerosis. The GIS was 1.26.+-.0.06
(FIG. 83) and significantly different in comparison to 1.50.+-.0.06
for Group 2 (P<0.01). This difference was mostly attributable to
higher percentage of Grade 1 glomeruli combined to a lower
percentage of Grade 2 glomeruli when compared to Group 2.
[1110] In Group 5 (PAN/XG-102, 1 i.v.; FIG. 85 M-O), the GIS was
comparable to Group 2 (1.53.+-.0.05, FIG. 83). At the histology
level, glomerular changes were often due to both hypercellularity
(mesangial cells) and an increase in mesengial matrix, as observed
in Group 2. The respective percentages of affected glomeruli in
each Grade (FIG. 84) were very comparable between the 2 groups.
[1111] Day 77 (groups 6-8).
[1112] As observed at Day 49, all naive control animals (Group 6:
saline/vehicle; FIG. 86 A-C) presented a high percentage of normal
glomeruli (>60-90% Grade 0, FIG. 84). Histologically, the
glomerular changes were identical to that observed in Group 1
(saline/vehicle, day 49) and consisted, when present, in a minimal
and segmental increase in both mesangial matrix and
cellularity.
[1113] Group 7 (PAN/vehicle; FIG. 86 D-F) showed a GIS of
1.39.+-.0.10 (FIG. 83) and significantly different compared to
Group 6 (saline/vehicle, P<0.001). Three animals of this group
(rats n.degree. 74, 111, and 115) were excluded due to a large
difference with the Group average (>2 SD from the mean).
Histologically, glomerular lesions ranged from a minimal to mild
segmental membranoproliferative glomerulosclerosis (Grade 1 and 2)
to a moderate to severe terminal glomerulosclerosis (Grade 3 and
4). The percentage of affected glomeruli (91%) was comparable to
that observed in Group 2 (90%) at day 49 (FIG. 84).
[1114] In comparison to Group 7 (PAN/vehicle), animals in Group 8
(PAN/XG-102, 1 i.v.) presented a significant decrease of GIS
(0.82.+-.0.04 vs 1.39.+-.0.10, FIG. 83; P<0.001). Group 8
(PAN/XG-102, 1 i.v.) presented also a lower percentage of affected
glomeruli (69%) in comparison to 91% of Group 7 (PAN/vehicle, FIG.
85; P<0.001). Histologically, glomerular changes when present in
Group 8 were characteristic of a segmental membrano-proliferative
glomerulosclerosis (FIG. 86 G-I), as described in Group 3
(PAN/XG-102, 4 i.v.) animals at day 49.
[1115] In summary, the glomerular changes observed in rats
receiving puromycin were histologically consistent to what has been
described in the literature (Hill, 1986) and in Example 21. The
lesions consisted of a membranoproliferative and progressive
glomerulopathy with evidence of increased mesangial cell number,
presence of large and pale cells, and increased mesangial matrix.
XG-102 significantly reduced the extent and severity of glomerular
changes when administered (i) by 4 i.v. (weekly, Group 3) and 2
i.v. (every 2-weeks, Group 4) compared to Group 2 (PAN/vehicle) at
day 49; and (ii) by 1 i.v. (Group 8) compared to Group 7
(PAN/vehicle) at day 77 (2 months after administration).
[1116] These results show that XG-102 has a curative effect: (i)
four (weekly administration) and two (every 2 weeks administration)
i.v. injections of XG-102 at the dose of 4 mg/kg significantly
reduced PAN-induced glomerulosclerosis in term of severity of
lesions (glomerular injury score) but also significantly decreased
glomerular damage incidence (percentage of injured glomeruli) at
day 49; (ii) single i.v. injection of XG-102 at the dose of 4 mg/kg
also lead to a strong effect on glomerulosclerosis in term of both
severity of lesions (glomerular injury score) and of glomerular
damage incidence (percentage of injured glomeruli) at day 77 (2
months after administration); and (iii) the duration of action of
XG-102 is considered to be up to 2 months. Taken together, even a
single injection of XG-102 caused a strong long-term effect
observed on day 77.
Example 42: Evaluation of XG-102 (SEQ ID NO: 11) in Langerhans
Islet Isolation and Transplantation
[1117] This study is based on the previous studies on porcine and
rat islet isolation and transplantation (cf. Examples 17, 22 and
40) and aims at determining the effects of XG-102 on human islet
function. To this end, the same hypoxia model was used as described
in Example 40 for rat islets.
[1118] Briefly, human islets were pre-treated or not with 100
microM XG-102 for 1 h and then submitted to hypoxia during 24 h
still in presence or not of the inhibitor XG-102.
[1119] As shown in FIG. 87 relating to the impact of ischemia and
XG-102 on islet viability, it was again observed that XG-102
decreases apoptosis and necrosis under hypoxia conditions. In
particular, FIG. 87A shows that XG-102 decreased necrosis either in
normoxic and hypoxic conditions. FIG. 87B shows that XG-102 also
decreases apoptosis induced by hypoxia. These results show that
XG-102 has a beneficial effect on islet viability in the hypoxia
model.
Example 43: Evaluation of the Action Duration of XG-102 (SEQ ID NO:
11) in a Rat Model of Endotoxin-Induced Uveitis Following
Subconjunctival Administration
[1120] Acute anterior uveitis is a recurrent inflammatory disease
of the eye that occurs commonly and may have potentially blinding
sequalae. The pathogenesis of this disease is poorly understood.
Patients suffering from acute anterior uveitis complain of
photophobia (light sensitivity), which is frequently sever. Other
symptoms may include redness of the eye, tearing and reduced
vision. Findings on examination are characteristic and include
congestion of vessels, cells and protein flare in aqueous humor,
and miosis. In severe cases a hypopion and or fibrin may form.
Clinically, chronic progressive or relapsing forms of
non-infectious uveitis are treated with topical and/or systemic
corticosteroids. However, long-term use of these drugs can result
in deleterious ocular and systemic side effects such as glaucoma,
cataract, osteoporosis, hypertension and diabetes. Use of
alternative steroid-sparing, immunosuppressive agents has also
shown clinical benefit, but in themselves carry adverse risks.
Given these restrictions, there is an obvious demand for
development of new therapeutic strategies. Recent advances in
knowledge of the mechanisms of inflammatory resolution and the
discovery of several inflammatory mediators has led to a whole new
range of potential therapeutic possibilities.
[1121] The Endotoxin-Induced Uveitis (EIU) in the rat is a useful
animal model for human anterior uveitis. The systemic
administration of LPS results in an acute inflammatory response in
the anterior and posterior segment of the eye with a breakdown of
blood-ocular barrier and inflammatory cell infiltration. Clinical
signs of EIU reflect the changes seen in human disease. The
characteristic protein flare and cells in the aqueous humor, miosis
and posterior synechiae occur, as do fibrin clots and hypopion.
[1122] The aim of this study was to evaluate the duration of action
of SDD-1002 following sub-conjunctival administration in a rat
model of EIU.
[1123] 90 male Lewis rats were used, age approximately 6-8 weeks
(at the induction), 4 weeks (at the injection for the Day-28), 5
weeks (at the injection for the Day-21), 6 weeks (at the injection
for the Day-14, Day-7 and for the Day 0), 7 weeks (at the injection
for Day-2 and Day-1), and housed by five in standard cages. Animals
were allocated to the following groups: Group No. Treatment Dose
Time-point
TABLE-US-00054 Group No. Treatment Dose Time-point 1 XG-102 20 Day
-28 2 microgram/eye Day -21 3 Day -14 4 Day -7 5 Day -2 6 Day -1 7
Day 0 8 Saline (0.9% NaCl) -- 9 Solumedrol .RTM. 20
microgram/eye
[1124] Thus, each animal received a single subconjunctival
injection of either XG-102 (20 .mu.g/eye), saline (0.9% NaCl)
vehicle control or Solumedrol.RTM. (20 .mu.g/eye) into each eye.
Methylprednisolone (Solumedrol.RTM.) is most commonly used in
uveitis as sub-conjunctival treatment.
[1125] The schedule of the study is shown in the following:
TABLE-US-00055 Study date Procedure Ocular Sampling Baseline
General clinical examination - Weighing Slit-lamp -- D -28 General
clinical examination - Weighing -- -- sub-conjunctival
administration of test item (group 1) (20 .mu.g/eye, both eyes) D
-21 General clinical examination - Weighing -- -- sub-conjunctival
administration of test item (group 2) (20 .mu.g/eye, both eyes) D
-14 General clinical examination - Weighing -- -- sub-conjunctival
administration of test item (group 3) (20 .mu.g/eye, both eyes) D
-7 General clinical examination - Weighing -- -- sub-conjunctival
administration of test item (group 4) (20 .mu.g/eye, both eyes) D
-2 General clinical examination - Weighing -- -- sub-conjunctival
administration of test item (group 5) (20 .mu.g/eye, both eyes) D
-1 General clinical examination - Weighing -- -- sub-conjunctival
administration of test item (group 6) (20 .mu.g/eye, both eyes) D 0
General clinical examination - Weighing Slit-lamp before --
sub-conjunctival administration of test item induction (group 7),
control item (group 8), reference item (group 9) (20 .mu.g/eye,
both eyes) Induction of ocular inflammation for all groups D 1
Ocular clinical examination - Slit-lamp Aqueous humor for Weighing
Euthanasia leucocyte count and protein level
[1126] On Day 0, ocular inflammation was induced by a single
footpad injection of liposaccharide (LPS, 1 mg/kg, 0.5 mL/kg Sigma
# L6511) on anesthetized animals. LPS powder was reconstituted the
day of induction. XG-102 was administered by a single injection (20
microgram/5 microL) in each eye on Day-28 or Day-21 or Day-14 or
Day-7 or Day-2 or Day-1 or Day 0 (immediately before induction).
Saline control and reference item (Solumedrol.RTM.; 20 .mu.g/eye)
were administered by a single injection in each eye on Day 0
(immediately before induction).
[1127] Animals were examined with a slit-lamp before XG-102
administration (baseline) before induction (Day 0) then 24 h after
induction (Day 1). The inflammation was graded using a scoring
system as described by Devos A., Van Haren M., Verhagen C., Hoek
Zema R., Kijlstra A: Systemic anti-tumor necrosis factor antibody
treatment exacerbates Endotoxin Induced Uveitis in the rat. Exp.
Eye. Res. 1995; 61: 667-675. Briefly, flare, miosis and hypopion
were scored for absence (0), or presence (1), iris hyperemia and
cells in the anterior chamber were scored for absence (0), or mild
(1) or severe presence (2). The maximum score (sum of the five
parameter scores) was 7.
[1128] At the end of the evaluation (24 h after induction), animals
were euthanized by intravenous injection of overdosed
pentobarbital. The aqueous humor was collected immediately for each
eye. For quantification of Cellular Infiltration in Aqueous Humor
(AH), the sample was diluted 10-fold with PBS before detection. The
number of infiltrated cells was manually counted after Giemsa
staining under microscope.
[1129] Results:
[1130] 1. Ocular Evaluation
[1131] The pathologic symptoms of EIU in Lewis rat eyes injected
with LPS and treated with vehicle, test item or reference were
graded in blinded fashion with a slit-lamp microscope to evaluate
its efficacy. The results are illustrated in FIG. 88A and
summarized below:
TABLE-US-00056 Mean .+-. Reduction SEM of clinical Treatment (n =
20) Median scores Vehicle (5 .mu.L/eye, both eyes treated on the
day of induction) 4.0 .+-. 0.2 4.0 -- Methylprednisolone (20
.mu.g/eye, both eyes treated on the day 2.0 .+-. 0.2 2.0 50% of
induction) SDD-1002 (20 .mu.g/eye, both eyes treated on the day of
2.8 .+-. 0.2 3.0 30% induction) SDD-1002 (20 .mu.g/eye, both eyes
treated 1 day before the 1.6 .+-. 0.1 1.5 60% induction) SDD-1002
(20 .mu.g/eye, both eyes treated 2 days before the 1.8 .+-. 0.2 2.0
55% induction) SDD-1002 (20 .mu.g/eye, both eyes treated 7 days
before the 3.3 .+-. 0.2 3.0 18% induction) SDD-1002 (20 .mu.g/eye,
both eyes treated 14 days before the 2.9 .+-. 0.2 3.0 28%
induction) SDD-1002 (20 .mu.g/eye, both eyes treated 21 days before
the 3.1 .+-. 0.3 3.0 23% induction) SDD-1002 (20 .mu.g/eye, both
eyes treated 28 days before the 3.3 .+-. 0.2 4.0 18% induction)
Reduction: (mean grade in vehicle-treated eye - mean grade in test
item-treated eye)/(mean grade in vehicle-treated eye)
[1132] Twenty-four hours after LPS induction, clinical scores for
the vehicle-treated rats were 4.0.+-.0.2 (mean.+-.SEM, n=20) with
median of 4 (range, 2-5).
[1133] A reduction in the severity of the ocular inflammation was
detected 24 hours after induction and treatment with XG-102. The
reduction was higher particularly as the delay between the
induction and the treatment is short. The maximal reduction was
observed when XG-102 was administered 1 day before induction. The
mean score was 1.6.+-.0.1 with median of 1.5 (-60%, p<0.001
compared with vehicle). The reduction was less marked (18 to 23%)
when XG-102 was administered 7, 21 or 28 days before, but was
significant when XG-102 was administered 14 days before induction
(28%, p<0.05). Sub-conjunctival treatment with
methylprednisolone (20 .mu.g/eye, both eyes treated), used as
positive control drugs also significantly reduced the clinical
scores by 50% (mean score: 2.0.+-.0.2, median: 2).
[1134] 2. Cellular Infiltration in Aqueous Humor
[1135] Twenty-four hours after LPS Induction, the number of
inflammatory cells that had infiltrated into the aqueous humor was
counted for each group. The results are illustrated in FIG. 88B and
summarized below:
TABLE-US-00057 Mean .+-. SEM Reduction of Treatment (n = 20) Median
leucocytes Vehicle (5 .mu.L/eye, both eyes treated on the day of
3236 .+-. 346 3215 -- induction) Methylprednisolone (20 .mu.g/eye,
both eyes treated on the 3170 .+-. 276 3385 2% day of induction)
SDD-1002 (20 .mu.g/eye, both eyes treated on the day of 2226 .+-.
192 2005 31% induction) SDD-1002 (20 .mu.g/eye, both eyes treated 1
day before the 1668 .+-. 149 1540 48% induction) SDD-1002 (20
.mu.g/eye, both eyes treated 2 days before the 1844 .+-. 232 1500
43% induction) SDD-1002 (20 .mu.g/eye, both eyes treated 7 days
before the 2878 .+-. 331 2473 11% induction) SDD-1002 (20
.mu.g/eye, both eyes treated 14 days before the 976 .+-. 143 648
70% induction) SDD-1002 (20 .mu.g/eye, both eyes treated 21 days
before the 1029 .+-. 164 1023 68% induction) SDD-1002 (20
.mu.g/eye, both eyes treated 28 days before the 1260 .+-. 263 915
61% induction)
[1136] The median value number of inflammatory cells in the aqueous
humor of vehicle-treated eyes was 3236 cells/.mu.L (range 270-6140
cells/.mu.L). The withdrawal of aqueous humor could not be
performed in 2 out of 20 injured eyes in vehicle group; the
formation of fibrin clot blocked the needle during the withdrawal
process. Rats treated with XG-102 showed a significantly reduced
number of infiltrating cells compared with that of vehicle whatever
the delay between treatment and the day of induction. Rats treated
with methylprednisolone did not have significant difference in the
number of infiltrating cells with that of vehicle. A dose similar
to dexamethasone (20 .mu.g) and to the test item was used.
Regarding to the leucocyte infiltration, methylprednisolone was
less potent than dexamethasone at the same dose (data from previous
studies). In clinic, methylprednisolone is used regionally with
typical doses ranging from 40-125 mg whereas dexamethasone acetate
is used with doses ranging from 4-8 mg.
[1137] Conclusion:
[1138] The result herein demonstrates that single sub-conjunctival
injection of XG-102 in both eyes partially prevented the
endotoxin-induced inflammation observed in the anterior chamber,
since a significant reduction of clinical scores and cellular
infiltration were observed. The XG-102 is active up to 28 days on
the inflammatory EIU model in the rat. The efficacy on clinical
scores was observed up to 4 weeks, with a marked effect the first
two days and on cellular infiltration in aqueous humor up to 4
weeks with a marked effect at 2, 3 and 4 weeks. The
methylprednisolone (20 .mu.g/eye, both eyes treated) could not show
any significant efficacy on cellular infiltration even if a
reduction of clinical scores was observed. This lack of efficacy
(compared to previous data with dexamethasone) may be related to
low administered dose.
Example 44: Effects of XG-102 (SEQ ID No. 11) in a Rat Model of
Diabetic Retinopathy
[1139] This study is based on the previous studies of XG-102 in
diabetic retinopathy as described in Examples 25, 26 and 39. The
objective of this study is to determine the action duration of
XG-102 on loss of visual acuity, ocular clinical signs, retinal
layer thickness, and cytokine profiling after repeated
sub-conjunctival administration on varying frequencies in a rat
model of streptozotocin (STZ)-induced diabetic retinopathy.
[1140] To this end, 36 rats (female, Brown Norway, 6-8 weeks at
time of STZ-treatment are assigned to the following 6 groups (6
animals per group):
TABLE-US-00058 Group STZ Treatment Assessment 1 - Vehicle Weekly
recording of body weight and NaCl 0.9% blood glucose levels (Weeks
1-16); 2 + Vehicle Weekly Draize scoring of chemosis, NaCl 0.9%
hyperemia, and discharge (Weeks 1-16); 3 + XG-102 Quantification of
contrast threshold at (200 .mu.g/eye at 3- Days 43, 57, 71, 85, 99,
and 106; week intervals) Quantification of spatial frequency 4 +
XG-102 threshold at Days 43, 57, 71, 85, 99, (200 .mu.g/eye at 4-
and 106; week intervals) Multiplex cytokine quantification of 5 +
XG-102 retinas using Bio-Rad rat 23-plex kit (200 .mu.g/eye at 6-
(n = 8 retinas/arm) week intervals) Enucleation of eyes for
quantitative 6 + XG-102 retinal histology (200 .mu.g/eye at 12- (n
= 4 eyes/arm - each eye from a week intervals) separate animal)
[1141] Groups 1, 2, and 5 were treated by bilateral
sub-conjunctival administration of vehicle or XG-102, respectively,
(cf. above) on Days 22 and 64. Group 3 was treated by bilateral
sub-conjunctival administration of XG-102 on Days 22, 43, 64 and
85. Group 4 was treated by bilateral sub-conjunctival
administration of XG-102 on Days 22, 50 and 78. Group 6 was treated
by bilateral sub-conjunctival administration of XG-102 on Day
22.
[1142] The experimental design is the following:
[1143] Day 1: IP injection of streptozotocin (groups 2-6)
[1144] Day 4: Blood glucose quantification
[1145] Day 22: Bilateral subconjunctival injection of vehicle or
test agent (Groups 1-6)
[1146] Day 43: OKT assessment of contrast sensitivity and spatial
frequency threshold
[1147] Day 43: Bilateral subconjunctival injection of vehicle or
test agent (Group 3)
[1148] Day 50: Bilateral subconjunctival injection of vehicle or
test agent (Group 4)
[1149] Day 57: OKT assessment of contrast sensitivity and spatial
frequency threshold
[1150] Day 64: Bilateral subconjunctival injection of vehicle or
test agent (Groups 1-3, and 5)
[1151] Day 71: OKT assessment of contrast sensitivity and spatial
frequency threshold
[1152] Day 78: Bilateral subconjunctival injection of vehicle or
test agent (Group 4)
[1153] Day 85: OKT assessment of contrast sensitivity and spatial
frequency threshold
[1154] Day 85: Bilateral subconjunctival injection of vehicle or
test agent (Group 3)
[1155] Day 99: OKT assessment of contrast sensitivity and spatial
frequency threshold
[1156] Day 106: OKT assessment of contrast sensitivity and spatial
frequency threshold
[1157] Day 107: Collection of tissue [1158] n=4 eyes/group
collected for quantitative retinal histology [1159] n=8
retinas/group collected multiplex cytokine analyses
[1160] *OKT: optokinetic tracking
[1161] For streptomycin administration, rats of the same age are
weighed the day prior to injections and are fasted overnight, and
cages are marked with yellow cards in animal facility. The weights
are averaged, and a single dose is calculated for all rats based
upon the average weight. No more than ten animals are injected with
a single preparation of STZ due to quick decrease of STZ activity
in solution, and the procedure is repeated for each batch of 10
animals. STZ powder is dissolved in 10 mM sodium citrate, pH 4.5,
immediately before injection and rats receive 50 mg/kg STZ
intraperitoneally in a volume of 1 mL using a 22 gauge syringe with
care to avoid stomach and any vital organs.
[1162] For subconjunctival administration, animals are anesthetized
with ketamine/xylazine (Ketamine and xylazine are mixed using a
U-100 syringe utilizing 20 units of ketamine (100 mg/mL) and 100
units of xylazine (20 mg/mL) and the anesthesia mixture is applied
via IP injection at 1 mL/kg (body weight)) and pupils are dilated
with topical administration of Cyclogel and/or Tropicamide.
Following sedation and dilation, a total volume of 30 .mu.L per eye
is injected into conjunctiva using a 31-gauge needle attached to an
insulin syringe.
[1163] For Draize scoring of hyperemia, chemosis, and discharge,
animals are restrained by hand and scores for chemosis, hyperemia,
and discharge are recorded by a masked observer using the "EyeCRO
ocular scoring system".
[1164] All optokinetic tracking experiments are performed using an
Optomotry designed for rodent use (Cerebra) Mechanics Inc.). In
this non-invasive assessment, rats are placed onto a platform
surrounded by 4 LCD screens which resides within a light-protected
box. Visual stimuli are then presented to the rats via the LCD
screens and a masked observer visualizes and scores optokinetic
tracking reflexes from a digital camcorder which is mounted on the
top of the box. For measurements of spatial frequency threshold,
the rats are tested at a range of spatial frequencies from 0.034 to
0.664 cycles/degree. The Optomotry device employs a proprietary
algorithm to accept the input from the masked observer and
automatically adjust the testing stimuli based upon whether the
animal exhibited the correct or incorrect tracking reflex. All
measurements of contrast threshold are performed at a spatial
frequency threshold of 0.064 cycles/degree.
[1165] For multiplex cytokine analysis, at study termination, the
retinas will be individually isolated and immediately snap-frozen
in liquid N.sub.2. The Bio-Rad "Bio-plex Pro Rat Cytokine 23-plex
assay (Cat# L80-01V11 S5) is used according to manufacturer's
specifications to quantify the protein expression of EPO, G-CSF,
GM-CSF, GRO/KC, IFN-.gamma., IL-1.alpha., IL-1.beta., IL-2, IL-4,
IL-5, IL-6, IL-7, IL-10, IL-12, p70, IL-13, IL-17A, IL-18, M-CSF,
MCP-1, MIP-3.alpha., RANTES, TNF-.alpha., and VEGF in each retina
isolated in the study.
Example 45: Effects of XG-102 (SEQ ID No. 11) in a Rat Model of
Kidney Bilateral Ischemia Reperfusion
[1166] This study is based on the previous study of XG-102 in renal
ischemia/reperfusion (Example 36). The aim of the study was to
evaluate the effect of XG-102 on histological damages in a rat
model of kidney bilateral ischemia reperfusion.
[1167] Ischemia reperfusion (IR) injury is a complex phenomenon,
which is often encountered in vascular surgery, organ procurement
and transplantation in humans. The experimental model of kidney
bilateral ischemia reperfusion (IR) in rodents leads to an acute
tubular injury characterized by impaired kidney function and
tubular degeneration. The present model is frequently used for
providing a rapid proof of concept for the use of drug candidates
in preventing renal IR damages.
[1168] Male Sprague-Dawley rats weighing 200-250 g at delivery were
used (Charles River Laboratories, L'Arbresle, France). Animals were
delivered to the laboratory at least 5 days before the experiments
during which time they were acclimatized to laboratory conditions.
This study included 3 groups of 11-12 rats each, as follow:
TABLE-US-00059 Number of Group IR surgery Treatment (i.v.)
animals/group 1 Sham-operated vehicle 12 2 yes vehicle 11 3 yes
XG-102 (2 mg/kg) 12
[1169] The study design is shown in FIG. 96.
[1170] The protocol of warm renal ischemia was similar to that
previously described (Pechman K R et al., 2009). Briefly, under
general anesthesia (pentobarbital; 60 mg/kg, i.p. and atropine; 1
mg/kg, i.p.), both renal pedicles were isolated and clamped for 40
minutes using atraumatic clamps. After this time, clamps were
released to start reperfusion. Animals were maintained at
37.degree. C. using a thermo-regulated system (TCAT-2LV Controller,
Physitemp Instruments, Clifton, N.J., USA) during the surgery. All
the animals were sacrificed 24 hours after the release of both
vascular clamps (reperfusion). Sham-operated animals underwent the
same surgical procedure without clamping of the kidney vessels.
[1171] XG-102 or vehicle (0.9% NaCl) were administered into the
tail vein (i.v.) at the dose of 2 mg/kg twenty minutes after the
release of the second vascular clamp. Intravenous administrations
into the tail vein were performed using the volume of 1 mL/kg.
[1172] After sacrifice, kidneys were removed, cleaned from all
connective tissue and capsule and weighted on an electronic balance
(VWR, France). One kidney was transferred in formalin solution 10%
(Sigma Aldrich, France) for at least 24 h and then transferred in
ethanol 70% for further histological analysis performed by Histalim
(Montpellier, France). Right and left kidneys were randomly chosen.
Kidney samples were fixed in 10% formalin during 72 hours,
transferred into 70% ethanol, then embedded in paraffin blocks by
Histalim (Montpellier, France). One longitudinal section (3 to 5
.mu.m) was made per block. Kidney sections of paraffin embedded
tissue were stained by hematoxylin and eosin (H&E). All the
slides were digitalized at .times.20 magnitude using Nanozoomer 2.0
HT from Hamamatsu (Hamamatsu, Japan). Each tissue section was
examined histologically in a blinded manner to determine if tubular
changes were present. The severity of each finding was then graded
as follows: Tubular damage score consisted of either
degeneration/necrosis, tubular epithelial vacuolation, regeneration
(basophil tubules), and tubular cast:
[1173] 0: <5% tubules affected (background)
[1174] 1: 5-20% of tubules affected
[1175] 2: 21-40% of tubules affected
[1176] 3: 41-75% of tubules affected
[1177] 4: >75% of tubules affected
[1178] As shown in FIG. 97, Group 2 (IR/Vehicle) animals showed a
significant increase of tubular damages including tubular
degeneration and necrosis, tubular cast formation, and basophilic
tubules compared to Sham/Vehicle animals. XG-102 showed significant
beneficial effects on tubular damages, specifically on tubular
degeneration, necrosis and tubular cast formation (FIG. 97) and on
the total tubular score (FIG. 98). The main difference in term of
tubular degeneration and necrosis between animals from XG-102
treated rats (Group 3) and vehicle (Group 2) animals is that the
number of tubules affected was lower, and the lesions were mostly
limited to the cortico-medullary junction and not extended to the
superficial cortex. Kidneys from Group 3 (IR/XG-102) presented also
a less severe score for tubular casts when compared to Group 2
(IR/Vehicle). Representative images of these histologicals changes
are included in FIG. 99.
[1179] In particular, tubular changes in Group 1 (Sham/Vehicle)
were limited to the presence of single to a few basophilic tubules
(Score 1) in 3/12 animals (FIG. 97). This incidence is within
expected normal limits in naive young adult control rats and was
considered as incidental in origin. Comparatively, all animals in
Group 2 (IR/Vehicle) presented moderate to marked (Score 3 and 4)
tubular epithelial degeneration and necrosis (3.45.+-.0.52). The
most affected tubules were concentrated at the cortico-medullary
junction and were histologically characterized by tubules
containing large clumps of sloughed and necrotic epithelial cells.
Tubular degenerative lesions were also present in most of the
cortex in animals with the most severe lesions (Score 4). In
addition to tubular degeneration, all animals showed a large number
of tubular casts in lumen (Score 3). The presence of small to
moderate number of basophilic tubules (Score 1 and 2,
mean=1.36.+-.0.67) was also observed throughout the cortex in 10/11
animals of Group 2 (IR/Vehicle). The basophilic tubules were
indicative of early epithelial regeneration in tubules. For Group 3
(IR/XG-102), tubular lesions were essentially of the same nature
and appearance to that observed in Group 2 (IR/Vehicle), but were
generally less severe in distribution.
[1180] More specifically, the mean tubular epithelial
degeneration/necrosis score was 2.67.+-.0.65 in Group 3
(IR/XG-102). The main difference between Group 2 (IR/Vehicle) and
Group 3 (IR/XG-102) was that several animals in the latter group
showed a score of 2 (5/12 in Group 3 and 0/11 in Group 2). Finally,
only 1/12 animal in Group 3 had a score of 4 comparatively to 5/11
for Group 2. Histologically, the main difference in term of tubular
degeneration and necrosis between animals from Group 3 (IR/XG-102)
in comparison to Group 2 (IR/Vehicle) was that the number of
tubules affected was lower, and the lesions were mostly limited to
the cortico-medullary junction and were not extended to the
superficial cortex. Group 3 (IR/XG-102) and kidneys presented also
a less severe score for tubular casts when compared to Group 2
(IR/Vehicle). Actually, tubular cast scores were 2.50.+-.0.52 in
Group 3 (IR/XG-102). In comparison, Group 2 (IR/vehicle) tubular
cast score was 3.00.+-.0.00. The number of basophilic tubules in
Group 3 (IR/XG-102) were very comparable to that observed in Group
2. The mean basophilic tubule score for Group 3 (IR/XG-102) was
1.33.+-.0.65; the score for Group 2 was 1.36.+-.0.67 (FIG. 97).
[1181] There was no tubular vacuolation observed in any of the four
experimental groups. Accordingly, the total tubular score in Group
1 (Sham/Vehicle) was very low as expected (0.25.+-.0.45) since only
few animals presented basophilic tubules without any other tubular
changes. In Group 2, the total tubular score was the highest among
the four experimental groups, and ranged from 6 to 9
(7.82.+-.0.98). Group 3 total tubular score was relatively lower to
that observed in Group 2 (IR/vehicle) with scores ranging from 5 to
8 (6.50.+-.0.80). The differences observed between Group 2
(IR/vehicle) and Group 3 (IR/XG-102) were considered to be
biologically significant.
[1182] Taken together, XG-102 showed significant beneficial effects
on tubular damages and specifically on tubular degeneration,
necrosis and tubular cast formation. The main difference in term of
tubular degeneration and necrosis between animals from XG-102
treated rats (Group 3) and vehicle (Group 2) IR animals is that the
number of tubules affected was lower, and the lesions were mostly
limited to the cortico-medullary junction and not extended to the
superficial cortex. Kidneys from Group 3 (IR/XG-102) presented also
a less severe score for tubular casts when compared to Group 2
(IR/Vehicle).
Example 46: Effects of XG-102 (SEQ ID No. 11) Administered
Intravesically on Acute Cystitis Model Induced by Cyclophosphamide
in Conscious Rats: Evaluation of Visceral Pain and Urinary Bladder
Inflammation
[1183] The aim of the present study was to evaluate the effects of
intravesical treatment with XG-102 (50 mg/mL) on urinary bladder
pain and inflammation in acute CYP-induced cystitis in female
Sprague-Dawley rats. This preclinical model is well-used to test
therapeutic approaches for the treatment of interstitial
cystitis/painful bladder syndrome (IC/PBS).
[1184] Adult female Sprague-Dawley rats (Janvier Labs, Le Genest
Saint Isle, France), weighing 215.+-.20 g at the beginning of the
experiments, were used. Animals were acclimatized to the laboratory
conditions for at least 3 days before the start of any experiments.
The animals were allocated to the following four experimental
groups (n=10 animals per group):
TABLE-US-00060 Group Injection (i.p.) Treatment (i.ves.) n 1 Saline
Vehicle (500 .mu.L, i.ves.) 10 2 CYP Vehicle (500 .mu.L, i.ves.) 10
3 CYP XG-102 (50 mg/mL, i.ves.) 10 4 CYP Ibuprofen (50 mg/mL,
i.ves.) 10
[1185] To induce acute cystitis, a single i.p. injection of CYP at
a dose of 150 mg/kg in a final volume of 5 mL/kg was performed.
Control rats received physiological saline under the same
experimental conditions as CYP (final volume of 5 mL/kg, i.p.).
[1186] On the day of each experiment, weight of rats was recorded.
Then, in a randomized manner, 500 .mu.L of XG-102 (50 mg/mL),
ibuprofen (50 mg/mL) or vehicle were intravesically infused during
30 min under isoflurane anesthesia (2%-3%).
[1187] Assessment of Referred Visceral Pain Using Von Frey
Filaments:
[1188] Standardized conditions including fixed time-of-day (a.m. to
minimize the potential circadian variations in the behaviours
responses) and single-experimenter testing of all animals were
applied to minimize variability behavior-based pain testing.
Visceral pain including allodynia and hyperalgesia was evaluated by
applying to the lower abdomen, close to the urinary bladder, a set
of 8 calibrated von Frey filaments of increasing forces (1, 2, 4,
6, 8, 10, 26 and 60 g) with an interstimulus interval of 5 seconds.
Prior testing, the abdominal area designed for mechanical
stimulation of each animal was shaved. Animals were then placed on
a raised wire mesh floor under individual transparent Plexiglas box
and acclimatized for at least 30 minutes before starting the von
Frey test. Filaments were then applied 1-2 seconds through the mesh
floor with enough strength to cause the filament to slightly bend.
Each filament was tested 3 times. Care was taken to stimulate
different areas within the lower abdominal region in the vicinity
of the urinary bladder to avoid desensitization.
[1189] Nociceptive behaviors were scored for each animal and each
filament as follows:
TABLE-US-00061 Score Behavior 0 no response 1 reaction of the
animal (e.g. retraction of the abdomen) 2 reaction of the animal
and change of position 3 reaction of the animal, change of position
and licking of the site stimulated with von Frey filaments
and/or
[1190] The study design is schematically shown in FIG. 100 A.
Birefly, acute cystitis was induced by CYP injection (i.p.) at D0
(as described above). XG-102, ibuprofen or vehicle was
intravesically administrated once just after CYP injection (as
described above). Von Frey testing was performed in a non-blinded
manner as follow: [1191] At D-1, rats were acclimatized to the
individual Plexiglas box for a minimum of 30 min and to the von
Frey filaments application, in order to decrease the level of
stress due to the new environment. [1192] At D0, von Frey testing
was performed 15 min before CYP or saline injection in order to
obtain basal values (D0, T=-15 min). [1193] At D1, von Frey testing
was performed 24 hours after CYP or saline injection in order to
analyze test compounds effect on CYP-induced visceral pain (D1,
T=+24 h). [1194] Just after von Frey testing (+24 h), rats were
anesthetized for blood samples collection, then sacrificed and
urinary bladders were collected as described below.
[1195] At the end of the experiment, rats were sacrificed by
injection of pentobarbital (54.7 mg/mL, 0.5 mL/rat, i.p.) followed
by cervical dislocation. Urinary bladders were rapidly collected
and cleaned from lipoid tissue. Urinary bladders were weighed, cut
at the bladder neck and haemorrhage scoring was performed (see
table below). Finally, wall thickness was measured using a digital
caliper by placing the bladder wall between the two outside jaws.
Urinary bladder haemorrhage scores were adapted from Gray's
criteria (Gray et at, 1986) as follows:
TABLE-US-00062 Scores Haemorrhage 0 absent - normal aspect 1
telangiectasia - dilatation of the mucosal blood vessels 2
petechial haemorrhages - mucosal pinpoint red dots (glomerulation)
3 Hemorrhagic spots with blood clots
[1196] Nociceptive parameters are expressed as follows:
TABLE-US-00063 Parameters Expression Description nociceptive g von
Frey filament for which a first threshold score of at least 1 (for
3 applications) is obtained nociceptive scores % % of the maximal
response (maximum score = 9) for 3 pooled applications area under
the % plot of individual percentage of curve (AUC) scores .times. g
nociceptive scores against 1-8 g (allodynia) von Frey forces from:
area under the 1 to 8 g or 8 to 60 g curve (AUC) 8-60 g
(hyperalgesia)
[1197] AUCs were calculated using GraphPad Prism.RTM. (GraphPad
Software Inc., La Jolla, Calif., USA). The AUCs method to assess
allodynia and hyperalgesia is schematically shown in FIG. 100
B.
[1198] Macroscopic parameters are expressed as follows:
TABLE-US-00064 Parameters Expression whole urinary bladder weight
mg and % of body weight haemorrhage scores urinary wall thickness
mm
[1199] Results:
[1200] Before CYP injection, no significant difference in the
nociceptive parameters were observed between the 3 different
CYP-injected groups. In order to analyse effect of XG-102 on
CYP-induced visceral pain, nociceptive parameters were compared
between the Vehicle- and the XG-102-treated groups. Twenty-four
hours after CYP injection, nociceptive threshold was significantly
increased by XG-102 treatment as compared to vehicle (p<0.01,
FIG. 101 A). XG-102 treatment also significantly decreased
nociceptive scores in CYP-injected rats as compared to vehicle
(p<0.001, FIG. 101 B). In addition, AUC 1-8 g was significantly
decreased by XG-102 treatment as compared to vehicle (p<0.001,
FIG. 101 C). Similarly, AUC 8-60 g was reduced by XG-102 treatment
as compared to vehicle (p<0.01, FIG. 101 D). In order to analyse
the effects of ibuprofen on CYP-induced visceral pain, nociceptive
parameters were compared between the Vehicle- and the
Ibuprofen-treated groups. Nociceptive threshold was significantly
increased by ibuprofen treatment as compared to vehicle in CYP
injected rats (p<0.01, FIG. 101 A). Similarly in the Ibuprofen
group significant decrease of nociceptive scores was observed as
compared to vehicle (p<0.01, FIG. 101 B). In addition, AUC 1-8 g
and AUC 8-60 g were significantly decreased by ibuprofen treatment
as compared to vehicle (p<0.001 and p<0.05, FIGS. 101 C and
101 D, respectively).
[1201] Moreover, urinary wall thickness was significantly decreased
in XG-102-treated rats (p<0.01, FIG. 102 A). Although XG-102
treatment also showed a trend towards decreased haemorrhage scores,
it did not reach statistical significance (FIG. 102 B). For
ibuprofen, also a significant decrease was observed in urinary
bladder wall thickness (p<0.001, FIG. 102 A). However, no
significant change was observed regarding haemorrhage scores
(p>0.05, FIG. 102 B) in the Ibuprofen-treated group. It is
noteworthy that reddish urine was noticed for some animal in the
Ibuprofen-treated group.
[1202] Taken together, intravesical treatment of XG-102 (50 mg/mL)
significantly reversed visceral pain induced by CYP, 24 h after its
injection. XG-102 efficiently inhibited both allodynia and
hyperalgesia. On analyzed inflammatory parameters, XG-102 decreased
urinary bladder inflammation (wall thickness). In conclusion,
administered intravesically, XG-102 displayed strong
antinociceptive effects and significant anti-inflammatory
properties in an experimental model of IC/PBS.
Example 47: Effects of XG-102 (SEQ ID No. 11) Administered
Intravenously on Acute Cystitis Model Induced by Cyclophosphamide
in Conscious Rats: Evaluation of Visceral Pain
[1203] The aim of the present study was to evaluate the effects of
intravenous treatment with XG-102 (2 mg/kg) on urinary bladder pain
in acute CYP-induced cystitis in female Sprague-Dawley rats. This
preclinical model is well-used to test therapeutic approaches for
the treatment of interstitial cystitis/painful bladder syndrome
(IC/PBS).
[1204] Adult female Sprague-Dawley rats (Janvier Labs, Le Genest
Saint Isle, France), weighing 215.+-.20 g at the beginning of the
experiments, were used. Animals were acclimatized to the laboratory
conditions for at least 3 days before the start of any experiments.
The animals were allocated to the following four experimental
groups (n=10 animals per group):
TABLE-US-00065 Group Injection (i.p.) Treatment (i.ves.) n 1 Saline
Vehicle (1 mL/kg, i.v.) 10 2 CYP Vehicle (1 mL/kg, i.v.) 10 3 CYP
XG-102 (2 mg/kg, i.v.) 10 4 CYP Ibuprofen (10 mg/kg, i.v.) 10
[1205] To induce acute cystitis, a single i.p. injection of CYP at
a dose of 150 mg/kg in a final volume of 5 m/kg was performed.
Control rats received physiological saline under the same
experimental conditions as CYP (final volume of 5 mL/kg, i.p.).
[1206] On the day of each experiment, weight of rats was recorded.
Then, in a randomized manner, XG-102 (2 mg/kg), ibuprofen (10
mg/kg) or vehicle were intravenously administered at a volume of 1
mL/kg.
[1207] Assessment of Referred Visceral Pain Using Von Frey
Filaments:
[1208] Standardized conditions including fixed time-of-day (a.m. to
minimize the potential circadian variations in the behaviours
responses) and single-experimenter testing of all animals were
applied to minimize variability behavior-based pain testing.
Visceral pain including allodynia and hyperalgesia was evaluated by
applying to the lower abdomen, close to the urinary bladder, a set
of 8 calibrated von Frey filaments of increasing forces (1, 2, 4,
6, 8, 10, 26 and 60 g) with an interstimulus interval of 5 seconds.
Prior testing, the abdominal area designed for mechanical
stimulation of each animal was shaved. Animals were then placed on
a raised wire mesh floor under individual transparent Plexiglas box
and acclimatized for at least 30 minutes before starting the von
Frey test. Filaments were then applied 1-2 seconds through the mesh
floor with enough strength to cause the filament to slightly bend.
Each filament was tested 3 times. Care was taken to stimulate
different areas within the lower abdominal region in the vicinity
of the urinary bladder to avoid desensitization.
[1209] Nociceptive behaviors were scored for each animal and each
filament as follows:
TABLE-US-00066 Score Behavior 0 no response 1 reaction of the
animal (e.g. retraction of the abdomen) 2 reaction of the animal
and change of position 3 reaction of the animal, change of position
and licking of the site stimulated with von Frey filaments
and/or
[1210] The study design differs from that of Example 46 (cf. FIG.
100 A) only in the route of administration (intravenously instead
of intravesically) and the doses as specified above. Birefly, acute
cystitis was induced by CYP injection (i.p.) at D0 (as described
above). XG-102, ibuprofen or vehicle was intravvenously
administrated once just after CYP injection (as described above).
Von Frey testing was performed in a non-blinded manner as follow:
[1211] At D-1, rats were acclimatized to the individual Plexiglas
box for a minimum of 30 min and to the von Frey filaments
application, in order to decrease the level of stress due to the
new environment. [1212] At D0, von Frey testing was performed 15
min before CYP or saline injection in order to obtain basal values
(D0, T=-15 min). [1213] At D1, von Frey testing was performed 24
hours after CYP or saline injection in order to analyze test
compounds effect on CYP-induced visceral pain (D1, T=+24 h). [1214]
Just after von Frey testing (+24 h), rats were anesthetized for
blood samples collection, then sacrificed and urinary bladders were
collected as described below.
[1215] Nociceptive parameters are expressed as follows:
TABLE-US-00067 Parameters Expression Description nociceptive g von
Frey filament for which a first threshold score of at least 1 (for
3 applications) is obtained nociceptive scores % % of the maximal
response (maximum score = 9) for 3 pooled applications area under
the % plot of individual percentage of curve (AUC) scores .times. g
nociceptive scores against von 1-8 g (allodynia) Frey forces from:
area under the 1 to 8 g or 8 to 60 g curve (AUC) 8-60 g
(hyperalgesia)
[1216] AUCs were calculated using GraphPad Prism.RTM. (GraphPad
Software Inc., La Jolla, Calif., USA). The AUCs method to assess
allodynia and hyperalgesia is schematically shown in FIG. 100
B.
[1217] Results:
[1218] Before CYP injection, no significant difference in the
nociceptive parameters was observed between the 3 different
CYP-injected groups. In order to analyse the effect of XG-102 on
CYP-induced visceral pain, nociceptive parameters were compared
between the Vehicle- and the XG-102-treated groups independently.
Twenty-four hours after CYP injection, nociceptive threshold was
significantly increased by XG-102 treatment as compared to vehicle
(p<0.01, FIG. 103 A). XG-102 treatment significantly decreased
nociceptive scores in CYP-injected rats as compared to vehicle
(p<0.001, FIG. 103 B). In addition, AUC 1-8 g was significantly
decreased by XG-102 treatment as compared to vehicle (p<0.001,
FIG. 103 C). Similarly, AUC 8-60 g was significantly reduced by
XG-102 treatment as compared to vehicle (p<0.001, FIG. 103 D).
In order to analyse effect of ibuprofen on CYP-induced visceral
pain, nociceptive parameters were compared between Vehicle- and
Ibuprofen-treated groups. Nociceptive threshold was significantly
increased by ibuprofen treatment as compared to vehicle in CYP
injected rats (p<0.01, FIG. 103 A). Ibuprofen treatment
significantly decrease nociceptive scores as compared to vehicle
(p<0.001, FIG. 103 B). In addition, AUC 1-8 g and AUC 8-60 g
were significantly reduced by ibuprofen treatment as compared to
vehicle (p<0.001, FIGS. 103 C and 103 D).
[1219] Taken together, intravenous treatment of XG-102 (2 mg/kg)
thus significantly reversed visceral pain induced by CYP, 24 h
after its injection. XG-102 efficiently inhibited both allodynia
and hyperalgesia. Similar effects were observed with intravenous
administration of ibuprofen (10 mg/kg). In conclusion, in the
experimental cystitis preclinical model, XG-102 displayed
significant anti-nociceptive properties.
Example 48: Effects of XG-102 (SEQ ID No. 11) Administered
Intravenously on Cystometric Parameters in Conscious Rats with
Acute Cystitis Induced by Cyclophosphamide
[1220] The aim of the present study was to evaluate the effects of
intravenous (i.v.) administration of XG-102 (2 mg/kg) on
cystometric parameters in CYP-induced cystitis in conscious female
Sprague-Dawley rats. This preclinical model is well-used to test
therapeutic approaches for the treatment of interstitial
cystitis/painful bladder syndrome (IC/PBS).
[1221] Female Sprague-Dawley rats (211-281 g) were used (Janvier
Labs, Le Genest Saint Isle, France). They were delivered to the
laboratory at least 5 days before the experiments in order to be
acclimatized to laboratory conditions. The animals were allocated
to the following three experimental groups:
TABLE-US-00068 Groups i.p. administration i.v. treatment dose n 1
Physiological saline Vehicle -- 11 2 CYP 150 mg/kg Vehicle -- 10 3
CYP 150 mg/kg XG-102 2 mg/kg 11
[1222] Rats were anesthetized with isoflurane (1.5-3%). After a
laparotomy, bladder was exteriorized and a polyethylene catheter
(0.58 and 0.96 mm of internal and outer diameter, respectively) was
implanted in the bladder through the dome and exteriorized at the
scapular level. A jugular polyethylene catheter (0.58 and 0.96 mm
of internal and outer diameter, respectively) was also implanted
and exteriorized at the scapular level for i.v. administrations. At
D-1 (24 hours after the surgery), a single dose of CYP at 150 mg/kg
or its vehicle (physiological saline: 0.9% NaCl) was administered
i.p. at 5 mL/kg.
[1223] The method evaluating the effects of test substances on
lower urinary tract function has been described by Lluel P, Barras
M, Palea S. Cholinergic and purinergic contribution to the
micturition reflex in conscious rats with long-term bladder outlet
obstruction. Neurourol Urodyn. 2002; 21: 142-153. Cystometric
investigations were performed in conscious rats 24 hours
postintraperitoneal injection of CYP or vehicle. On the day of
experiment, animals were held under partial restraint in a
restraining device. The bladder catheter was connected via a T-tube
to a pressure transducer to measure the intravesical pressure and
to an injection pump to fill the bladder at a rate of 2 mL/hr.
Vesical pressure was recorded continuously for 120 min: a 60 min as
a basal period before intravenous administration and a 60 min
period post-administration.
[1224] XG-102 or vehicle (1 mL in 5 min) was administered
intravenously after 1 hour of basal period.
[1225] The study design is schematically shown in FIG. 104 A.
[1226] The following cystometric parameters were analysed (see FIG.
104 B): [1227] Threshold Pressure (ThP, mmHg), pressure just before
micturition, [1228] Amplitude of micturition (AM), i.e. pressure
between threshold pressure (ThP) and maximal pressure of
micturition (MP) (mmHg), [1229] Intercontraction interval (ICI),
i.e. time between two subsequent micturitions (sec), and [1230]
Bladder capacity (BC), i.e. ICI.times.infusion rate (mL).
[1231] Results:
[1232] No effects of vehicle (i.v.) was observed on the cystometric
parameters ICI, BC, ThP and AM parameters in conscious rats treated
with CYP, compared to basal values (FIGS. 105 A, B, C and D). In
contrats, XG-102 (2 mg/kg, i.v.) significantly increased ICI and BC
30-60 min post-administration in CYP-treated rats, compared to
basal values (P<0.01, FIGS. 106 A and B). This increase was
associated with a significant decrease in ThP at the same time
point (P<0.01, FIG. 106 C).
[1233] Taken together, intravenous treatment of XG-102 (2 mg/kg)
significantly increased ICI and BC and decreased ThP for the period
of 30-60 min post administration.
Example 49: Effects of XG-102 (SEQ ID No. 11) on
.beta.-Amyloid-Induced Neuronal Apoptosis (Alzheimer's Disease
Model)
[1234] The effect of the JNK inhibitor XG-102 on JNK activation and
on neuronal apoptosis was investigated in two experiments. In a
first experiment the effect of different doses of XG-102 on JNK
activation after induction of oxidative stress was determined. In a
second experiment the effect of XG-102 on JNK activation and
neuronal apoptosis after A.beta..sub.42 cell stress was
determined.
[1235] In experiment 1, primary mouse cortical neuron cultures were
exposed to 1 mM of hydrogen peroxide (H.sub.2O.sub.2) for 15
minutes to induce oxidative stress. Neurons were pre-treated with
or without 5 .mu.M or 10 .mu.M of the specific inhibitor of JNK,
XG-102 (SEQ ID No. 11). Levels of phosphorylated JNK (pJNK), total
JNK (JNK) and Tubulin (control) were determined. The ratio of
pJNK/JNK served as a measure of JNK activity.
[1236] Results of the immunoblot analysis of the primary mouse
cortical neuron cultures pre-treated with or without 5 .mu.M or 10
.mu.M of XG-102 and exposed to 1 mM of hydrogen peroxide
(H.sub.2O.sub.2) during 15 minutes are shown in FIG. 108 (A). In
FIG. 108 (B), the corresponding histogram is depicted with the
ratio of phosphorylated JNK on total JNK (pJNK/JNK) for the
different experimental groups. As can be retrieved from this
histogram, after induction of the oxidative stress JNK activity
increased by 34% ("Controls" vs. "H.sub.2O.sub.2"). Pre-treatment
of cortical neurons with the inhibitor XG-102 prevented JNK
activity when used at 5 .mu.M. A decreased JNK activity (45% of
controls) is noted with a concentration of 10 .mu.M, in oxidative
stress conditions.
[1237] In experiment 2, primary mouse cortical neuron cultures were
exposed to 2 .mu.M of .beta.-amyloid 1-42 (A.beta..sub.42) for 5
hours to induce A.beta..sub.42 cell stress. Neurons were
pre-treated with or without 10 .mu.M of the specific inhibitor of
JNK, XG-102 (SEQ ID No. 11). Levels of phosphorylated JNK (pJNK),
total JNK (JNK), c-Jun, cleaved PARP and Tubulin (control) were
determined. The ratio of pJNK/JNK served as a measure of JNK
activity. The level of cleaved protein PARP, which is known to
increase during apoptosis, served as a measure of neuronal
apoptosis.
[1238] Results of the immunoblot analysis of the primary mouse
cortical neuron cultures pre-treated with or without 10 .mu.M of
XG-102 and exposed to 2 .mu.M of .beta.-amyloid 1-42
(A.beta..sub.42) during 5 hours are shown in FIG. 109 (A). In FIG.
109 (B and C), the corresponding histograms are depicted showing
the ratio of phosphorylated JNK on total JNK (pJNK/JNK) for the
different experimental groups (B) and the level of cleaved protein
PARP (C). Interestingly, in the condition of A.beta..sub.42 cell
stress, no modification of JNK activity was observed, neither with
nor without XG-102 pre-treatment (FIG. 109 B). Neuronal apoptosis
was measured by the level of cleaved protein PARP, which is
increased during apoptosis (FIG. 109 C). Accordingly, 1-amyloid
1-42 (A.beta..sub.42) treatment resulted in a 40% increase of
cleaved PARP, indicating A.beta..sub.42 stress induced apoptosis.
However, if cultures were pre-treated with XG-102 (10 .mu.M),
apoptosis was decreased by 37%.
[1239] Taken together, XG-102 thus prevented JNK activity in
oxidative stress conditions produced by H.sub.2O.sub.2 and
decreased neuronal apoptosis induced by A.beta..sub.42.
Example 50: Effects of XG-102 (SEQ ID No. 11) on Brain Lesions and
Apoptosis in 5.times.FAD Mice (Mouse Model of Alzheimer's
Disease)
[1240] The aim of this study is to analyze the modulation of brain
lesions and apoptosis with the injection of JNK peptide inhibitor
XG-102 in a mouse model of Alzheimer's disease (AD), the
5.times.FAD mice.
[1241] To this end, male 3 months-old C57BI/65.times.FAD, C57BI/6
wildtype littermates, and C57BI/6 5.times.FAD/PKR knockout mice are
used. The mice of each genotype are randomly divided into 10 groups
of 5 animals each. 25 animals are treated with XG-102 and 25
animals are the saline controls. The effect of XG-102 is evaluated
after 3 months or 6 months of repeated injections in the caudal
vein of the tail (every 21 days) at 10 mg/kg. The table below
summarizes the random allocation:
TABLE-US-00069 Number Group Route of of N.sup.o Mice Treatment
Duration administration animal 1 WT Saline 3 months i.v. injection
5 2 5XFAD Saline 3 months every 3 5 3 5XFAD/ Saline 3 months weeks
5 PKR KO (caudal vein) 4 WT XG-102 3 months 5 10 mg/kg 5 5XFAD
XG-102 3 months 5 10 mg/kg 6 5XFAD/ XG-102 3 months 5 PKR KO 10
mg/kg 7 WT Saline 6 months 5 8 5XFAD Saline 6 months 5 9 WT XG-102
6 months 5 10 mg/kg 10 5XFAD XG-102 6 months 5 10 mg/kg
[1242] Administrations are performed by intravenous injections in
the caudal vein (tail). Each aliquot is diluted 10 times in NaCl
0.9% to obtain a solution at 1.4 mg/mL. The volume injected does
not exceed 200 .mu.L, and it is adjusted according to the mouse
weight. The dose volume is 7.1 mL/kg.
[1243] At the end the experiments, after 3 or 6 months of
injections, mice are anesthetized by intraperitoneal injection of
sodium pentobarbital (50 mg/kg) and sacrificed. Brains are then
removed and dissected on ice then placed in 4% (v/v)
paraformaldehyde in PBS for immunohistochemistry or immediately
frozen in liquid nitrogen for immunoblotting and ELISA studies. For
immunoblot and ELISA analyses, brains samples are homogenized and
sonicated in a radio immune precipitation assay buffer (RIPA).
[1244] JNK activity, A.beta. pathway (A.beta., sAPP.alpha.,
sAPP.beta., BACE1, NEP), tau pathway (tau phosphorylation, CDK5
activation, GSK3 activation, p35, p25) and apoptosis (cleaved PARP,
cleaved caspase 3) is analyzed by immunoblot. A.beta. production
and caspase 3 activity is analyzed by ELISA. The number and size of
senile plaques, inflammation (GFAP, IBA1), and apoptosis (Tunnel,
NeuN, caspase 3) are analyzed by immunohistochemistry.
Example 51: Effects of XG-102 (SEQ ID No. 11) Alone or in
Combination with PKR Down-Regulation on .beta.-Amyloid-Induced
Neuronal Apoptosis (Alzheimer's Disease Model)
[1245] To obtain primary cortical neuronal cultures, E15.5 mice
embryos were dissected in PBS (Phosphate Buffered Saline) 6%
glucose, on ice. Embryos cortices were minced into small pieces and
treated with PBS glucose trypsin (Sigma Aldrich, Saint-Louis, USA)
for 20 min at 37.degree. C. Dissociated cortical cells were
cultured in Neurobasal media complemented with B27, Glutamax and
penicillin-streptomycin (Gibco). Neurons were cultured at
37.degree. C., 5% CO.sub.2 on pre-coated with poly-L-lysin (Sigma
Aldrich) petri dishes. Neurons were cultured to maturity (7 days)
before use.
[1246] To induce A.beta..sub.42 stress 2 .mu.M of A.beta.1-42
(Thermo Fisher Scientific, MA, USA) were used during 5 h on
cortical neurons. A.beta.42-1 inversed peptide (Thermo Fisher
Scientific) was used as negative control. A.beta.1-42 and
A.beta.42-1 were dissolved in pure water and incubated at
37.degree. C. for 48 h before use.
[1247] To inhibit JNK, cortical neurons were pre-treated with 10
.mu.M of XG-102 1 h before cell-stress treatment.
[1248] For immunoblot analysis cells were lysed on ice in a lysis
buffer containing 10 nM NaPi pH 7.8, 59 nM NaCl, 1% Triton, 0.5%
DOC, 0.1% SDS, 10% glycerol, 0.1 .mu.M calyculin A, 1 mM Na3VO4 and
1.times. of a protease inhibitor cocktail (Sigma Aldrich). Lysates
were sonicated and centrifugated 10 min at 15000 g at 4.degree. C.
The supernatant protein concentration was determined with the Micro
BCA protein assay kit (Thermo Scientific). Thirty micrograms of
proteins were resolved on SDS-PAGE and transferred onto
nitrocellulose membrane. After blocking with TBS 5% skim milk, the
membranes were probed with primary antibodies to JNK full, c-Jun,
PKR, eIF2.alpha. (Santa Cruz, Danvers, USA), pJNK (Millipore,
Billerica, USA), phosphor eIF2.alpha. (Thermo Fisher Scientific),
PARP and tubulin (Cell Signaling, Danvers, USA). IR Dyes 800 and
700 (Rockland Immunochemical Inc, Gilbertsville, USA) antibodies
were used as secondary antibodies. Blots were reveled with Odyssey
imaging system (LI-COR Biosciences, Lincoln, USA).
[1249] For caspase 3 activity analysis culture cell supernatants
containing degenerating and dead neurons, and cell medium were
collected in parallel of adhesive neurons lysis. Culture cell
supernatants were centrifugated 10 min at 15000 g at 4.degree. C.
Pellets were then resuspended in lysis buffer and caspase 3
activity was measured by using the Caspase 3 Assay kit reagents and
protocol (Abcam, Cambridge, UK).
[1250] Results:
[1251] Decrease of JNK and c-JNK Activations with XG-102 in
A.beta..sub.42-Stressed WT and PKR.sup.-/- Neurons
[1252] In the neuronal cultures stressed by A.beta..sub.42
peptides, the efficacy of XG-102 was investigated. XG-102 was used
at 10N.sub.1M, and added to cell medium 1 hour before the induction
of A.beta..sub.42 stress. In WT neurons, JNK activation is only
reduced after JNKi exposure (-60%, FIG. 3A) in A.beta..sub.42
stressed cultures. Both peptides showed efficacy in order to
decrease c-Jun phosphorylation: -74% with XG-102 (FIG. 2C) and -29%
with JNKi (FIG. 3C), and c-Jun expression: -65% with XG-102 (FIG.
2D) and -62% (FIG. 3D), compared to stressed WT neurons without
peptides. In PKR.sup.-/- neurons, JNK activation is reduced by
XG-102 (-35%, FIG. 2A) and JNKi (-60%, FIG. 3A) in A.beta..sub.42
stressed cultures. In PKR.sup.-/- cultures, the use of both
peptides does not modified c-Jun activation (FIGS. 2C and 3C), but
the use of JNKi showed a decrease by 62% of c-Jun protein
expression after A.beta..sub.42 stress induction (FIG. 3D).
[1253] XG-102 showed -74% efficacy in order to decrease c-Jun
phosphorylation (FIG. 110 C) and -65% efficacy in order to decrease
c-Jun expression (FIG. 110 D), compared to stressed WT neurons
without peptides.
[1254] In PKR.sup.-/- neurons, JNK activation is reduced by XG-102
(-35%, FIG. 110 A) in A.beta..sub.42 stressed cultures. In
PKR.sup.-/- cultures, the use of XG-102 does not modify c-Jun
activation (FIG. 110 C).
[1255] Decrease of Neuronal Apoptosis after JNK Inhibition in
A.beta..sub.42-Stressed WT Neurons
[1256] In WT neuronal cultures treated by A.beta..sub.42 peptides,
the use of XG-102 decreased apoptosis. With XG-102 it was noted a
93% reduction of cleaved caspase 3 expression level (FIG. 110 E), a
71% decrease of caspase 3 activity (FIG. 110 F), and a 55% decrease
of cleaved PARP expression level (FIG. 110 G) compared to
A.beta..sub.42 treated WT neurons.
[1257] Neuronal Death Due to A.beta..sub.42 Drastically Reduced
after Dual Inhibition of PKR and JNK in Neurons
[1258] In PKR.sup.-/- neurons treated by A.beta..sub.42 and XG-102,
the efficacy of the dual inhibition of PKR and JNK was assessed for
neuronal apoptosis. In neurons dually inhibited for PKR and JNK,
cleaved caspase 3, caspase 3 activity and PARP expression levels
decreased respectively by 83%, 87% and 93% compared to treated WT
neurons.
Sequence CWU 1
1
105119PRTArtificialDescription of sequence Peptide L-IB1(s) (see
Table 1) 1Arg Pro Lys Arg Pro Thr Thr Leu Asn Leu Phe Pro Gln Val
Pro Arg 1 5 10 15 Ser Gln Asp 219PRTArtificialDescription of
sequence Peptide D-IB1(s) (see Table 1) 2Asp Gln Ser Arg Pro Val
Gln Pro Phe Leu Asn Leu Thr Thr Pro Arg 1 5 10 15 Lys Pro Arg
319PRTArtificialDescription of sequence Peptide L-IB (generic) (s)
(see Table 1) 3Xaa Xaa Arg Pro Thr Thr Leu Xaa Leu Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Gln Asp Xaa 419PRTArtificialDescription of
sequence Peptide D-IB (generic) (s) (see Table 1) 4Xaa Asp Gln Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Leu Thr Thr Pro 1 5 10 15 Arg Xaa
Xaa 510PRTArtificialDescription of sequence Peptide L-TAT (see
Table 1) 5Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10
610PRTArtificialDescription of sequence Peptide D-TAT (see Table 1)
6Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 1 5 10
711PRTArtificialDescription of sequence Peptide L-generic-TAT (s)
(see Table 1) 7Xaa Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa 1 5 10
811PRTArtificialDescription of sequence Peptide D-generic-TAT (s)
(see Table 1) 8Xaa Arg Arg Arg Gln Arg Arg Lys Lys Arg Xaa 1 5 10
931PRTArtificialDescription of sequence L-TAT-IB1 (s) (see Table 1)
9Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Arg Pro Lys Arg 1
5 10 15 Pro Thr Thr Leu Asn Leu Phe Pro Gln Val Pro Arg Ser Gln Asp
20 25 30 1029PRTArtificialDescription of sequence Peptide L-TAT
(generic) (s) (see Table 1) 10Xaa Arg Lys Lys Arg Arg Gln Arg Arg
Arg Xaa Xaa Arg Pro Thr Thr 1 5 10 15 Leu Xaa Leu Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Gln Asp Xaa 20 25 1131PRTArtificialDescription of
sequence Peptid D-TAT-IB1 (s) (see Table 1) 11Asp Gln Ser Arg Pro
Val Gln Pro Phe Leu Asn Leu Thr Thr Pro Arg 1 5 10 15 Lys Pro Arg
Pro Pro Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 20 25 30
1229PRTArtificialDescription of sequence Peptid D-TAT (generic) (s)
(see Table 1) 12Xaa Asp Gln Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Leu
Thr Thr Pro 1 5 10 15 Arg Xaa Xaa Arg Arg Arg Gln Arg Arg Lys Lys
Arg Xaa 20 25 1329PRTArtificialDescription of sequence peptide
IB1-long (see Table 1) 13Pro Gly Thr Gly Cys Gly Asp Thr Tyr Arg
Pro Lys Arg Pro Thr Thr 1 5 10 15 Leu Asn Leu Phe Pro Gln Val Pro
Arg Ser Gln Asp Thr 20 25 1427PRTArtificialDescription of sequence
Peptide IB2-long (see Table 1) 14Ile Pro Ser Pro Ser Val Glu Glu
Pro His Lys His Arg Pro Thr Thr 1 5 10 15 Leu Arg Leu Thr Thr Leu
Gly Ala Gln Asp Ser 20 25 1529PRTArtificialDescription of sequence
Peptide derived from c-Jun (see Table 1) 15Gly Ala Tyr Gly Tyr Ser
Asn Pro Lys Ile Leu Lys Gln Ser Met Thr 1 5 10 15 Leu Asn Leu Ala
Asp Pro Val Gly Asn Leu Lys Pro His 20 25
1629PRTArtificialDescription of sequence Peptide derived from ATF2
(see Table 1) 16Thr Asn Glu Asp His Leu Ala Val His Lys His Lys His
Glu Met Thr 1 5 10 15 Leu Lys Phe Gly Pro Ala Arg Asn Asp Ser Val
Ile Val 20 25 1723PRTArtificialDescription of sequence Peptide
L-IB1 (see Table 1) 17Asp Thr Tyr Arg Pro Lys Arg Pro Thr Thr Leu
Asn Leu Phe Pro Gln 1 5 10 15 Val Pro Arg Ser Gln Asp Thr 20
1823PRTArtificialDescription of sequence Peptide D-IB1 (see Table
1) 18Thr Asp Gln Ser Arg Pro Val Gln Pro Phe Leu Asn Leu Thr Thr
Pro 1 5 10 15 Arg Lys Pro Arg Tyr Thr Asp 20
1919PRTArtificialDescription of sequence Peptide L-IB (generic)
(see Table 1) 19Xaa Arg Pro Thr Thr Leu Xaa Leu Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Gln 1 5 10 15 Asp Xaa Xaa 2019PRTArtificialDescription of
sequence Peptide D-IB (generic) (see Table 1) 20Xaa Xaa Asp Gln Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Leu Thr Thr 1 5 10 15 Pro Arg Xaa
2117PRTArtificialDescription of sequence Peptide L-generic-TAT (see
Table 1) 21Xaa Xaa Xaa Xaa Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa
Xaa Xaa 1 5 10 15 Xaa 2217PRTArtificialDescription of sequence
Peptide D-generic-TAT (see Table 1) 22Xaa Xaa Xaa Xaa Arg Arg Arg
Gln Arg Arg Lys Lys Arg Xaa Xaa Xaa 1 5 10 15 Xaa
2335PRTArtificialDescription of sequence Peptide L-TAT-IB1 (see
Table 1) 23Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Asp Thr
Tyr Arg 1 5 10 15 Pro Lys Arg Pro Thr Thr Leu Asn Leu Phe Pro Gln
Val Pro Arg Ser 20 25 30 Gln Asp Thr 35
2442PRTArtificialDescription of sequence Peptide L-TAT IB (generic)
(see Table 1) 24Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Lys Lys Arg Arg Gln
Arg Arg Arg 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Pro Thr
Thr Leu Xaa Leu Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Gln Asp Xaa
Xaa 35 40 2535PRTArtificialDescription of sequence Peptide
D-TAT-IB1 (see Table 1) 25Thr Asp Gln Ser Arg Pro Val Gln Pro Phe
Leu Asn Leu Thr Thr Pro 1 5 10 15 Arg Lys Pro Arg Tyr Thr Asp Pro
Pro Arg Arg Arg Gln Arg Arg Lys 20 25 30 Lys Arg Gly 35
2642PRTArtificialDescription of sequence Peptide D-TAT IB (generic)
(see Table 1) 26Xaa Xaa Asp Gln Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa
Leu Thr Thr 1 5 10 15 Pro Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg
Arg Arg Gln Arg Arg 20 25 30 Lys Lys Arg Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 35 40 2730PRTArtificialDescription of sequence chimeric peptide
sequence L-TAT-IB1(s1) (see Table 1) 27Arg Lys Lys Arg Arg Gln Arg
Arg Arg Pro Pro Arg Pro Lys Arg Pro 1 5 10 15 Thr Thr Leu Asn Leu
Phe Pro Gln Val Pro Arg Ser Gln Asp 20 25 30
2830PRTArtificialDescription of sequence chimeric peptide sequence
L-TAT-IB1(s2) (see Table 1) 28Gly Arg Lys Lys Arg Arg Gln Arg Arg
Arg Xaa Arg Pro Lys Arg Pro 1 5 10 15 Thr Thr Leu Asn Leu Phe Pro
Gln Val Pro Arg Ser Gln Asp 20 25 30 2929PRTArtificialDescription
of sequence chimeric peptide sequence L-TAT-IB1(s3) (see Table 1)
29Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Arg Pro Lys Arg Pro Thr 1
5 10 15 Thr Leu Asn Leu Phe Pro Gln Val Pro Arg Ser Gln Asp 20 25
3030PRTArtificialDescription of sequence chimeric peptide sequence
D-TAT-IB1(s1) (see Table 1) 30Asp Gln Ser Arg Pro Val Gln Pro Phe
Leu Asn Leu Thr Thr Pro Arg 1 5 10 15 Lys Pro Arg Pro Pro Arg Arg
Arg Gln Arg Arg Lys Lys Arg 20 25 30 3130PRTArtificialDescription
of sequence chimeric peptide sequence D-TAT-IB1(s2) (see Table 1)
31Asp Gln Ser Arg Pro Val Gln Pro Phe Leu Asn Leu Thr Thr Pro Arg 1
5 10 15 Lys Pro Arg Xaa Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 20
25 30 3229PRTArtificialDescription of sequence chimeric peptide
sequence D-TAT-IB1(s3) (see Table 1) 32Asp Gln Ser Arg Pro Val Gln
Pro Phe Leu Asn Leu Thr Thr Pro Arg 1 5 10 15 Lys Pro Arg Xaa Arg
Arg Arg Gln Arg Arg Lys Lys Arg 20 25 3313PRTArtificialDescription
of sequence L-IB1(s1) (see Table 1) 33Thr Leu Asn Leu Phe Pro Gln
Val Pro Arg Ser Gln Asp 1 5 10 3413PRTArtificialDescription of
sequence L-IB1(s2) (see Table 1) 34Thr Thr Leu Asn Leu Phe Pro Gln
Val Pro Arg Ser Gln 1 5 10 3513PRTArtificialDescription of sequence
L-IB1(s3) (see Table 1) 35Pro Thr Thr Leu Asn Leu Phe Pro Gln Val
Pro Arg Ser 1 5 10 3613PRTArtificialDescription of sequence
L-IB1(s4) (see Table 1) 36Arg Pro Thr Thr Leu Asn Leu Phe Pro Gln
Val Pro Arg 1 5 10 3713PRTArtificialDescription of sequence
L-IB1(s5) (see Table 1) 37Lys Arg Pro Thr Thr Leu Asn Leu Phe Pro
Gln Val Pro 1 5 10 3813PRTArtificialDescription of sequence
L-IB1(s6) (see Table 1) 38Pro Lys Arg Pro Thr Thr Leu Asn Leu Phe
Pro Gln Val 1 5 10 3913PRTArtificialDescription of sequence
L-IB1(s7) (see Table 1) 39Arg Pro Lys Arg Pro Thr Thr Leu Asn Leu
Phe Pro Gln 1 5 10 4012PRTArtificialDescription of sequence
L-IB1(s8) (see Table 1) 40Leu Asn Leu Phe Pro Gln Val Pro Arg Ser
Gln Asp 1 5 10 4112PRTArtificialDescription of sequence L-IB1(s9)
(see Table 1) 41Thr Leu Asn Leu Phe Pro Gln Val Pro Arg Ser Gln 1 5
10 4212PRTArtificialDescription of sequence L-IB1(s10) (see Table
1) 42Thr Thr Leu Asn Leu Phe Pro Gln Val Pro Arg Ser 1 5 10
4312PRTArtificialDescription of sequence L-IB1(s11) (see Table 1)
43Pro Thr Thr Leu Asn Leu Phe Pro Gln Val Pro Arg 1 5 10
4412PRTArtificialDescription of sequence L-IB1(s12) (see Table 1)
44Arg Pro Thr Thr Leu Asn Leu Phe Pro Gln Val Pro 1 5 10
4512PRTArtificialDescription of sequence L-IB1(s13) (see Table 1)
45Lys Arg Pro Thr Thr Leu Asn Leu Phe Pro Gln Val 1 5 10
4612PRTArtificialDescription of sequence L-IB1(s14) (see Table 1)
46Pro Lys Arg Pro Thr Thr Leu Asn Leu Phe Pro Gln 1 5 10
4712PRTArtificialDescription of sequence L-IB1(s15) (see Table 1)
47Arg Pro Lys Arg Pro Thr Thr Leu Asn Leu Phe Pro 1 5 10
4811PRTArtificialDescription of sequence L-IB1(s16) (see Table 1)
48Asn Leu Phe Pro Gln Val Pro Arg Ser Gln Asp 1 5 10
4911PRTArtificialDescription of sequence L-IB1(s17) (see Table 1)
49Leu Asn Leu Phe Pro Gln Val Pro Arg Ser Gln 1 5 10
5011PRTArtificialDescription of sequence L-IB1(s18) (see Table 1)
50Thr Leu Asn Leu Phe Pro Gln Val Pro Arg Ser 1 5 10
5111PRTArtificialDescription of sequence L-IB1(s19) (see Table 1)
51Thr Thr Leu Asn Leu Phe Pro Gln Val Pro Arg 1 5 10
5211PRTArtificialDescription of sequence L-IB1(s20) (see Table 1)
52Pro Thr Thr Leu Asn Leu Phe Pro Gln Val Pro 1 5 10
5311PRTArtificialDescription of sequence L-IB1(s21) (see Table 1)
53Arg Pro Thr Thr Leu Asn Leu Phe Pro Gln Val 1 5 10
5411PRTArtificialDescription of sequence L-IB1(s22) (see Table 1)
54Lys Arg Pro Thr Thr Leu Asn Leu Phe Pro Gln 1 5 10
5511PRTArtificialDescription of sequence L-IB1(s23) (see Table 1)
55Pro Lys Arg Pro Thr Thr Leu Asn Leu Phe Pro 1 5 10
5611PRTArtificialDescription of sequence L-IB1(s24) (see Table 1)
56Arg Pro Lys Arg Pro Thr Thr Leu Asn Leu Phe 1 5 10
5710PRTArtificialDescription of sequence L-IB1(s25) (see Table 1)
57Leu Phe Pro Gln Val Pro Arg Ser Gln Asp 1 5 10
5810PRTArtificialDescription of sequence L-IB1(s26) (see Table 1)
58Asn Leu Phe Pro Gln Val Pro Arg Ser Gln 1 5 10
5910PRTArtificialDescription of sequence L-IB1(s27) (see Table 1)
59Leu Asn Leu Phe Pro Gln Val Pro Arg Ser 1 5 10
6010PRTArtificialDescription of sequence L-IB1(s28) (see Table 1)
60Thr Leu Asn Leu Phe Pro Gln Val Pro Arg 1 5 10
6110PRTArtificialDescription of sequence L-IB1(s29) (see Table 1)
61Thr Thr Leu Asn Leu Phe Pro Gln Val Pro 1 5 10
6210PRTArtificialDescription of sequence L-IB1(s30) (see Table 1)
62Pro Thr Thr Leu Asn Leu Phe Pro Gln Val 1 5 10
6310PRTArtificialDescription of sequence L-IB1(s31) (see Table 1)
63Arg Pro Thr Thr Leu Asn Leu Phe Pro Gln 1 5 10
6410PRTArtificialDescription of sequence L-IB1(s32) (see Table 1)
64Lys Arg Pro Thr Thr Leu Asn Leu Phe Pro 1 5 10
6510PRTArtificialDescription of sequence L-IB1(s33) (see Table 1)
65Pro Lys Arg Pro Thr Thr Leu Asn Leu Phe 1 5 10
6610PRTArtificialDescription of sequence L-IB1(s34) (see Table 1)
66Arg Pro Lys Arg Pro Thr Thr Leu Asn Leu 1 5 10
6713PRTArtificialDescription of sequence D-IB1(s1) (see Table 1)
67Gln Pro Phe Leu Asn Leu Thr Thr Pro Arg Lys Pro Arg 1 5 10
6813PRTArtificialDescription of sequence D-IB1(s2) (see Table 1)
68Val Gln Pro Phe Leu Asn Leu Thr Thr Pro Arg Lys Pro 1 5 10
6913PRTArtificialDescription of sequence D-IB1(s3) (see Table 1)
69Pro Val Gln Pro Phe Leu Asn Leu Thr Thr Pro Arg Lys 1 5 10
7013PRTArtificialDescription of sequence D-IB1(s4) (see Table 1)
70Arg Pro Val Gln Pro Phe Leu Asn Leu Thr Thr Pro Arg 1 5 10
7113PRTArtificialDescription of sequence D-IB1(s5) (see Table 1)
71Ser Arg Pro Val Gln Pro Phe Leu Asn Leu Thr Thr Pro 1 5 10
7213PRTArtificialDescription of sequence D-IB1(s6) (see Table 1)
72Gln Ser Arg Pro Val Gln Pro Phe Leu Asn Leu Thr Thr 1 5 10
7313PRTArtificialDescription of sequence D-IB1(s7) (see Table 1)
73Asp Gln Ser Arg Pro Val Gln Pro Phe Leu Asn Leu Thr 1 5 10
7412PRTArtificialDescription of sequence D-IB1(s8) (see Table 1)
74Pro Phe Leu Asn Leu Thr Thr Pro Arg Lys Pro Arg 1 5 10
7512PRTArtificialDescription of sequence D-IB1(s9) (see Table 1)
75Gln Pro Phe Leu Asn Leu Thr Thr Pro Arg Lys Pro 1 5 10
7612PRTArtificialDescription of sequence D-IB1(s10) (see Table 1)
76Val Gln Pro Phe Leu Asn Leu Thr Thr Pro Arg Lys 1 5 10
7712PRTArtificialDescription of sequence D-IB1(s11) (see Table 1)
77Pro Val Gln Pro Phe Leu Asn Leu Thr Thr Pro Arg 1 5 10
7812PRTArtificialDescription of sequence D-IB1(s12) (see Table 1)
78Arg Pro Val Gln Pro Phe Leu Asn Leu Thr Thr Pro 1 5 10
7912PRTArtificialDescription of sequence D-IB1(s13) (see Table 1)
79Ser Arg Pro Val Gln Pro Phe Leu Asn Leu Thr Thr 1 5 10
8012PRTArtificialDescription of sequence D-IB1(s14) (see Table 1)
80Gln Ser Arg Pro Val Gln Pro Phe Leu Asn Leu Thr 1 5 10
8112PRTArtificialDescription of sequence D-IB1(s15) (see Table 1)
81Asp Gln Ser Arg Pro Val Gln Pro Phe Leu Asn Leu 1 5 10
8211PRTArtificialDescription of sequence D-IB1(s16) (see Table 1)
82Phe Leu Asn Leu Thr Thr Pro Arg Lys Pro Arg 1 5 10
8311PRTArtificialDescription of sequence D-IB1(s17) (see Table 1)
83Pro Phe Leu Asn Leu Thr Thr Pro Arg Lys Pro 1 5 10
8411PRTArtificialDescription of sequence D-IB1(s18) (see Table 1)
84Gln Pro Phe Leu Asn Leu Thr Thr Pro Arg
Lys 1 5 10 8511PRTArtificialDescription of sequence D-IB1(s19) (see
Table 1) 85Val Gln Pro Phe Leu Asn Leu Thr Thr Pro Arg 1 5 10
8611PRTArtificialDescription of sequence D-IB1(s20) (see Table 1)
86Pro Val Gln Pro Phe Leu Asn Leu Thr Thr Pro 1 5 10
8711PRTArtificialDescription of sequence D-IB1(s21) (see Table 1)
87Arg Pro Val Gln Pro Phe Leu Asn Leu Thr Thr 1 5 10
8811PRTArtificialDescription of sequence D-IB1(s22) (see Table 1)
88Ser Arg Pro Val Gln Pro Phe Leu Asn Leu Thr 1 5 10
8911PRTArtificialDescription of sequence D-IB1(s23) (see Table 1)
89Gln Ser Arg Pro Val Gln Pro Phe Leu Asn Leu 1 5 10
9011PRTArtificialDescription of sequence D-IB1(s24) (see Table 1)
90Asp Gln Ser Arg Pro Val Gln Pro Phe Leu Asn 1 5 10
9110PRTArtificialDescription of sequence D-IB1(s25) (see Table 1)
91Asp Gln Ser Arg Pro Val Gln Pro Phe Leu 1 5 10
9210PRTArtificialDescription of sequence D-IB1(s26) (see Table 1)
92Gln Ser Arg Pro Val Gln Pro Phe Leu Asn 1 5 10
9310PRTArtificialDescription of sequence D-IB1(s27) (see Table 1)
93Ser Arg Pro Val Gln Pro Phe Leu Asn Leu 1 5 10
9410PRTArtificialDescription of sequence D-IB1(s28) (see Table 1)
94Arg Pro Val Gln Pro Phe Leu Asn Leu Thr 1 5 10
9510PRTArtificialDescription of sequence D-IB1(s29) (see Table 1)
95Pro Val Gln Pro Phe Leu Asn Leu Thr Thr 1 5 10
9610PRTArtificialDescription of sequence D-IB1(s30) (see Table 1)
96Val Gln Pro Phe Leu Asn Leu Thr Thr Pro 1 5 10
9710PRTArtificialDescription of sequence D-IB1(s31) (see Table 1)
97Gln Pro Phe Leu Asn Leu Thr Thr Pro Arg 1 5 10
9810PRTArtificialDescription of sequence D-IB1(s32) (see Table 1)
98Pro Phe Leu Asn Leu Thr Thr Pro Arg Lys 1 5 10
9910PRTArtificialDescription of sequence D-IB1(s33) (see Table 1)
99Phe Leu Asn Leu Thr Thr Pro Arg Lys Pro 1 5 10
10010PRTArtificialDescription of sequence D-IB1(s34) (see Table 1)
100Leu Asn Leu Thr Thr Pro Arg Lys Pro Arg 1 5 10
10121DNAArtificialDescription of sequence ap-1 doubled labeled
probe (see p. 66) 101cgcttgatga gtcagccgga a
211022953DNAArtificialdescription of sequence rat IB1 cDNA sequence
and its predicted amino acid sequence (see Figure 1) 102ccgccccagc
tcagtccgaa ccccgcggcg gcggcggcct cctccacacg cctccacctc 60cgccgccgcc
gccgccgccg ccgcctcccg cgccgctctc cgcccgg atg gcc agg 116 Met Ala
Arg 1 ctg agc ccg gga atg gcg gag cga gag agc ggc ctg agc ggg ggt
gcc 164Leu Ser Pro Gly Met Ala Glu Arg Glu Ser Gly Leu Ser Gly Gly
Ala 5 10 15 gcg tcc cca ccg gcc gct tcc cca ttc ctg gga ctg cac atc
gcg tcg 212Ala Ser Pro Pro Ala Ala Ser Pro Phe Leu Gly Leu His Ile
Ala Ser 20 25 30 35 cct ccc aat ttc agg ctc acc cat gat atc agc ctg
gag gag ttt gag 260Pro Pro Asn Phe Arg Leu Thr His Asp Ile Ser Leu
Glu Glu Phe Glu 40 45 50 gat gaa gac ctt tcg gag atc act gat gag
tgt ggc atc agc ctg cag 308Asp Glu Asp Leu Ser Glu Ile Thr Asp Glu
Cys Gly Ile Ser Leu Gln 55 60 65 tgc aaa gac acc ttg tct ctc cgg
ccc ccg cgc gcc ggg cta ctg tct 356Cys Lys Asp Thr Leu Ser Leu Arg
Pro Pro Arg Ala Gly Leu Leu Ser 70 75 80 gcg ggt agc agc ggt agc
gcg ggg agc cgg ctg cag gcg gag atg ctg 404Ala Gly Ser Ser Gly Ser
Ala Gly Ser Arg Leu Gln Ala Glu Met Leu 85 90 95 cag atg gac ctg
atc gac gcg gca agt gac act ccg ggc gcc gag gac 452Gln Met Asp Leu
Ile Asp Ala Ala Ser Asp Thr Pro Gly Ala Glu Asp 100 105 110 115 gac
gaa gag gac gac gac gag ctc gct gcc caa cgg cca gga gtg ggg 500Asp
Glu Glu Asp Asp Asp Glu Leu Ala Ala Gln Arg Pro Gly Val Gly 120 125
130 cct tcc aaa gcc gag tct ggc cag gag ccg gcg tct cgc agc cag ggt
548Pro Ser Lys Ala Glu Ser Gly Gln Glu Pro Ala Ser Arg Ser Gln Gly
135 140 145 cag ggc cag ggc ccc ggc aca ggc tgc gga gac acc tac cgg
ccc aag 596Gln Gly Gln Gly Pro Gly Thr Gly Cys Gly Asp Thr Tyr Arg
Pro Lys 150 155 160 agg cct acc acg ctc aac ctt ttc ccg cag gtg ccg
cgg tct cag gac 644Arg Pro Thr Thr Leu Asn Leu Phe Pro Gln Val Pro
Arg Ser Gln Asp 165 170 175 acg ctg aat aat aac tct tta ggc aaa aag
cac agt tgg cag gac cgt 692Thr Leu Asn Asn Asn Ser Leu Gly Lys Lys
His Ser Trp Gln Asp Arg 180 185 190 195 gtg tct cga tca tcc tcc cct
ctg aag aca ggg gag cag acg cct cca 740Val Ser Arg Ser Ser Ser Pro
Leu Lys Thr Gly Glu Gln Thr Pro Pro 200 205 210 cat gaa cat atc tgc
ctg agt gat gag ctg ccg ccc cag ggc agt cct 788His Glu His Ile Cys
Leu Ser Asp Glu Leu Pro Pro Gln Gly Ser Pro 215 220 225 gtt ccc acc
cag gat cgt ggc act tcc acc gac agc cct tgt cgc cgt 836Val Pro Thr
Gln Asp Arg Gly Thr Ser Thr Asp Ser Pro Cys Arg Arg 230 235 240 act
gca gcc acc cag atg gca cct cca agt ggt ccc cct gcc act gca 884Thr
Ala Ala Thr Gln Met Ala Pro Pro Ser Gly Pro Pro Ala Thr Ala 245 250
255 cct ggt ggc cgg ggc cac tcc cat cga gat cgg tcc ata tca gca gat
932Pro Gly Gly Arg Gly His Ser His Arg Asp Arg Ser Ile Ser Ala Asp
260 265 270 275 gtg cgg ctc gag gcg act gag gag atc tac ctg acc cca
gtg cag agg 980Val Arg Leu Glu Ala Thr Glu Glu Ile Tyr Leu Thr Pro
Val Gln Arg 280 285 290 ccc cca gac cct gca gaa ccc acc tcc acc ttc
ttg cca ccc act gag 1028Pro Pro Asp Pro Ala Glu Pro Thr Ser Thr Phe
Leu Pro Pro Thr Glu 295 300 305 agc cgg atg tct gtc agc tcg gat cct
gac cct gcc gct tac tct gta 1076Ser Arg Met Ser Val Ser Ser Asp Pro
Asp Pro Ala Ala Tyr Ser Val 310 315 320 act gca ggg cga ccg cac cct
tcc atc agt gaa gag gat gag ggc ttc 1124Thr Ala Gly Arg Pro His Pro
Ser Ile Ser Glu Glu Asp Glu Gly Phe 325 330 335 gac tgt ctg tca tcc
cca gag caa gct gag cca cca ggt gga ggg tgg 1172Asp Cys Leu Ser Ser
Pro Glu Gln Ala Glu Pro Pro Gly Gly Gly Trp 340 345 350 355 cgg gga
agc ctc ggg gag cca cca ccg cct cca cgg gcc tca ctg agc 1220Arg Gly
Ser Leu Gly Glu Pro Pro Pro Pro Pro Arg Ala Ser Leu Ser 360 365 370
tcg gac acc agc gca ctg tcc tac gac tct gtc aag tac aca ctg gtg
1268Ser Asp Thr Ser Ala Leu Ser Tyr Asp Ser Val Lys Tyr Thr Leu Val
375 380 385 gtg gat gag cat gcc cag ctt gag ttg gtg agc ctg cgg cca
tgt ttt 1316Val Asp Glu His Ala Gln Leu Glu Leu Val Ser Leu Arg Pro
Cys Phe 390 395 400 gga gat tac agt gac gaa agc gac tct gcc act gtc
tat gac aac tgt 1364Gly Asp Tyr Ser Asp Glu Ser Asp Ser Ala Thr Val
Tyr Asp Asn Cys 405 410 415 gcc tct gcc tcc tcg ccc tac gag tca gcc
att ggt gag gaa tat gag 1412Ala Ser Ala Ser Ser Pro Tyr Glu Ser Ala
Ile Gly Glu Glu Tyr Glu 420 425 430 435 gag gcc cct caa ccc cgg cct
ccc acc tgc ctg tca gag gac tcc aca 1460Glu Ala Pro Gln Pro Arg Pro
Pro Thr Cys Leu Ser Glu Asp Ser Thr 440 445 450 ccg gat gag cct gac
gtc cac ttc tct aag aag ttt ctg aat gtc ttc 1508Pro Asp Glu Pro Asp
Val His Phe Ser Lys Lys Phe Leu Asn Val Phe 455 460 465 atg agt ggc
cgc tct cgt tcc tcc agt gcc gag tcc ttt ggg ctg ttc 1556Met Ser Gly
Arg Ser Arg Ser Ser Ser Ala Glu Ser Phe Gly Leu Phe 470 475 480 tcc
tgt gtc atc aat ggg gag gag cat gag caa acc cat cgg gct ata 1604Ser
Cys Val Ile Asn Gly Glu Glu His Glu Gln Thr His Arg Ala Ile 485 490
495 ttc agg ttt gtg cct cgg cat gaa gat gaa ctt gag ctg gaa gtg gac
1652Phe Arg Phe Val Pro Arg His Glu Asp Glu Leu Glu Leu Glu Val Asp
500 505 510 515 gac cct ctg ctg gtg gag ctg cag gca gaa gac tat tgg
tat gag gcc 1700Asp Pro Leu Leu Val Glu Leu Gln Ala Glu Asp Tyr Trp
Tyr Glu Ala 520 525 530 tat aac atg cgc act gga gcc cgt ggt gtc ttt
cct gcc tac tat gcc 1748Tyr Asn Met Arg Thr Gly Ala Arg Gly Val Phe
Pro Ala Tyr Tyr Ala 535 540 545 att gag gtc acc aag gag cct gag cac
atg gca gcc ctt gcc aaa aac 1796Ile Glu Val Thr Lys Glu Pro Glu His
Met Ala Ala Leu Ala Lys Asn 550 555 560 agc gac tgg att gac cag ttc
cgg gtg aag ttc ctg ggc tct gtc cag 1844Ser Asp Trp Ile Asp Gln Phe
Arg Val Lys Phe Leu Gly Ser Val Gln 565 570 575 gtt cct tat cac aag
ggc aat gat gtc ctc tgt gct gct atg caa aag 1892Val Pro Tyr His Lys
Gly Asn Asp Val Leu Cys Ala Ala Met Gln Lys 580 585 590 595 atc gcc
acc acc cgc cgg ctc acc gtg cac ttt aac ccg ccc tcc agc 1940Ile Ala
Thr Thr Arg Arg Leu Thr Val His Phe Asn Pro Pro Ser Ser 600 605 610
tgt gtc ctt gaa atc agc gtt agg ggt gtc aag ata ggt gtc aaa gct
1988Cys Val Leu Glu Ile Ser Val Arg Gly Val Lys Ile Gly Val Lys Ala
615 620 625 gat gaa gct cag gag gcc aag gga aat aaa tgt agc cac ttt
ttc cag 2036Asp Glu Ala Gln Glu Ala Lys Gly Asn Lys Cys Ser His Phe
Phe Gln 630 635 640 cta aaa aac atc tct ttc tgt ggg tac cat cca aag
aac aac aag tac 2084Leu Lys Asn Ile Ser Phe Cys Gly Tyr His Pro Lys
Asn Asn Lys Tyr 645 650 655 ttt ggg ttt atc act aag cac cct gct gac
cac cgg ttt gcc tgc cat 2132Phe Gly Phe Ile Thr Lys His Pro Ala Asp
His Arg Phe Ala Cys His 660 665 670 675 gtc ttt gtg tct gaa gat tcc
acc aaa gcc ctg gca gag tct gtg ggg 2180Val Phe Val Ser Glu Asp Ser
Thr Lys Ala Leu Ala Glu Ser Val Gly 680 685 690 cgt gca ttt cag cag
ttc tac aag caa ttt gtg gaa tat acc tgt cct 2228Arg Ala Phe Gln Gln
Phe Tyr Lys Gln Phe Val Glu Tyr Thr Cys Pro 695 700 705 aca gaa gat
atc tac ttg gag tag cagcaacccc cctctctgca gcccctcagc 2282Thr Glu
Asp Ile Tyr Leu Glu 710 cccaggccag tactaggaca gctgactgct gacaggatgt
tgtactgcca cgagagaatg 2342ggggagtgag ggctgttggg gtcggggggc
aggggtttgg ggagaggcag atgcagttta 2402ttgtaatata tggggttaga
ttaatctatg gaggacagta caggctctct cggggctggg 2462gaagggcagg
gctggggtgg gggtcaggca tctggccaca aaggggtccc ctagggacag
2522aggcgctgca ccatcctggg cttgtttcat actagaggcc ctggctttct
ggctcttggg 2582tcctgccttg acaaagccca gccacctgga agtgtcacct
tcccttgtcc acctcaccca 2642gtgccctgag ctcatgctga gcccaagcac
ctccgaagga ctttccagta aggaaatggc 2702aacatgtgac agtgagaccc
tgttctcatc tgtggggctc cggcagctcc gacccccagc 2762ctggccagca
cgctgaccct ggcaagcttg tgtgttcaaa gaaggagagg gccacagcaa
2822gccctgcctg ccagggaagg ttccctctca gctggcccca gccaactggt
cactgtcttg 2882tcacctggct actactatta aagtgccatt tcttgtctga
aaaaaaaaaa aaaaaaaaaa 2942aaaaactcga g
2953103714PRTArtificialdescription of sequence Protein encoded by
Exon-Intron Boundary of the rIB1 Gene - Splice donor 103Met Ala Arg
Leu Ser Pro Gly Met Ala Glu Arg Glu Ser Gly Leu Ser 1 5 10 15 Gly
Gly Ala Ala Ser Pro Pro Ala Ala Ser Pro Phe Leu Gly Leu His 20 25
30 Ile Ala Ser Pro Pro Asn Phe Arg Leu Thr His Asp Ile Ser Leu Glu
35 40 45 Glu Phe Glu Asp Glu Asp Leu Ser Glu Ile Thr Asp Glu Cys
Gly Ile 50 55 60 Ser Leu Gln Cys Lys Asp Thr Leu Ser Leu Arg Pro
Pro Arg Ala Gly 65 70 75 80 Leu Leu Ser Ala Gly Ser Ser Gly Ser Ala
Gly Ser Arg Leu Gln Ala 85 90 95 Glu Met Leu Gln Met Asp Leu Ile
Asp Ala Ala Ser Asp Thr Pro Gly 100 105 110 Ala Glu Asp Asp Glu Glu
Asp Asp Asp Glu Leu Ala Ala Gln Arg Pro 115 120 125 Gly Val Gly Pro
Ser Lys Ala Glu Ser Gly Gln Glu Pro Ala Ser Arg 130 135 140 Ser Gln
Gly Gln Gly Gln Gly Pro Gly Thr Gly Cys Gly Asp Thr Tyr 145 150 155
160 Arg Pro Lys Arg Pro Thr Thr Leu Asn Leu Phe Pro Gln Val Pro Arg
165 170 175 Ser Gln Asp Thr Leu Asn Asn Asn Ser Leu Gly Lys Lys His
Ser Trp 180 185 190 Gln Asp Arg Val Ser Arg Ser Ser Ser Pro Leu Lys
Thr Gly Glu Gln 195 200 205 Thr Pro Pro His Glu His Ile Cys Leu Ser
Asp Glu Leu Pro Pro Gln 210 215 220 Gly Ser Pro Val Pro Thr Gln Asp
Arg Gly Thr Ser Thr Asp Ser Pro 225 230 235 240 Cys Arg Arg Thr Ala
Ala Thr Gln Met Ala Pro Pro Ser Gly Pro Pro 245 250 255 Ala Thr Ala
Pro Gly Gly Arg Gly His Ser His Arg Asp Arg Ser Ile 260 265 270 Ser
Ala Asp Val Arg Leu Glu Ala Thr Glu Glu Ile Tyr Leu Thr Pro 275 280
285 Val Gln Arg Pro Pro Asp Pro Ala Glu Pro Thr Ser Thr Phe Leu Pro
290 295 300 Pro Thr Glu Ser Arg Met Ser Val Ser Ser Asp Pro Asp Pro
Ala Ala 305 310 315 320 Tyr Ser Val Thr Ala Gly Arg Pro His Pro Ser
Ile Ser Glu Glu Asp 325 330 335 Glu Gly Phe Asp Cys Leu Ser Ser Pro
Glu Gln Ala Glu Pro Pro Gly 340 345 350 Gly Gly Trp Arg Gly Ser Leu
Gly Glu Pro Pro Pro Pro Pro Arg Ala 355 360 365 Ser Leu Ser Ser Asp
Thr Ser Ala Leu Ser Tyr Asp Ser Val Lys Tyr 370 375 380 Thr Leu Val
Val Asp Glu His Ala Gln Leu Glu Leu Val Ser Leu Arg 385 390 395 400
Pro Cys Phe Gly Asp Tyr Ser Asp Glu Ser Asp Ser Ala Thr Val Tyr 405
410 415 Asp Asn Cys Ala Ser Ala Ser Ser Pro Tyr Glu Ser Ala Ile Gly
Glu 420 425 430 Glu Tyr Glu Glu Ala Pro Gln Pro Arg Pro Pro Thr Cys
Leu Ser Glu 435 440 445 Asp Ser Thr Pro Asp Glu Pro Asp Val His Phe
Ser Lys Lys Phe Leu 450 455 460 Asn Val Phe Met Ser Gly Arg Ser Arg
Ser Ser Ser Ala Glu Ser Phe 465 470 475 480 Gly Leu Phe Ser Cys Val
Ile Asn Gly Glu Glu His Glu Gln Thr His 485 490 495 Arg Ala Ile Phe
Arg Phe Val Pro Arg His Glu Asp Glu Leu Glu Leu 500 505 510 Glu Val
Asp Asp Pro Leu Leu Val Glu Leu Gln Ala Glu Asp Tyr Trp 515 520 525
Tyr Glu Ala Tyr Asn Met Arg Thr Gly Ala Arg Gly Val Phe
Pro Ala 530 535 540 Tyr Tyr Ala Ile Glu Val Thr Lys Glu Pro Glu His
Met Ala Ala Leu 545 550 555 560 Ala Lys Asn Ser Asp Trp Ile Asp Gln
Phe Arg Val Lys Phe Leu Gly 565 570 575 Ser Val Gln Val Pro Tyr His
Lys Gly Asn Asp Val Leu Cys Ala Ala 580 585 590 Met Gln Lys Ile Ala
Thr Thr Arg Arg Leu Thr Val His Phe Asn Pro 595 600 605 Pro Ser Ser
Cys Val Leu Glu Ile Ser Val Arg Gly Val Lys Ile Gly 610 615 620 Val
Lys Ala Asp Glu Ala Gln Glu Ala Lys Gly Asn Lys Cys Ser His 625 630
635 640 Phe Phe Gln Leu Lys Asn Ile Ser Phe Cys Gly Tyr His Pro Lys
Asn 645 650 655 Asn Lys Tyr Phe Gly Phe Ile Thr Lys His Pro Ala Asp
His Arg Phe 660 665 670 Ala Cys His Val Phe Val Ser Glu Asp Ser Thr
Lys Ala Leu Ala Glu 675 680 685 Ser Val Gly Arg Ala Phe Gln Gln Phe
Tyr Lys Gln Phe Val Glu Tyr 690 695 700 Thr Cys Pro Thr Glu Asp Ile
Tyr Leu Glu 705 710 104711PRTHomo sapiensdescription of sequence
human IB1 protein sequence 104Met Ala Glu Arg Glu Ser Gly Gly Leu
Gly Gly Gly Ala Ala Ser Pro 1 5 10 15 Pro Ala Ala Ser Pro Phe Leu
Gly Leu His Ile Ala Ser Pro Pro Asn 20 25 30 Phe Arg Leu Thr His
Asp Ile Ser Leu Glu Glu Phe Glu Asp Glu Asp 35 40 45 Leu Ser Glu
Ile Thr Asp Glu Cys Gly Ile Ser Leu Gln Cys Lys Asp 50 55 60 Thr
Leu Ser Leu Arg Pro Pro Arg Ala Gly Leu Leu Ser Ala Gly Gly 65 70
75 80 Gly Gly Ala Gly Ser Arg Leu Gln Ala Glu Met Leu Gln Met Asp
Leu 85 90 95 Ile Asp Ala Thr Gly Asp Thr Pro Gly Ala Glu Asp Asp
Glu Glu Asp 100 105 110 Asp Asp Glu Glu Arg Ala Ala Arg Arg Pro Gly
Ala Gly Pro Pro Lys 115 120 125 Ala Glu Ser Gly Gln Glu Pro Ala Ser
Arg Gly Gln Gly Gln Ser Gln 130 135 140 Gly Gln Ser Gln Gly Pro Gly
Ser Gly Asp Thr Tyr Arg Pro Lys Arg 145 150 155 160 Pro Thr Thr Leu
Asn Leu Phe Pro Gln Val Pro Arg Ser Gln Asp Thr 165 170 175 Leu Asn
Asn Asn Ser Leu Gly Lys Lys His Ser Trp Gln Asp Arg Val 180 185 190
Ser Arg Ser Ser Ser Pro Leu Lys Thr Gly Glu Gln Thr Pro Pro His 195
200 205 Glu His Ile Cys Leu Ser Asp Glu Leu Pro Pro Gln Ser Gly Pro
Ala 210 215 220 Pro Thr Thr Asp Arg Gly Thr Ser Thr Asp Ser Pro Cys
Arg Arg Ser 225 230 235 240 Thr Ala Thr Gln Met Ala Pro Pro Gly Gly
Pro Pro Ala Ala Pro Pro 245 250 255 Gly Gly Arg Gly His Ser His Arg
Asp Arg Ile His Tyr Gln Ala Asp 260 265 270 Val Arg Leu Glu Ala Thr
Glu Glu Ile Tyr Leu Thr Pro Val Gln Arg 275 280 285 Pro Pro Asp Ala
Ala Glu Pro Thr Ser Ala Phe Leu Pro Pro Thr Glu 290 295 300 Ser Arg
Met Ser Val Ser Ser Asp Pro Asp Pro Ala Ala Tyr Pro Ser 305 310 315
320 Thr Ala Gly Arg Pro His Pro Ser Ile Ser Glu Glu Glu Glu Gly Phe
325 330 335 Asp Cys Leu Ser Ser Pro Glu Arg Ala Glu Pro Pro Gly Gly
Gly Trp 340 345 350 Arg Gly Ser Leu Gly Glu Pro Pro Pro Pro Pro Arg
Ala Ser Leu Ser 355 360 365 Ser Asp Thr Ser Ala Leu Ser Tyr Asp Ser
Val Lys Tyr Thr Leu Val 370 375 380 Val Asp Glu His Ala Gln Leu Glu
Leu Val Ser Leu Arg Pro Cys Phe 385 390 395 400 Gly Asp Tyr Ser Asp
Glu Ser Asp Ser Ala Thr Val Tyr Asp Asn Cys 405 410 415 Ala Ser Val
Ser Ser Pro Tyr Glu Ser Ala Ile Gly Glu Glu Tyr Glu 420 425 430 Glu
Ala Pro Arg Pro Gln Pro Pro Ala Cys Leu Ser Glu Asp Ser Thr 435 440
445 Pro Asp Glu Pro Asp Val His Phe Ser Lys Lys Phe Leu Asn Val Phe
450 455 460 Met Ser Gly Arg Ser Arg Ser Ser Ser Ala Glu Ser Phe Gly
Leu Phe 465 470 475 480 Ser Cys Ile Ile Asn Gly Glu Glu Gln Glu Gln
Thr His Arg Ala Ile 485 490 495 Phe Arg Phe Val Pro Arg His Glu Asp
Glu Leu Glu Leu Glu Val Asp 500 505 510 Asp Pro Leu Leu Val Glu Leu
Gln Ala Glu Asp Tyr Trp Tyr Glu Ala 515 520 525 Tyr Asn Met Arg Thr
Gly Ala Arg Gly Val Phe Pro Ala Tyr Tyr Ala 530 535 540 Ile Glu Val
Thr Lys Glu Pro Glu His Met Ala Ala Leu Ala Lys Asn 545 550 555 560
Ser Asp Trp Val Asp Gln Phe Arg Val Lys Phe Leu Gly Ser Val Gln 565
570 575 Val Pro Tyr His Lys Gly Asn Asp Val Leu Cys Ala Ala Met Gln
Lys 580 585 590 Ile Ala Thr Thr Arg Arg Leu Thr Val His Phe Asn Pro
Pro Ser Ser 595 600 605 Cys Val Leu Glu Ile Ser Val Arg Gly Val Lys
Ile Gly Val Lys Ala 610 615 620 Asp Asp Ser Gln Glu Ala Lys Gly Asn
Lys Cys Ser His Phe Phe Gln 625 630 635 640 Leu Lys Asn Ile Ser Phe
Cys Gly Tyr His Pro Lys Asn Asn Lys Tyr 645 650 655 Phe Gly Phe Ile
Thr Lys His Pro Ala Asp His Arg Phe Ala Cys His 660 665 670 Val Phe
Val Ser Glu Asp Ser Thr Lys Ala Leu Ala Glu Ser Val Gly 675 680 685
Arg Ala Phe Gln Gln Phe Tyr Lys Gln Phe Val Glu Tyr Thr Cys Pro 690
695 700 Thr Glu Asp Ile Tyr Leu Glu 705 710 1052136DNAHomo
sapiensdescription of sequence nucleic acid sequence encoding human
IB1 protein 105atggcggagc gagaaagcgg cggcctggga gggggggccg
cgtccccgcc cgccgcctcc 60ccgttcctgg ggctgcacat cgcttcgcct cccaatttca
ggctcaccca tgacatcagc 120ctggaggagt ttgaggatga agacctctcg
gagatcactg atgagtgtgg catcagctta 180cagtgcaaag acaccctgtc
cttacggccc ccgcgcgccg ggctgctctc tgcgggcggc 240ggcggcgcgg
ggagccggtt gcaggccgag atgctgcaga tggacctgat cgacgcgacg
300ggggacactc ccggggccga ggacgacgag gaggacgacg acgaggagcg
cgcggcccgg 360cggccgggag cggggccgcc caaggccgag tccggccagg
agccggcgtc ccgcggccag 420ggccagagcc aaggccagag ccagggcccg
ggcagcgggg acacgtaccg gcccaagcgg 480cccaccacgc tcaacctctt
tccgcaggtg ccgcggtctc aggacacact gaataataat 540tctctgggca
aaaagcacag ttggcaggat cgggtgtctc gatcatcctc acccctgaag
600acaggggagc agacaccacc gcatgaacac atctgcctga gcgatgagct
gcccccccag 660agcggccccg cccccaccac agatcgaggc acctccaccg
acagcccttg ccgccgcagc 720acagccaccc agatggcacc tccgggtggt
ccccctgctg ccccgcctgg gggtcggggc 780cactcgcatc gagaccgaat
ccactaccag gccgatgtgc gactagaggc cactgaggag 840atctacctga
ccccagtgca gaggccccca gacgctgcag agcccacctc cgccttcctg
900ccgcccactg agagccggat gtcagtcagc tccgatccag accctgccgc
ctacccctcc 960acggcagggc ggccgcaccc ctccatcagt gaagaggaag
agggcttcga ctgcctgtcg 1020tccccagagc gggctgagcc cccaggcgga
gggtggcggg ggagcctggg ggagccgccg 1080ccacctccac gggcctctct
gagctcggac accagcgccc tgtcctatga ctctgtcaag 1140tacacgctgg
tggtagatga gcatgcacag ctggagctgg tgagcctgcg gccgtgcttc
1200ggagactaca gtgacgagag tgactctgcc accgtctatg acaactgtgc
ctccgtctcc 1260tcgccctatg agtcggccat cggagaggaa tatgaggagg
ccccgcggcc ccagccccct 1320gcctgcctct ccgaggactc cacgcctgat
gaacccgacg tccatttctc caagaaattc 1380ctgaacgtct tcatgagtgg
ccgctcccgc tcctccagtg ctgagtcctt cgggctgttc 1440tcctgcatca
tcaacgggga ggagcaggag cagacccacc gggccatatt caggtttgtg
1500cctcgacacg aagacgaact tgagctggaa gtggatgacc ctctgctagt
ggagctccag 1560gctgaagact actggtacga ggcctacaac atgcgcactg
gtgcccgggg tgtctttcct 1620gcctattacg ccatcgaggt caccaaggag
cccgagcaca tggcagccct ggccaaaaac 1680agtgactggg tggaccagtt
ccgggtgaag ttcctgggct cagtccaggt tccctatcac 1740aagggcaatg
acgtcctctg tgctgctatg caaaagattg ccaccacccg ccggctcacc
1800gtgcacttta acccgccctc cagctgtgtc ctggagatca gcgtgcgggg
tgtgaagata 1860ggcgtcaagg ccgatgactc ccaggaggcc aaggggaata
aatgtagcca ctttttccag 1920ttaaaaaaca tctctttctg cggatatcat
ccaaagaaca acaagtactt tgggttcatc 1980accaagcacc ccgccgacca
ccggtttgcc tgccacgtct ttgtgtctga agactccacc 2040aaagccctgg
cagagtccgt ggggagagca ttccagcagt tctacaagca gtttgtggag
2100tacacctgcc ccacagaaga tatctacctg gagtag 2136
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