U.S. patent application number 13/709727 was filed with the patent office on 2013-10-17 for method for treatment of inflammatory disease and disorder.
The applicant listed for this patent is HEALOR LTD.. Invention is credited to Moshe BEN-HAMO, Liora BRAIMAN-WIKSMAN, Ephraim BRENER, Marina GARTSBEIN, Liat HAMMER, Yuval SAGIV, Tamar TENNENBAUM.
Application Number | 20130274196 13/709727 |
Document ID | / |
Family ID | 44305895 |
Filed Date | 2013-10-17 |
United States Patent
Application |
20130274196 |
Kind Code |
A1 |
BRAIMAN-WIKSMAN; Liora ; et
al. |
October 17, 2013 |
METHOD FOR TREATMENT OF INFLAMMATORY DISEASE AND DISORDER
Abstract
The present disclosure provides a method, composition and kit
for treatment of inflammatory disease and disorder using PKC
isoform modulators.
Inventors: |
BRAIMAN-WIKSMAN; Liora;
(Rishon Le-Zion, IL) ; TENNENBAUM; Tamar;
(Jerusalem, IL) ; SAGIV; Yuval; (Gedera, IL)
; GARTSBEIN; Marina; (Petach Tikva, IL) ; BRENER;
Ephraim; (Rishon Le Zion, IL) ; BEN-HAMO; Moshe;
(Bene Braq, IL) ; HAMMER; Liat; (Modiin,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HEALOR LTD. |
Rehovot |
IL |
US |
|
|
Family ID: |
44305895 |
Appl. No.: |
13/709727 |
Filed: |
December 10, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13508636 |
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PCT/IL2011/000034 |
Jan 11, 2011 |
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13709727 |
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61293794 |
Jan 11, 2010 |
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61405509 |
Oct 21, 2010 |
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Current U.S.
Class: |
514/17.9 ;
514/18.7 |
Current CPC
Class: |
A61P 11/06 20180101;
A61P 37/02 20180101; A61P 5/14 20180101; A61P 21/04 20180101; A61P
13/12 20180101; A61P 19/02 20180101; A61K 38/08 20130101; A61P
29/00 20180101; A61P 17/08 20180101; C12N 9/12 20130101; A61K 38/10
20130101; C07K 14/4703 20130101; A61P 1/00 20180101; A61P 3/10
20180101; C12Y 207/11013 20130101; A61K 38/4866 20130101; A61P 1/16
20180101; A61P 17/00 20180101; A61P 37/06 20180101; A61P 17/04
20180101; A61P 17/06 20180101; A61P 27/02 20180101; A61P 1/04
20180101; A61P 25/00 20180101; A61P 11/00 20180101; A61P 37/08
20180101 |
Class at
Publication: |
514/17.9 ;
514/18.7 |
International
Class: |
A61K 38/10 20060101
A61K038/10; A61K 38/08 20060101 A61K038/08 |
Claims
1. A method of treating an inflammatory disease or disorder in a
subject comprising, administering to the subject an inhibitor of
PKC, thereby treating the inflammatory disease or disorder in the
subject.
2-33. (canceled)
Description
BACKGROUND OF THE DISCLOSURE
[0001] 1. Field of the Disclosure
[0002] The disclosure relates generally to methods of treating
disease and more specifically to treatment of inflammatory disease
and disorder.
[0003] 2. Background Information
[0004] Initiation of inflammation begins with an inflammatory
response and leads to the activation of neutrophils, granulocytes,
monocytes, macrophages, as well as other immunomodulatory cells.
This may result in a topical or systemic inflammatory cascade
involving inflammatory cytokines and mediators, such as
interleukins, TNF.alpha., and prostaglandins. This complex
inflammatory mediated cascade triggers a whole range of responses,
such as cellular chemotaxis and endothelial injury and leads to the
recruitment of additional cells from the innate and adaptive immune
systems.
[0005] The skin serves as an important boundary between the
internal body and the environment, preventing contact with
potentially harmful pathogens. In the case of antigen/pathogen
penetration, an inflammatory response is often induced to eliminate
the antigen. This response leads to a dermal infiltrate that
consists predominantly of T cells, polymorphonuclear cells, and
macrophages.
[0006] The inflammatory response is not necessarily associated with
external stimuli, or may be caused by a non-harmful environmental
substances (in case of allergies). In both cases an over-expression
of proinflammatory cytokines without proper controls leads to
inflammation which is the hallmark of topical and systemic
inflammation generally, as well as a variety of inflammatory
diseases and disorders. Inflammation is associated with a variety
of disorders such as eczema and dermatitis, including for example,
atopic dermatitis, seborrheic dermatitis, dyshidrotic eczema,
nummular dermatitis, stasis dermatitis, allergic dermatitis,
psoriasis, pruritis, multiple sclerosis, cutaneous inflammation,
cicatricial pemphigoid, scleroderma, hidradenitis suppurativa,
toxic epidermal necrolysis, acne, osteitis, graft vs. host disease
(GvHD), pyroderma gangrenosum, and Behcet's Syndrome.
[0007] Not surprisingly, over production of proinflammatory
cytokines has been implicated in many inflammatory and autoimmune
diseases. For example, the secretion of cytokines such as
TNF.alpha. and Interleukin (IL)-23, which stimulates survival and
proliferation of Th17 cells, are highly associated with psoriasis,
where IL-6 is required for Th17 development in addition to its
general role as proinflammatory cytokine. Other cytokines like
IL-12 and IP-10 are initiators and involves in Th1 pathway which is
typical to psoriasis and other autoimmune diseases. Interleukin 5
(IL-5), a cytokine that increases the production of eosinophils, is
over-expressed in asthma resulting in accumulation of eosinophils
in the asthmatic bronchial mucosa, a hallmark of allergic
inflammation. Interleukin 4 (IL-4) and interleukin 13 (IL-13) are
known mediators of the hypercontractility of smooth muscle found in
inflammatory bowel disease and asthma. Additionally, as discussed
further below, inflammatory cytokines have been shown to be
implicated in, by way of example, psoriasis, multiple sclerosis,
arthritis, ischemia, septic shock, and organ transplant
rejection.
[0008] Similarly, granulocyte macrophage-colony stimulating factor
(GM-CSF) is a regulator of maturation of granulocyte and macrophage
lineage population and has been implicated as a key factor in many
inflammatory and autoimmune diseases. For example, antibodies that
inhibit GM-CSF secretion have been shown to ameliorate autoimmune
disease.
[0009] Thus, development of therapeutics that reduce secretion of
proinflammatory cytokines and/or regulate immunomodulators would be
beneficial in alleviating topical and systemic inflammation
generally, as well as a host of inflammatory and/or autoimmune
diseases as discussed herein. Several lines of evidence point to
modulators of PKC isoforms as useful in achieving these
results.
[0010] Several in vivo studies have shown the involvement of T
helper (Th) 17 cells as well as secretion of cytokines such as
interleukins and TNF.alpha., by skin associated cells such as
keratinocytes, dendritic and T helper cells, as key players in the
development of the inflammatory response involved in the
pathogenesis of psoriasis and other autoimmune inflammatory
diseases. As used herein, in vivo (Latin for "within the living")
is experimentation using a whole, living organism as opposed to a
partial or dead organism, or an in vitro ("within the glass", for
instance, in a test tube or petri dish) controlled environment. The
secretion of cytokines such as TNF.alpha. and Interleukin (IL)-23,
which stimulates survival and proliferation of Th17 cells, also
serves as a key master cytokine regulator for these diseases.
(Fitch et al. (2007) Curr Rheumatol Rep. 9:461-7). Th17 cells
within dermis in turn, induce secretion of IL-17A and IL-22. IL-22,
in particular, derive keratinocyte hyperproliferation and augment
the inflammatory response in psoriasis (Fitch et al. (2007) Curr
Rheumatol Rep 9:461-7).
[0011] The protein kinase C (PKC) family represents a group of
phospholipid dependent enzymes catalyzing the covalent transfer of
phosphate from ATP to serine and threonine residues of proteins.
The family is currently considered as composed of at least 12
individual isoforms which belong to 3 distinct categories based on
their activation by calcium ion(s) and other factors. The PKC
family consists of at least ten members, usually divided into three
subgroups: classical, novel and atypical PKCs (FIG. 1). The
specific cofactor requirements, tissue distribution, and cellular
compartmentalization suggest differential functions and the tuning
of specific signaling cascades for each isoform. Thus, specific
stimuli can lead to differential responses via isoform specific PKC
signaling regulated by their factors, such as: expression,
localization, and/or phosphorylation status in particular
biological settings. PKC isoforms are activated by a variety of
extracellular signals and, in turn, modify the activities of
cellular proteins including receptors, enzymes, cytoskeletal
proteins, and transcription factors. Accordingly, the PKC family
plays a central role in cellular signal processing including
regulation of cell proliferation, differentiation, survival and
death.
[0012] PKC.alpha., which is highly abundant in skin, is the major
conventional, Ca2+ responsive, PKC isoform in epidermis and it was
initially the only cPKC detected in the keratinocytes in vitro and
in vivo (Dlugosz et al. (1992) Biomed Pharmacother 46:304; Wang et
al. (1993) J Cancer Res Clin Oncol 119:279-287). Therefore,
PKC.alpha. had been proposed as a key player in Ca2+ induced
differentiation (Denning et al. (1995) Cell Growth Differ
6:149-157; Dlugosz et al. (1992) Biomed Pharmacother 46:304). Being
in epidermis and mainly restricted to suprabasal layers (Denning et
al. (2004) Int J Biochem Cell Biol 36:1141-1146), PKC.alpha. is
involved in cell cycle withdrawal and primarily associated with the
keratin cytoskeleton and desmosomal cell-cell junctions (Jansen et
al. (2001) Int J Cancer 93:635-643; Tibudan et al. (2002) J Invest
Dermatol. 119:1282-1289). Since, upon exposure to the classical PKC
activator TPA (12-O-tetradecanoylphorbol-13-acetate), spinous
markers were suppressed, PKC.alpha. was thought to be largely
responsible for the shift from spinous to granular differentiation
as a result of TPA activation (Dlugosz and Yuspa (1993) J Cell Biol
120:217-225; Lee et al. (1998) J Invest Dermatol 111:762-766;
Matsui et al. (1992) J Invest Dermatol 99:565-571; Punnonen et al.
(1993) J Invest Dermatol 101:719-726). Indeed, blocking PKC.alpha.
activity or its synthesis by antisense oligonucleotides appeared to
abolished granular markers and revive spinous markers like K1 and
K10. Likewise, implementation of dominant negative PKC.alpha.
appeared to restore the (late) spinous marker involucrin (Deucher
et al. (2002) J Biol Chem 277:17032-17040). Accordingly, defective
differentiation in skin cancer (Tennenbaum et al. (1993) Cancer Res
3:4803-4810; Tomakidi et al. (2003) J Pathol 200:298-307)
correlates with elevated PKC.alpha. activity, also observed in
tumor cells in vitro (Dlugosz et al. (1992) Biomed Pharmacother
46:304; Yang et al. (2003) J Cell Physiol. 195:249-259). However,
over-expression of PKC.alpha. in normal human keratinocytes did not
appear to alter their differentiation pattern (Deucher et al.
(2002) J Biol Chem 277:17032-17040). The influence of PKC.alpha. on
the cellular traffic and membrane recruitment of .beta.1-integrin
during migration (Ng et al. (1999) EMBO J. 18:3909-3923) may well
promote both wound reepithelialization and tumor cell invasion.
[0013] Over-expression of PKC.alpha. in transgenic mice has
appeared to induce a striking inflammatory response, increased
epidermal thickening and edema correlated with neutrophil
infiltration, multiple micro-abscesses, and a marked increase of
inflammatory cytokines and chemokines, such as TNF.alpha., MIP-2,
COX-2 or macrophage inflammatory protein (MIP). These results
implicate PKC.alpha. in the epidermal inflammatory response (Wang
and Smart (1999) J Cell Sci 112:3497-3506). Treatment with TPA (a
PKC.alpha. activator) apparently caused epidermal hyperplasia,
intra-epidermal inflammation, and massive apoptosis (Cataisson et
al. (2003) J Immunol 171:2703-2713; Jansen et al. (2001) Int J
Cancer 93:635-643). In addition, recent in vivo studies in PKC
isoenzyme-selective knockout and transgenic mice appear to have
highlighted distinct functions of individual PKCs in the immune
system. These genetic analyses, along with biochemical studies
appear to indicate that PKC-regulated signaling pathways play a
significant role in many aspects of the immune responses. For
example, members of the PKC family appear crucial in T cell
signaling pathways. Particularly, PKC.alpha., isotype appears to
determine the nature of lymphocyte-specific in vivo effector.
PKC.alpha. is also discussed as being involved in macrophages
activation and was apparently shown to be involved in mast cell
signaling (Cataisson et al. (2005) J Immunol 174:1686-1692).
Therefore, PKC isotypes are validated drug targets in adaptive
immunity.
[0014] One example of an inflammatory disease is psoriasis. There
are two main hypotheses about the basic pathology leading to
psoriasis development. The first considers psoriasis as primarily a
disorder of excessive growth and reproduction of skin cells. The
second hypothesis considers psoriasis as an immune-mediated
disorder in which the excessive reproduction of skin cells is
secondary to factors produced by the immune system. Accordingly,
most drugs for psoriasis target one component of the disease,
either the hyper-proliferative state of skin cells, or the skin
inflammatory response as presented in psoriasis plaques.
[0015] Recent data support the notion that both pathways underlie
the pathology of the diseases through a cross talk between skin
cells and immunological milieu (encompassing environment,
surroundings, location and/or setting). Classic genome wide linkage
analysis has identified nine locations (loci) on different
chromosomes associated with tendency to develop psoriasis named
psoriasis susceptibility 1 through 9 (PSORS 1 through PSORS9) loci.
In these locations several genes were characterized and found to
encode for proteins expressed in epidermal cells such as
corneodesmosin, expressed in the granular and cornified layers of
the epidermis and upregulated in psoriasis. On the other hand,
other psoriasis linked genes encode for proteins involved in
modulation of the immune system where characterized such as IL12B
on chromosome 5q which expresses interleukin-12B (Frank et al.
(2009) N Engl J Med 361:496-509).
[0016] Another example of inflammatory disease is Multiple
Sclerosis (MS). MS is a chronic and unpredictable inflammatory
disease of the CNS, which may affect the brain and spinal cord that
commonly affects young adults (Hafler et al. (2005) Immunol Rev
204:208-31). It is currently alleged to be the most common
neurological disease of young adults, and usually initiates between
the ages of 20 and 40, with the tendency to occur in women at
almost double the probability in comparison to men.
[0017] In MS, the myelin sheath, the material that surrounds and
protects the nerve cells, and/or its ability for production is
damaged, this is referred to as "demyelization". This damage has
the effect of slowing down or blocking messages between the brain
and the body, leading to the symptoms observed with MS.
Demyelization and scarring or other lesions in areas disseminated
in the brain and/or spinal cord are considered characteristic of
the disease (Beeton et al. (2007) Journal of Visualized Experiments
594-604). These lesions appear to alter nerve conduction and induce
the disabling neurological deficits that vary with the location of
the demyelinated plaques in the CNS (Beeton et al. (2007) Journal
of Visualized Experiments 594-604). Its clinical signs and symptoms
are variable and depend on the parts of the CNS it affects, and may
include motor, sensory, autonomic and cognitive disabilities
(Noseworthy et al. (2000) N Engl J Med 343:938-52).
[0018] Some common symptoms of MS include: 1) overwhelming sense of
tiredness; 2) balance--walking and co-ordination difficulties; 3)
visual problems--double vision and loss of sight; 4) numbness and
tingling in hands and feet; 5) pain--both mild and severe; loss of
muscle strength; 6) stiffness and spasms in muscles; 7) mood
swings--depression and anxiety; 8) memory and concentration
problems; speech problems (The National MS Society Web Site).
[0019] Progressive disability is the fate of most patients with MS,
especially when a 25-year perspective is included. Half of MS
patients require a cane to walk within 15 years of disease onset.
MS is a major cause of neurologic disability in young and
middle-aged adults and, until the past decade, has had no known
beneficial treatments. MS is difficult to diagnose because of the
non-specific clinical findings, which led to the development of
highly structured diagnostic criteria that include several
technological advances, consisting of MRI scans, evoked potentials,
and cerebrospinal fluid (CSF) studies. Diagnostic criteria
generally rely upon the general principles of scattered lesions in
the central white matter occurring at different times and not
explained by other etiologies such as infection, vascular disorder,
or autoimmune disorder.
[0020] MS is widely considered an autoimmune disease whereby an
unknown agent or agents triggers a T-cell-mediated inflammatory
attack, causing demyelization of CNS (central nervous system)
tissue (Weiner et al. (2004) Arch Neurol 61:1613-1615). The
evidence for an autoimmune reaction targeting myelin is strong but
not definitive. There are, for example, descriptions of primary
oligodendrocyte apoptosis with microglial activation in early
multiple sclerosis lesions in the absence of lymphocytes or myelin
phagocytosis (Manuel et al. (2006) Brain).
[0021] MS characteristically is reported as having four patterns of
disease: relapsing-remitting MS (RRMS), primary progressive MS
(PPMS), progressive relapsing MS (PRMS); and secondary progressive
MS (SPMS). An estimated 50% of patients with RRMS will develop SPMS
in 10 years, and up to 90% of RRMS patients will eventually develop
SPMS. Each pattern of disease may present as mild, moderate or
severe. Persons with RRMS present defined attacks of worsening
neurologic function. These attacks are followed by partial or
complete recovery periods (remissions), during which no disease
progression occurs, (about 85% of people are initially diagnosed
with RRMS). PPMS is characterized by slowly worsening neurologic
function from the beginning, with no distinct relapses or
remissions (about 10% of people are diagnosed with PPMS). In SPMS,
following an initial period of RRMS, many people develop a
Secondary-Progressive disease course in which the disease worsens
more steadily, (about 50% of people with RRMS develop this form of
the disease within 10 years). In PRMS people experience steadily
worsening disease symptoms from the beginning, but with clear
attacks of worsening neurologic function along the way, while the
disease appears to progress without remissions (5%) (The National
MS Society web site).
[0022] There is currently no cure for MS although several
treatments that attempt to reduce disease activity and disease
progression are available. Six drugs in four classes are approved
in the United States for the treatment of MS. FDA-Approved disease
treatments include the following: interferon class, IFN-beta-1a
(REBIF.RTM. and AVONEX.RTM.) and IFN-beta-1b (BETASERON.RTM.);
glatiramer acetate (COPAXONE.RTM.), a polypeptide; natalizumab
(TYSABRI.RTM.); and mitoxantrone (NOVANTRONE.RTM.), a cytotoxic
agent. Other drugs have been used with varying degrees of success,
including corticosteroids, methotrexate, cyclophosphamide,
azathioprine, and intravenous (IV) immunoglobulin. The benefits of
currently approved treatments are relatively modest for relapse
rate and prevention of disability in MS.
[0023] REBIF.RTM. (interferon beta 1a) is a medication manufactured
by a biotechnological process that produces the same interferon
beta as found in the human body. REBIF.RTM. is reportedly given
three times a week subcutaneously. (From the FDA approved
prescription information for REBIF.RTM.).
[0024] AVONEX.RTM. (interferon beta 1a) is a medication
manufactured by a biotechnological process that produces the same
interferon beta as found in the human body. AVONEX.RTM. is
reportedly given as a once a week intramuscular injection. (From
the FDA approved prescription information for AVONEX.RTM.).
[0025] BETASERON.RTM. (interferon beta 1b) is a medication
manufactured by a biotechnological process that made up the same
interferon beta as found in the human body. BETASERON.RTM. is
reportedly injected subcutaneously every other day. (From the FDA
approved prescription information for BETASERON.RTM.).
[0026] COPAXONE.RTM. (glatiramer acetate) is a synthetic protein
that simulates myelin basic protein. Through a mechanism that is
not completely understood, this drug seems to block myelin damaging
T cells by acting as a myelin decoy. COPAXONE.RTM. is reportedly
injected subcutaneously once a day. (From the FDA approved
prescription information for COPAXONE.RTM.).
[0027] TYSABRI.RTM. (natalizumab) is a laboratory produced
monoclonal antibody. It is designed to hamper movement of
potentially damaging immune cells from the bloodstream, across the
"blood-brain barrier" into the brain and spinal cord. TYSABRI.RTM.
is reportedly given once every four weeks by intravenous infusion.
(From the FDA approved prescription information for
TYSABRI.RTM.).
[0028] NOVANTRONE.RTM. (mitoxantrone) belongs to the general group
of medicines called antineoplastics. It has been used to treat
certain forms of cancer. It reportedly acts in MS treatment by
suppressing the activity of T cells, B cells and macrophages that
are presumed to lead the attack on the myelin sheath. (From the FDA
approved prescription information for NOVANTRONE.RTM.).
[0029] Current therapies for combating inflammatory diseases
generally fail to provide a multi-component approach targeting
multiple components of pathogenesis. For example, many treatments
for autoimmune diseases involve targeting a single component of a
disease, either by blocking cellular proliferation, or by
suppressing the immune response in order to block inflammation.
Consequently, there is a strong need to provide effective
therapeutics which target multiple components of inflammatory
disease pathogenesis by targeting and modulating PCK isoform
activity. Specifically targeted therapeutics that are capable of
selective inhibition or activation of specific PKC isoforms are
necessary and would provide for a therapeutic approach that targets
multiple components of inflammatory disease pathogenesis, while
retaining a low level of side effects, for example, when topically
administered. Thus, development of therapeutics that reduce
secretion of proinflammatory cytokines and/or regulate
immunomodulators via PKC isoform modulation would be beneficial in
alleviating topical and systemic inflammation generally, as well as
a host of inflammatory and/or autoimmune diseases as discussed
herein.
SUMMARY OF THE DISCLOSURE
[0030] The present disclosure relates to treatment of inflammatory
disease and disorder by administering to a subject a modulator of
PKC, such as an inhibitor of PKC.epsilon., or PKC.eta., or
activator of PKC.delta..
[0031] Accordingly, in one aspect, the present disclosure provides
a method of treating an inflammatory disease or disorder in a
subject. The method includes administering to the subject an
inhibitor of PKC, thereby treating the inflammatory disease or
disorder in the subject. In exemplary embodiments, the inhibitor is
a polypeptide which selectively inhibits PKC.alpha., PKC.epsilon.
or PKC.eta., such as the polypeptides of SEQ ID NOs: 1-29.
[0032] In another aspect, the present disclosure provides a method
of treating an inflammatory disease or disorder in a subject. The
method includes administering to the subject an activator of
PKC.delta., thereby treating the inflammatory disease or disorder
in the subject. In various embodiments, the activator is a
polypeptide which selectively activates PKC.delta., such as the
polypeptides of SEQ ID NOs: 30-37.
[0033] In another aspect, the present disclosure provides a method
of treating pruritus in a subject. The method includes
administering to the subject an inhibitor of PKC, thereby treating
pruritus in the subject. In various embodiments, the inhibitor is
an inhibitor of PKC.alpha., PKC.epsilon. or PKC.eta.. In exemplary
embodiments, the inhibitor is a polypeptide which selectively
inhibits PKC.alpha., PKC.epsilon. or PKC.eta., such as the
polypeptides of SEQ ID NOs: 1-29.
[0034] In another aspect, the present disclosure provides a method
of treating pruritus in a subject. The method includes
administering to the subject an activator of PKC.delta., thereby
treating pruritus in the subject. In various embodiments, the
activator is a polypeptide which selectively activates PKC6, such
as the polypeptides of SEQ ID NOs: 30-37.
[0035] In another aspect, the present disclosure provides a method
of treating multiple sclerosis in a subject. The method includes
administering to the subject an inhibitor of PKC.alpha.. PKC.eta.,
PKC.epsilon., or PKC.epsilon. thereby treating multiple sclerosis
in the subject. In exemplary embodiments, the inhibitor is a
polypeptide which selectively inhibits PKC.alpha. or PKC.eta., such
as the polypeptides of SEQ ID NOs: 1-13 and 26-29.
[0036] In various aspects, the present disclosure provides a kit
for carrying out the method of the disclosure. In one embodiment,
the kit includes an inhibitor of PKC, such as an inhibitor of
PKC.alpha., PKC.epsilon. or PKC.eta., or an activator of
PKC.delta., as well as instructions for administering the inhibitor
or activator.
[0037] In another aspect, the present disclosure provides an
isolated polypeptide of SEQ ID NO: 3 or a physiologically
acceptable salt thereof, wherein the polypeptide is
N-myristoylated. In an exemplary embodiment, the polypeptide is SEQ
ID NO: 12.
[0038] The present disclosure further provides a pharmaceutical
composition that includes the polypeptide of SEQ ID NO: 3 or a
physiologically acceptable salt thereof, wherein the polypeptide is
N-myristoylated; and a pharmaceutically acceptable vehicle.
[0039] In another aspect, the present disclosure provides an
isolated polypeptide including the amino acid sequence of SEQ ID
NO: 4 or a physiologically acceptable salt thereof. In exemplary
embodiments, the isolated polypeptide is that of SEQ ID NO: 10 or
SEQ ID NO: 13.
[0040] The present disclosure further provides a pharmaceutical
composition that includes an isolated polypeptide including the
amino acid sequence of SEQ ID NO: 4 or a physiologically acceptable
salt thereof.
[0041] In another aspect, the present disclosure provides an
isolated polypeptide selected from SEQ ID NOs: 30-33 or a
physiologically acceptable salt thereof. In exemplary embodiments,
the isolated polypeptide is that of SEQ ID NO: 34-37.
[0042] The present disclosure further provides a pharmaceutical
composition that includes an isolated polypeptide including the
amino acid sequence of SEQ ID NOs: 30-33 or a physiologically
acceptable salt thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIG. 1 is a pictorial representation depicting various
members of the PKC family of isoforms.
[0044] FIG. 2 is a series of pictorial representations depicting
inhibition of PKC.alpha. which regulates keratinocytes structure
integrity characteristic to psoriasis. Skin tissues were paraffin
embedded and stained for hematoxiline and eosine (H&E) general
histological staining or distinct markers for the various skin
layers including Keratin 14 (K14) for basal layer, Keratin 1 (K1)
for spinous layer, Keratin 6 (K6) for keratinocytes migration and
PCNA for keratinocytes proliferation. The results demonstrate
normalization of skin properties following PKC.alpha. inhibition
(left column is WT, right column is PKC.alpha. knock out).
[0045] FIG. 3 is a histogram comparing severity of scaling in
different knock out mice as compared to control after treatment
with IMQ.
[0046] FIG. 4 is a series of pictorial representations showing
scaling in knock out mice as compared with control after treatment
with IMQ.
[0047] FIG. 5 is a series of pictorial representations showing
expression of Filaggrin (Fil), Loricrin (Lor) and Keratin 1
(K1).
[0048] FIGS. 6A-B are a series of pictorial and graphical
representations assessing keratinocytes proliferation in vitro and
in vivo. FIG. 6A is a pictorial representation showing expression
of PCNA. FIG. 6B is a histogram comparing the percentage of PCNA
positive cells treated with HO/02/10 and control.
[0049] FIG. 7 is a series of pictorial representations showing
expression of Filaggrin (Fil), Loricrin (Lor), Keratin 1 (K1), PCNA
and Keratin 14 (K14).
[0050] FIG. 8 is graphical representation presenting a summary of
protein expression data in keratinocytes for various peptide
PKC.alpha. inhibitors.
[0051] FIG. 9 is a histogram comparing the bursting pressure of
skin samples treated with HO/02/10 and control.
[0052] FIG. 10 is a histogram comparing the anti-inflammatory
effect of HO/02/10 on skin wound in B57BL/6J mice after 4 and 9
days post wounds.
[0053] FIG. 11 is a histogram comparing cytokine secretion in
splenocytes treated with HO/02/10.
[0054] FIG. 12 is a series of pictorial representations showing
ICAM expression in basal keratinocytes and endothelial cells in
blood vessels of the skin.
[0055] FIG. 13 is a series of pictorial representations showing
ICAM expression in basal keratinocytes and endothelial cells in
blood vessels of the skin.
[0056] FIG. 14 is a histogram comparing the percent of mice
exhibiting positive ICAM-1 staining at wound edges.
[0057] FIG. 15 is a histogram comparing the number of cells per
field of Iba-1 positively stained cells.
[0058] FIGS. 16A-B are a series of pictorial and graphical
representations showing MAC-2 expression in keratinocytes. FIG. 16A
is a series of stains showing MAC-2 expression. FIG. 16B is a
histogram comparing the number of cells per field of MAC-2
positively stained cells with control, 1, 10 and 100 micrograms per
mL PKC.alpha. inhibitor (from left).
[0059] FIGS. 17A-D are a series of histograms comparing cytokine
secretion in LPS activated keratinocytes treated with HO/02/10.
FIG. 17A compares secretion of IL-6, IL-1.alpha., and GM-CSF. FIG.
17B compares secretion of G-CSF. FIG. 17C compares secretion of
MIP-2. FIG. 17D compares secretion of KC.
[0060] FIGS. 18A-C are a series of histograms comparing cytokine
secretion in LPS activated macrophages treated with HO/02/10. FIG.
18A compares secretion of G-CSF, KC and MIP-2. FIG. 18B compares
secretion of IL1.alpha. (left bars of histogram pairs) and
TNF.alpha. (right bars of histogram pairs). FIG. 18C compares
secretion of IL1.beta. (left bars of histogram pairs) and IL12
(right bars of histogram pairs).
[0061] FIG. 19 is a histogram comparing cytokine secretion in LPS
activated keratinocytes treated with peptide PKC.alpha.
inhibitors.
[0062] FIG. 20 is a histogram comparing cytokine secretion in LPS
activated keratinocytes treated with peptide PKC.alpha.
inhibitors.
[0063] FIGS. 21A-B are histograms comparing cytokine secretion in
TNF.alpha. activated keratinocytes treated with peptide PKC.alpha.
inhibitors. FIG. 21A compares secretion of IL-1A. FIG. 21B compares
secretion of IL-6.
[0064] FIGS. 22A-B are histograms comparing cytokine secretion in
TNF.alpha. activated keratinocytes treated with peptide PKC.alpha.
inhibitors. FIG. 22A compares secretion of G-CSF. FIG. 22B compares
secretion of GM-CSF.
[0065] FIGS. 23A-B are histograms comparing cytokine secretion in
TNF.alpha. activated keratinocytes treated with peptide PKC.alpha.
inhibitors. FIG. 23A compares secretion of MIP-2. FIG. 22B compares
secretion of IP-10.
[0066] FIGS. 24A-B are histograms comparing cytokine secretion in
IL-17A activated keratinocytes treated with peptide PKC.alpha.
inhibitors. FIG. 24A compares secretion of IL-1A. FIG. 24B compares
secretion of IL-6.
[0067] FIGS. 25A-B are histograms comparing cytokine secretion in
IL-17A activated keratinocytes treated with peptide PKC.alpha.
inhibitors. FIG. 25A compares secretion of TNF.alpha.. FIG. 25B
compares secretion of IP-10.
[0068] FIGS. 26A-B are histograms comparing cytokine secretion in
IL-17A activated keratinocytes treated with peptide PKC.alpha.
inhibitors. FIG. 26A compares secretion of G-CSF. FIG. 26B compares
secretion of GM-CSF.
[0069] FIG. 27A-B are histograms comparing cytokine secretion in
IL-17A activated keratinocytes treated with peptide PKC.alpha.
inhibitors. FIG. 27A compares secretion of KC. FIG. 27B compares
secretion of MIP-2.
[0070] FIG. 28 is a series of pictorial and graphical
representations showing down regulation of T cell infiltration to
the dermis and epidermis during the inflammatory stage after
treatment with HO/02/10. FIG. 28A is a series of stains using
anti-CD3 antibodies. FIG. 28B is a histogram comparing the number
of cells per field of CD3 positively stained cells.
[0071] FIG. 29 is a graphical representation presenting a summary
of the effects of treatment using the peptide PKC.alpha. inhibitor
MPDY-1 on different cell types.
[0072] FIG. 30 is a graphical representation showing a schema of
the overall effect of HO/02/10 on the psoriatic related
pathway.
[0073] FIGS. 31A-B are a series of pictorial and graphical
representations showing down regulation of neutrophil infiltration
to the dermis and epidermis during the inflammatory stage after
treatment with HO/02/10. FIG. 31A is a stain using neutrophil
specific antibodies. FIG. 31B is a histogram comparing the number
of cells per field of neutrophil specific positively stained
cells.
[0074] FIG. 32 is a pictorial representation of an SDS PAGE stained
with Ser176/180 antibody.
[0075] FIG. 33 is a graphical representation showing EAE score over
time course of treatment.
[0076] FIG. 34 is a graphical representation showing EAE score over
time course of treatment.
[0077] FIG. 35 is a graphical representation showing EAE score over
time course of treatment.
[0078] FIG. 36 is a graphical representation showing EAE score over
time course of treatment.
[0079] FIG. 37 is a graphical representation showing EAE score over
time course of treatment.
[0080] FIG. 38 is a pictorial representation of the mechanism of
action of histamine that is used in a prick test model to assess
MPDY-1 effect on pruritus.
[0081] FIG. 39 is a pictorial representation showing subject's
forearms injected with histamine and treated with or without
MPDY-1.
[0082] FIG. 40 is a pictorial representation showing subject's
forearms injected with histamine and treated with or without
MPDY-1.
[0083] FIG. 41 is a pictorial representation showing subject's
forearms injected with histamine and treated with or without
MPDY-1.
[0084] FIG. 42 is a pictorial representation showing subject's
forearms injected with histamine and treated with or without
MPDY-1.
[0085] FIG. 43 is a table of data collected in in vitro
immunological tests for PKC.alpha. inhibitor MPDY-1 and PKCS
activator DAP-1 (SEQ ID NO: 34) (all data not presented).
[0086] FIG. 44 is a tabular summary of results for PKC.delta.
activator DAP-1 (SEQ ID NO: 34) of cytokine secretion in
keratinocytes treated with TNF.alpha. and inhibitor.
[0087] FIG. 45 is a histogram showing comparing cytokine secretion
in keratinocytes treated with LPS or TNF.alpha. and various
PKC.epsilon. inhibitors.
[0088] FIG. 46 is a histogram showing comparing cytokine secretion
in keratinocytes treated with LPS or TNF.alpha. and various
PKC.epsilon. inhibitors.
[0089] FIG. 47 is a histogram showing comparing cytokine secretion
in keratinocytes treated with LPS or TNF.alpha. and various PKCe
inhibitors.
[0090] FIG. 48 is a histogram showing comparing cytokine secretion
in keratinocytes treated with LPS or TNF.alpha. and various
PKC.epsilon. inhibitors.
[0091] FIG. 49 is a tabular summary of results for various PKCE
inhibitors of cytokine secretion in keratinocytes treated with LPS
or TNF.alpha. and inhibitor.
[0092] FIG. 50 is a histogram comparing cytokine secretion in LPS
activated keratinocytes treated with peptide PKC.alpha. inhibitors
including MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO. 7), AIP-2 (SEQ
ID NO: 8), AIP-1 (SEQ ID NO: 9), and PPDY (SEQ ID NO: 10).
[0093] FIG. 51 is a histogram comparing cytokine secretion in LPS
activated keratinocytes treated with peptide PKC.alpha. inhibitors
including MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO. 7), AIP-2 (SEQ
ID NO: 8), AIP-1 (SEQ ID NO: 9), and PPDY (SEQ ID NO: 10).
[0094] FIG. 52 is a histogram comparing cytokine secretion in LPS
activated keratinocytes treated with peptide PKC.alpha. inhibitors
including MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO. 7), AIP-2 (SEQ
ID NO: 8), AIP-1 (SEQ ID NO: 9), and PPDY (SEQ ID NO: 10).
[0095] FIG. 53 is a histogram comparing cytokine secretion in LPS
activated keratinocytes treated with peptide PKC.alpha. inhibitors
including MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO. 7), AIP-2 (SEQ
ID NO: 8), AIP-1 (SEQ ID NO: 9), and PPDY (SEQ ID NO: 10).
[0096] FIG. 54 is a histogram comparing cytokine secretion in LPS
activated keratinocytes treated with peptide PKC.alpha. inhibitors
including MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO. 7), AIP-2 (SEQ
ID NO: 8), AIP-1 (SEQ ID NO: 9), and PPDY (SEQ ID NO: 10).
[0097] FIG. 55 is a histogram comparing cytokine secretion in
TNF.alpha. activated keratinocytes treated with peptide PKC.alpha.
inhibitors including MPDY-1 (SEQ ID NO: 6). AWOT-1 (SEQ ID NO. 7),
AIP-2 (SEQ ID NO: 8), AIP-1 (SEQ ID NO: 9), and PPDY (SEQ ID NO:
10).
[0098] FIG. 56 is a histogram comparing cytokine secretion in
TNF.alpha. activated keratinocytes treated with peptide PKC.alpha.
inhibitors including MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO. 7),
AIP-2 (SEQ ID NO: 8), AIP-1 (SEQ ID NO: 9), and PPDY (SEQ ID NO:
10).
[0099] FIG. 57 is a histogram comparing cytokine secretion in
TNF.alpha. activated keratinocytes treated with peptide PKC.alpha.
inhibitors including MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO. 7),
AIP-2 (SEQ ID NO: 8), AIP-1 (SEQ ID NO: 9), and PPDY (SEQ ID NO:
10).
[0100] FIG. 58 is a histogram comparing cytokine secretion in LPS
activated keratinocytes treated with peptide PKC.alpha. inhibitors
including MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO. 7), AIP-2 (SEQ
ID NO: 8), AIP-1 (SEQ ID NO: 9), and PPDY (SEQ ID NO: 10).
[0101] FIG. 59 is a histogram comparing cytokine secretion in LPS
activated keratinocytes treated with peptide PKC.alpha. inhibitor
MPDY-1 (SEQ ID NO: 6).
[0102] FIG. 60 is a histogram comparing cytokine secretion in LPS
activated keratinocytes treated with peptide PKC.alpha. inhibitor
MPDY-1 (SEQ ID NO: 6).
[0103] FIG. 61 is a histogram comparing cytokine secretion in LPS
activated keratinocytes treated with peptide PKC.alpha. inhibitor
MPDY-1 (SEQ ID NO: 6).
[0104] FIG. 62 is a histogram comparing cytokine secretion in LPS
activated keratinocytes treated with peptide PKC.alpha. inhibitor
AWOT-1 (SEQ ID NO: 7).
[0105] FIG. 63 is a histogram comparing cytokine secretion in LPS
activated keratinocytes treated with peptide PKC.alpha. inhibitors
MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2 (SEQ ID NO:
8).
[0106] FIG. 64 is a histogram comparing cytokine secretion in LPS
activated keratinocytes treated with peptide PKC.alpha. inhibitors
MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2 (SEQ ID NO:
8).
[0107] FIG. 65 is a histogram comparing cytokine secretion in
IL-17A activated keratinocytes treated with peptide PKC.alpha.
inhibitors MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2
(SEQ ID NO: 8).
[0108] FIG. 66 is a histogram comparing cytokine secretion in
TNF.alpha. activated keratinocytes treated with peptide PKC.alpha.
inhibitors MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2
(SEQ ID NO: 8).
[0109] FIG. 67 is a histogram comparing cytokine secretion in
TNF.alpha. activated keratinocytes treated with peptide PKC.alpha.
inhibitors MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2
(SEQ ID NO: 8).
[0110] FIG. 68 is a histogram comparing cytokine secretion in
TNF.alpha. activated keratinocytes treated with peptide PKC.alpha.
inhibitors MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2
(SEQ ID NO: 8).
[0111] FIG. 69 is a histogram comparing cytokine secretion in
TNF.alpha. activated keratinocytes treated with peptide PKC.alpha.
inhibitors MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2
(SEQ ID NO: 8).
[0112] FIG. 70 is a histogram comparing cytokine secretion in
TNF.alpha. activated keratinocytes treated with peptide PKC.alpha.
inhibitors MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2
(SEQ ID NO: 8).
[0113] FIG. 71 is a histogram comparing cytokine secretion in
IL-17A activated keratinocytes treated with peptide PKC.alpha.
inhibitors MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2
(SEQ ID NO: 8).
[0114] FIG. 72 is a histogram comparing cytokine secretion in
IL-17A activated keratinocytes treated with peptide PKC.alpha.
inhibitors MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2
(SEQ ID NO: 8).
[0115] FIG. 73 is a histogram comparing cytokine secretion in
IL-17A activated keratinocytes treated with peptide PKC.alpha.
inhibitors MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2
(SEQ ID NO: 8).
[0116] FIG. 74 is a histogram comparing cytokine secretion in
IL-17A activated keratinocytes treated with peptide PKC.alpha.
inhibitors MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2
(SEQ ID NO: 8).
[0117] FIG. 75 is a histogram comparing cytokine secretion in
IL-17A activated keratinocytes treated with peptide PKC.alpha.
inhibitors MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2
(SEQ ID NO: 8).
[0118] FIG. 76 is a histogram comparing cytokine secretion in
IL-17A activated keratinocytes treated with peptide PKC.alpha.
inhibitors MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2
(SEQ ID NO: 8).
[0119] FIG. 77 is a histogram comparing cytokine secretion in
IL-17A activated keratinocytes treated with peptide PKC.alpha.
inhibitors MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2
(SEQ ID NO: 8).
[0120] FIG. 78 is a histogram comparing cytokine secretion in
IL-17A activated keratinocytes treated with peptide PKC.alpha.
inhibitors MPDY-1 (SEQ ID NO: 6), AWOT-1 (SEQ ID NO: 7), and AIP-2
(SEQ ID NO: 8).
[0121] FIG. 79 is a histogram comparing cytokine secretion in LPS
activated keratinocytes treated with peptide PKC.alpha. inhibitors
MPDY-1 (SEQ ID NO: 6) and PDY-1 (SEQ ID NO: 13).
[0122] FIG. 80 is a tabular summary of results for various
PKC.alpha. inhibitors of cytokine secretion in keratinocytes
treated with LPS, TNF.alpha. or IL-17A and inhibitor.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0123] The present disclosure is based on the seminal discovery
that modulators of PKC isoforms may be administered as effective
treatments for inflammatory disease and disorder. The involvement
of PKC isoforms in major cellular processes of skin cells, as well
as many components of the immune system, marks it as a potential
target for the treatment of inflammatory pathologies. The data
presented herein, demonstrate that PKC family isoforms regulate
activation processes in skin and immune cells associated with
inflammation and inflammatory diseases.
[0124] It is to be understood that this disclosure is not limited
to particular compositions, methods, and experimental conditions
described, as such methods and conditions may vary. It is also to
be understood that the terminology used herein is for purposes of
describing particular embodiments only, and is not intended to be
limiting, as the scope of the present disclosure will be limited
only in the appended claims.
[0125] The principles and operation of the methods according to the
present disclosure may be better understood with reference to the
figures and accompanying descriptions.
[0126] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example,
references to "the method" includes one or more methods, and/or
steps of the type described herein which will become apparent to
those persons skilled in the art upon reading this disclosure and
so forth.
[0127] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
skill in the art to which this disclosure belongs. Although any
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the disclosure,
some preferred methods and materials are now described.
[0128] As used herein, the term "subject" refers to a mammalian
subject. As such, treatment of any animal in the order mammalian is
envisioned. Such animals include, but are not limited to horses,
cats, dogs, rabbits, mice, goats, sheep, non-human primates and
humans. Thus, the method of the present disclosure is contemplated
for use in veterinary applications as well as human use.
[0129] "Treatment" of a subject herein refers to both therapeutic
treatment and prophylactic or preventative measures. Those in need
of treatment include those already with an inflammatory disease or
disorder as well as those in which it is to be prevented. Hence,
the subject may have been diagnosed as having an inflammatory
disease or disorder or may be predisposed or susceptible to an
inflammatory disease or disorder.
[0130] As used herein, an "inflammatory disease or disorder" is
intended to include any disease and disorder having etiologies
associated with PKC family isoform regulation. Such diseases
include, and are not limited to, pruritus, skin inflammation,
psoriasis, multiple sclerosis, rheumatoid arthritis,
osteoarthritis, systemic lupus erythematosus, Hashimoto's
thyroidis, myasthenia gravis, diabetes type I or II, asthma,
inflammatory lung injury, inflammatory liver injury, inflammatory
glomerular injury, atopic dermatitis, allergic contact dermatitis,
irritant contact dermatitis, seborrhoeic dermatitis, Sjoegren's
syndrome, keratoconjunctivitis, uveitis, inflammatory bowel
disease, Crohn's disease, ulcerative colitis, an inflammatory
disease of the joints, skin, or muscle, acute or chronic idiopathic
inflammatory arthritis, myositis, a demyelinating disease, chronic
obstructive pulmonary disease, interstitial lung disease,
interstitial nephritis and chronic active hepatitis.
[0131] A "symptom" of an inflammatory disease or disorder is any
morbid phenomenon or departure from the normal in structure,
function, or sensation, experienced by the subject and indicative
of an inflammatory disease or disorder.
[0132] The expression "effective amount" refers to an amount of an
inhibitor or activator of a PKC isoform, such as the polypeptides
of SEQ ID NOs: 1-37, that is effective for preventing, ameliorating
or treating an inflammatory disease or disorder. Such an effective
amount will generally result in an improvement in the signs,
symptoms and/or other indicators of an inflammatory disease or
disorder. For example, in skin inflammation, an effective amount
results in the reduction of swelling and/or inflammation and/or
clearance of redness. For pruritus, an effective amount may result
in the clearance of redness and/or itchiness. For MS, an effective
amount, may result in reducing relapse rate, preventing disability,
reducing number and/or volume of brain MRI lesions, improving timed
25-foot walk, extending the time to disease progression, and the
like.
[0133] As used herein, the term "PKC isoform" as used herein
encompasses all PKC isoforms including PKC.alpha., PKC.beta.,
PKC.delta., PKC.epsilon., PKC.eta., PKC.zeta., PKC.gamma.,
PKC.theta., and PKC.lamda..
[0134] The phrase "modulating expression and/or activity of a PKC
isoform" relates to an increased or reduced expression and/or
activity of a PKC isoform. Increase of the expression leads to
increased production of the PKC isoform.
[0135] The term "activator" is used herein to describe a molecule
that enhances expression and/or activity of a PKC isoform. The term
"inhibitor" is used herein to describe a molecule that inhibits
expression and/or activity of a PKC isoform. Among others, the
phosphoryl transfer region, the pseudosubstrate domain, the
phorbolester binding sequences, and the phosphorylation sites may
be targets for modulation of isoenzyme-specific PKC activity.
[0136] The "pseudosubstrate region" or autoinhibitory domain of a
PKC isoform is herein defined as a consensus sequence of substrates
for the kinase with essentially no phosphorylatable residue. The
pseudosubstrate domain is based in the regulatory region, closely
resembling the substrate recognition motif, which blocks the
recognition site and prevents phosphorylation. Thus, inhibitory
peptides of PKC isoforms, such as the polypeptides of the present
disclosure, are obtained as by replacing a phosphorylatable residue
of serine (S) or tyrosine (T) by alanine (A). PKC is the only PKC
isoform known to have additional binding site enabling the
isoform's activation on the C2 domain, the conserved domain 2 of
PKC6.
[0137] PKC is a major signaling pathway, which mediates
keratinocyte proliferation, migration and differentiation. Many PKC
isoforms are known to be expressed in skin tissue and their
expression/activity appears to play a role in cell proliferation
and/or cell migration and/or cell differentiation. However, their
specific modulation of expression and activity to effectuate
treatment of inflammatory diseases was previously unknown and is
demonstrated in the present disclosure.
[0138] Overall, the results presented herein demonstrate that
modulating expression and/or activity of distinct PKC isoforms is
effective in treatment of inflammation and inflammatory
disease.
[0139] Thus, in one aspect, the present disclosure provides a
method of treating an inflammatory disease or disorder in a
subject. The method includes administering to the subject an
inhibitor of PKC, thereby treating the inflammatory disease or
disorder in the subject. In exemplary embodiments, the inhibitor is
a polypeptide which selectively inhibits PKC.alpha., PKC.epsilon.
or PKC.eta., such as the polypeptides of SEQ ID NOs: 1-29.
[0140] As disclosed in the Examples, administration of PKC isoform
inhibitors has been shown to reduce secretion of pro-inflammatory
cytokines, chemokines and Th1 cytokines in a variety of different
skin cell types (not just skin cells i.e. macrophages are found and
active in other tissues). In addition, administration of PKC
isoform reduce the expression of activating factors such as ICAM-1
on keratinocytes and endothelial cells and mac-2 on macrophages.
Additionally, PKC.alpha. inhibitors have been found effective in
the treatment of skin inflammation and to attenuate the
inflammatory symptoms in inflammatory skin models of psoriasis. As
discussed further in the Examples, the mechanism of action of
inhibitors of PCK isoforms has been elucidated implicating their
use as an effective therapy for inflammatory diseases and
disorders. For example, peptide inhibitors of PCK isoforms have
been shown to: 1) normalize epidermal differentiation marker
expression by reducing terminal differentiation; 2) attenuate
abnormal hyper-proliferation; 3) regulate skin structure and
augment skin strength; and/or 4) down-regulate inflammation by
differentially affecting different cell type recruitment and
activation in various steps of the inflammatory process as
summarized, for example, in FIG. 30.
[0141] Also, as disclosed in the Examples activators of PKC.delta.
have also been shown to reduce secretion of pro-inflammatory
cytokines in a variety of different skin cell types. Thus, in
another aspect, the present disclosure provides a method of
treating an inflammatory disease or disorder in a subject by
administering to the subject an activator of PKC.delta., thereby
treating the inflammatory disease or disorder in the subject. In
various embodiments, the activator is a polypeptide which
selectively activates PKC.delta., such as the polypeptides of SEQ
ID NOs: 30-37.
[0142] Additionally, as disclosed in the Examples, administration
of PKC.alpha. inhibitors and PKC.eta. inhibitors has been found to
attenuate the symptoms of MS. As such, in another aspect, the
present disclosure provides a method of treating multiple sclerosis
in a subject. The method includes administering to the subject an
inhibitor of PKC.alpha. or PKC.eta., thereby treating multiple
sclerosis in the subject.
[0143] Further, administration of PKC isoform inhibitors has been
found effective in the treatment of pruritus. As such, in another
aspect, the present disclosure provides a method of treating an
pruritus in a subject. The method includes administering to the
subject an inhibitor of PKC, thereby treating pruritus in the
subject.
[0144] The Examples and Figures present data showing the ability of
activators of PKC.delta. to inhibit the secretion of major
pro-inflammatory cytokines, such as IL-1, IL-6 and TNF.alpha..
Similar data is shown for a variety of PKC isoform inhibitors,
including PKC.alpha., PKC.epsilon. and PKC.eta.. As shown in the
Examples, formulations including the PKC inhibitors and activators
of the present disclosure, have been shown to inhibit the secretion
of major pro-inflammatory cytokines. As regards psoriasis, without
being bound to a particular theory, it is believed that reducing
the level of pro-inflammatory agents prevents the activation of
endothelial cells in near-by blood vessels, and thus the
recruitment of neutrophiles, macrophages and T cells to the
psoriatic plaque. Moreover, TH1 and TH17 cells were shown to be
implicated in the pathogenesis of psoriasis by the secretion of
specific cytokines, which appear to enhance inflammation or drive
keratinocyte hyperproliferation, respectively. The above mentioned
pro-inflammatory cytokines appear essential for the development of
these TH17 cells (Mangan et al. (2006) Nature 441:231-234; Bettelli
et al. (2006) Nature 441:235-238) and for TH1 cell activity. The
decrease of their secretion by PKC inhibitors and activators
implicates their use in the effective treatment of inflammatory
disorders and pruritus.
[0145] In various embodiments, the inhibitors of PKC isoforms are
inhibitors of the pseudosubstrate region of PKC and are
polypeptides, while the activators of PKC isoforms are also
polypeptides. The terms "polypeptide", "peptide", or "protein" are
used interchangeably herein to designate a linear series of amino
acid residues connected one to the other by peptide bonds between
the alpha-amino and carboxy groups of adjacent residues.
[0146] In various embodiments, Examples of peptide PKC activators
and inhibitors that can be used include, without being limited to,
peptides of SEQ ID NOs: 1-5, 14-19, 26, 27 and 30-33 as shown in
Table 1 or physiologically acceptable salts thereof, as well as the
peptides of SEQ ID NOs: 6-13, 20-25, 28, 29, 34-37 of Table 1 which
are shown having particular modifications or terminal protecting
groups.
TABLE-US-00001 TABLE 1 PKC Isoform Inhibitor and Activator Peptides
Amino Acid Sequence SEQ ID NO PKC.alpha. Inhibitors
Phe-Ala-Arg-Lys-Gly-Ala 1 Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln 2
Phe-Ala-Arg-Lys-Gly-Ala-Leu 3 Phe-Ala-Arg-Lys-Gly-Ala-Arg-Gln 4
Thr-Leu-Asn-Pro-Gln-Trp-Glu-Ser 5
Myristoyl-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-OH 6
H-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-OH 7
Myristoyl-Phe-Ala-Arg-Lys-Gly-Ala-Leu-OH-trifluoracetate salt 8
H-Thr-Leu-Asn-Pro-Gln-Trp-Glu-Ser-OH 9
Palmitoyl-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-OH-acetate salt 10
Palmitoyl-Phe-Ala-Arg-Lys-Gly-Ala-Arg-Gln-OH 11
Myristoyl-Phe-Ala-Arg-Lys-Gly-Ala-Leu-OH 12
H-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-OH-acetate salt 13
PKC.epsilon. Inhibitors Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr 14
Pro-Tyr-Ile-Ala-Leu-Asn-Val-Asp 15 Pro-Ala-Trp-His-Asp 16
Leu-Glu-Pro-Glu-Ala-Ala-Ala-Ala-Ala-Ala-Gly-Lys 17
His-Phe-Glu-Asp-Trp-Ile-Asp 18 Val-Tyr-Val-Ile-Ile-Asp-Leu 19
H-Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr-OH 20
H-Pro-Tyr-Ile-Ala-Leu-Asn-Val-Asp-OH 21 H-Pro-Ala-Trp-His-Asp-OH 22
H-Leu-Glu-Pro-Glu-Ala-Ala-Ala-Ala-Ala-Ala-Gly-Lys-OH 23
H-His-Phe-Glu-Asp-Trp-Ile-Asp-OH 24
H-Val-Tyr-Val-Ile-Ile-Asp-Leu-OH 25 PKC.eta. Inhibitors
Thr-Arg-Lys-Arg-Gln-Arg-Ala-Met-Arg-Arg-Arg-Val-His-Gln-Ile- 26
Asn-Gly Lys-Arg-Thr-Leu-Arg 27
Myristoyl-Thru-Arg-Lys-Arg-Gln-Arg-Ala-Met-Arg-Arg-Arg-Val-His- 28
Gln-Ile-Asn-Gly-OH Myristoyl-Lys-Arg-Thr-Leu-Arg-OH 29 PKC.delta.
Activators His-Phe-Glu-Asp-Trp-Ile-Asp 30
His-Phe-Glu-Asp-Trp-Ile-Asp-His-Phe-Glu-Asp-Trp-Ile-Asp 31
Met-Arg-Ala-Ala-Glu-Ala-Ala-Ala-Ala-Glu-Pro-Met 32
Val-Tyr-Val-Ile-Ile-Asp-Leu-His-Phe-Glu-Asp-Trp-Ile-Asp 33
H-His-Phe-Glu-Asp-Trp-Ile-Asp-His-Phe-Glu-Asp-Trp-Ile-Asp-OH 34
H-Met-Arg-Ala-Ala-Glu-Ala-Ala-Ala-Ala-Glu-Pro-Met-OH 35
H-His-Phe-Glu-Asp-Trp-Ile-Asp-OH 36
H-Val-Tyr-Val-Ile-Ile-Asp-Leu-His-Phe-Glu-Asp-Trp-Ile-Asp-OH 37
PKC.zeta. Inhibitor
Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu 38
Myristoyl-Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu-OH
39
[0147] In various embodiments, the peptide PKC inhibitors or
activators typically contain between 6 and 12 amino acids, but may
be longer or shorter in length. In various embodiment, a peptide
PKC inhibitor or activator may range in length from 6 to 45, 6 to
40, 6 to 35, 6 to 30, 6 to 25, 6 to 20, 6 to 15, or 6 to 10 amino
acids. In one embodiment the peptide includes 6, 7, 8, 9, 10, 11,
12, 13, 14 or 15 amino acids.
[0148] In various embodiments, the peptide PKC inhibitors or
activators may be N-acylated, preferably by an acyl group derived
from a C12-C20 fatty acid, such as C14 acyl (myristoyl) or C16 acyl
(palmitoyl).
[0149] In general, peptide PKC.alpha. inhibitors include the common
motif sequence Phe-Ala-Arg-Lys-Gly-Ala (SEQ ID NO: 1).
Alternatively, in another embodiment, PKC.alpha. inhibitors include
the common motif sequence Thr-Leu-Asn-Pro-Gln-Trp-Glu-Ser (SEQ ID
NO: 5).
[0150] While the peptide PKC inhibitors and activators may be
defined by exact sequence or motif sequences, one skilled in the
art would understand that peptides that have similar sequences may
have similar functions. Therefore, peptides having substantially
the same sequence or having a sequence that is substantially
identical or similar to a PKC inhibitor or activator of Table 1 are
intended to be encompassed. As used herein, the term "substantially
the same sequence" includes a peptide including a sequence that has
at least 60+% (meaning sixty percent or more), preferably 70+%,
more preferably 80+%, and most preferably 90+%, 95+%, or 98+%
sequence identity with the sequences defined by SEQ ID NOs: 1-37
and inhibit or activate PKC isoform activity.
[0151] A further indication that two polypeptides are substantially
identical is that one polypeptide is immunologically cross reactive
with that of the second. Thus, a polypeptide is typically
substantially identical to a second polypeptide, for example, where
the two peptides differ only by conservative substitutions.
[0152] The term "conservative substitution" is used in reference to
proteins or peptides to reflect amino acid substitutions that do
not substantially alter the activity (for example, antimicrobial
activity) of the molecule. Typically conservative amino acid
substitutions involve substitution of one amino acid for another
amino acid with similar chemical properties (for example, charge or
hydrophobicity). The following six groups each contain amino acids
that are typical conservative substitutions for one another: 1)
Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D),
Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine
(R), Lysine (K) 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan
(W).
[0153] The term "amino acid" is used in its broadest sense to
include naturally occurring amino acids as well as non-naturally
occurring amino acids including amino acid analogs. In view of this
broad definition, one skilled in the art would know that reference
herein to an amino acid includes, for example, naturally occurring
proteogenic (L)-amino acids, (D)-amino acids, chemically modified
amino acids such as amino acid analogs, naturally occurring
non-proteogenic amino acids such as norleucine, and chemically
synthesized compounds having properties known in the art to be
characteristic of an amino acid. As used herein, the term
"proteogenic" indicates that the amino acid can be incorporated
into a protein in a cell through a metabolic pathway.
[0154] The terms "identical" or percent "identity" in the context
of two polypeptide sequences, refer to two or more sequences or
subsequences that are the same or have a specified percentage of
amino acid residues that are the same, when compared and aligned
for maximum correspondence, as measured using a sequence comparison
algorithm or by visual inspection.
[0155] The phrase "substantially identical," in the context of two
polypeptides, refers to two or more sequences or subsequences that
have at least 60+%, preferably 80+%, most preferably 90-95+% amino
acid residue identity, when compared and aligned for maximum
correspondence, as measured using a sequence comparison algorithm
or by visual inspection.
[0156] As is generally known in the art, optimal alignment of
sequences for comparison can be conducted, for example, by the
local homology algorithm of Smith & Waterman ((1981) Adv Appl
Math 2:482), by the homology alignment algorithm of Needleman &
Wunsch ((1970) J Mol Biol 48:443), by the search for similarity
method of Pearson & Lipman ((1988) Proc Natl Acad Sci USA
85:2444), by computerized implementations of these algorithms by
visual inspection, or other effective methods.
[0157] Peptide PKC inhibitors or activators may have modified amino
acid sequences or non-naturally occurring termini modifications.
Modifications to the peptide sequence can include, for example,
additions, deletions or substitutions of amino acids, provided the
peptide produced by such modifications retains PKC.alpha.
inhibitory activity. Additionally, the peptides can be present in
the formulation with free termini or with amino-protected (such as
N-protected) and/or carboxy-protected (such as C-protected)
termini. Protecting groups include: (a) aromatic urethane-type
protecting groups which include benzyloxycarbonyl,
2-chlorobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl,
isonicotinyloxycarbonyl and 4-methoxybenzyloxycarbonyl; (b)
aliphatic urethane-type protecting groups which include
t-butoxycarbonyl, t-amyloxycarbonyl, isopropyloxycarbonyl,
2-(4-biphenyl)-2-propyloxycarbonyl, allyloxycarbonyl and
methylsulfonylethoxycarbonyl; (c) cycloalkyl urethane-type
protecting groups which include adamantyloxycarbonyl,
cyclopentyloxycarbonyl, cyclohexyloxycarbonyl and
isobornyloxycarbonyl; (d) acyl protecting groups or sulfonyl
protecting groups. Additional protecting groups include
benzyloxycarbonyl, t-butoxycarbonyl, acetyl, 2-propylpentanoyl,
4-methylpentanoyl, t-butylacetyl, 3-cyclohexylpropionyl,
n-butanesulfonyl, benzylsulfonyl, 4-methylbenzenesulfonyl,
2-naphthalenesulfonyl, 3-naphthalenesulfonyl and
1-camphorsulfonyl.
[0158] In various embodiments, peptide PKC isoform inhibitors and
activators may be administered by any suitable means, including
topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary,
intranasal, intravenous, and/or intralesional administration in
order to treat the subject. However, in exemplary embodiments, the
peptides are formulated for topical application, such as in the
form of a liquid, cream, gel, ointment, foam spray or the like.
[0159] Therapeutic formulations of the PKC isoform inhibitor or
activator used in accordance with the present disclosure are
prepared, for example, by mixing a PKC isoform inhibitor or
activator having the desired degree of purity with optional
pharmaceutically acceptable carriers, excipients and/or stabilizers
(see, for example: Remington's Pharmaceutical Sciences, 16th
edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or
stabilizers are nontoxic to recipients at the dosages and
concentrations employed, and may include buffers such as phosphate,
citrate, and other organic acids; antioxidants including ascorbic
acid and methionine; preservatives (such as octadecyldimethylbenzyl
ammonium chloride; hexamethonium chloride; benzalkonium chloride,
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (for example, Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0160] In exemplary embodiments, the PKC isoform inhibitor or
activator, are formulated in a cream. The inhibitors and activators
of PKC isoforms are ideal for topical treatment of skin
inflammation and other inflammatory disorders since the activity of
PKC enzymes, may be specifically targeted. Inhibition or activation
of specific PKC enzymes is achieved by the ability to selectively
modulate a PKC isoform in lower concentrations, without affecting
other PKC isoforms.
[0161] An exemplary formulation for topical administration is
disclosed in Example 4, in which the peptide MPDY-1 (SEQ ID NO: 6)
is formulated as a cream for topical administration. However, one
skilled in the art would understand that alterations of the
formulation may be made while retaining the essential
characteristics of the cream, such as viscosity, stabilization,
non-toxicity and the like. Also, one skilled in the art would
recognize that the formulation may be used as a vehicle for any of
the peptide PKC inhibitors or activators of the present
disclosure.
[0162] In another embodiment, an article of manufacture, such as a
kit containing materials useful for carrying out the treatment
method of the disclosure is provided. In various embodiments, the
kit includes a PKC isoform activator or inhibitor, namely a peptide
PKC isoform inhibitor or activator as disclosed herein, and
instructions for administering the activator or inhibitor to the
subject.
[0163] The term "instructions" or "package insert" is used to refer
to instructions customarily included in commercial packages of
therapeutic products, that contain information about the
indications, usage, dosage, administration, contraindications,
other therapeutic products to be combined with the packaged
product, and/or warnings concerning the use of such therapeutic
products, and the like.
[0164] As disclosed herein, the inhibitor of PKC.alpha. may be
formulated for a specific route of administration. As such, the kit
may include a formulation including an inhibitor of PKC.alpha. that
is contained in a suitable container, such as, for example, tubes,
bottles, vials, syringes, and the like. The containers may be
formed from a variety of materials such as glass or plastic. The
container holds or contains a composition that is effective for
treating the inflammatory disease and may have a sterile access
port (for example the container may be an intravenous solution bag
or a vial having a stopper pierceable by a hypodermic injection
needle). At least one component in the formulation is an inhibitor
or activator of a PKC isoform. The label or package insert
indicates that the composition is used for treating inflammatory
disease in a subject suffering therefrom with specific guidance
regarding dosing amounts and intervals for providing the
formulation including an inhibitor or activator of a PKC isoform.
The article of manufacture may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0165] It will be understood, that the specific dose level and
frequency of dosage for any particular subject in need of treatment
may be varied and will depend upon a variety of factors including
the activity of the PKC isoform inhibitor or activator employed,
the metabolic stability and length of action of that compound, the
age, body weight, general health, sex, diet, mode and time of
administration, the severity of the particular condition, and the
host undergoing therapy. Generally however, dosage will approximate
that which is typical for known methods of administration of the
specific PKC isoform inhibitor or activator. Persons of skill in
the art can easily determine optimum dosages, dosing methodologies
and repetition rates. The exact formulation and dosage can be
chosen by the individual physician in view of the patient's
condition (Fingl et al. "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1 (1975)).
[0166] Thus, depending on the severity and responsiveness of the
condition to be treated, dosing can be a single or repetitive
administration, with course of treatment lasting from several days
to several weeks or until cure is effected or diminution of the
disorder is achieved.
[0167] In various embodiments where the PKC isoform inhibitor or
activator is a peptide, the peptide is provided in the composition
at a concentration of between 0.001 and 100 .mu.g/ml. For example,
the concentration may be between 0.001 and 100, 0.01 and 50, 0.01
and 10, 0.01 and 1, and 0.01 and 0.5 .mu.g/ml.
[0168] In one dosing protocol, the method comprises administering a
peptide PKC isoform inhibitor or activator to the subject
topically, for example as a cream. The peptide is topically applied
at a concentration of from about 1 .mu.g/ml to about 1000 .mu.g/ml,
1 .mu.g/ml to about 500 .mu.g/ml, 1 .mu.g/ml to about 100 .mu.g/ml,
1 .mu.g/ml to about 10 .mu.g/ml, or 10 .mu.g/ml to about 100
.mu.g/ml. The peptide is administered at least once daily until the
condition is treated.
[0169] In another dosing protocol, the method comprises
administering a peptide PKC isoform inhibitor or activator to the
subject parentally, subcutaneously or intravenously. The peptide is
applied in a concentration of from about 1 .mu.g/ml to about 1000
.mu.g/ml, 1 .mu.g/ml to about 500 .mu.g/ml, 1 .mu.g/ml to about 100
.mu.g/ml, 1 .mu.g/ml to about 10 .mu.g/ml, or 10 .mu.g/ml to about
100 .mu.g/ml. The peptide is administered at least once daily,
weekly, biweekly, or monthly until the condition is treated.
[0170] The following examples are provided to further illustrate
the embodiments of the present disclosure, but are not intended to
limit the scope. While they are typical of those that might be
used, other procedures, methodologies, or techniques known to those
skilled in the art may alternatively be used.
Example 1
Inhibition of PKC.alpha. Regulates Keratinocyte Structure Integrity
Characteristic to Inflammatory Skin Disorder Psoriasis
[0171] Inhibition of PKC.alpha. was shown to regulate keratinocyte
structure integrity characteristic to psoriasis. Skin tissues were
paraffin embedded and stained for H&E (hematoxiline and eosine)
general histological staining or for distinct markers for the
various skin layers including Keratin 14 (K14) for basal layer,
Keratin 1 (K1) for spinous layer, Keratin 6 (K6) for keratinocytes
migration and PCNA for keratinocytes proliferation. The results
demonstrate normalization of skin properties following PKC.alpha.
inhibition (FIG. 2).
Example 2
Models for Assessing In Vivo and Ex Vivo Treatment of Inflammation
Via Psoriasis Models
[0172] Numerous animal models have been previously used to study
psoriasis, however, none of these models were sufficient to
adequately mimic the human disease pathology characterized by
excessive skin production, formation of new blood vessels, and
severe immune dysfunction. In general, to be considered as a useful
model of psoriasis, the model has to share some histopathology
features with psoriasis, exhibit similar pathogenesis and/or
disease mechanism, and respond similarly to therapeutic agents for
the treatment of the disease Existing models exhibit several
characteristics including acanthosis, altered epidermal
differentiation, increase in vascularization, and Leukocytic/T cell
infiltration. However, among the existing mice models, not many
respond to existing drugs and therapies. As such, existing models
were used to develop new in-vitro, ex-vivo and in-vivo models to
assess psoriasis treatment which were utilized in the following
Examples.
In Vitro Models
[0173] Developed models included cell culture studies using cells
lines and primary cultures of skin derived cells as well as immune
cells, utilizing constructs and tools to over-express and
inactivate STAT3 and PKC.alpha. mediated signaling pathways. A vast
set of techniques for the study of skin cell proliferation,
migration, differentiation, inflammation and signaling were
utilized and proved useful in studying the mechanism of psoriasis
development and to study the therapeutic effect of PKC.alpha.
inhibition in psoriasis.
In Vivo Models
[0174] A PKC.alpha. over-expressing and knockout mouse models were
used. Over-expression of PKC.alpha. in keratinocytes using a
K5-PKC.alpha. transgenic mice, was shown to exhibit severe
intra-epidermal neutrophil infiltration and disruption of the
epidermis that mimic conditions such as pustular psoriasis. Both
PKC.alpha. and DN forms of transgenic mice were established which
were studied in vivo by sub-dermal application. In addition,
PKC.alpha. knockout mice are also used to study the effects of
PKC.alpha. inactivation on skin structure and function.
[0175] A STAT3 over-expressing mouse model used. Among the leading
mice models for psoriasis, in terms of similarity to human
psoriasis, is a transgenic mouse in which Stat3, is over-expressed
in epidermal keratinocytes. These mice, develop psoriasiform
epidermal acanthosis and have a cutaneous lymphocytic infiltrate
that is predominantly CD4+ in the dermis, and CD8+ in the
epidermis, all are features that are similar to psoriasis in
human.
[0176] Wound as a model for skin inflammation and hyperplasia. A
screening methodology was developed to detect and quantitatively
assess inflammation in skin lesions in a wound setting which allows
to follow cutaneous inflammatory response in the different skin
compartments and identify agents that affect this response.
Ex Vivo Models
[0177] Psoriatic skin grafting on Chick Chorioalantoic Membrane
(CAM). A technique of psoriatic skin grafting on Chick
Chorioalantoic Membrane (CAM) was developed for the purpose of
testing ex-vivo treatment applications. While this technique is
commonly used for skin tumor studies and angiogenesis experiments,
it was adopted and used for psoriasis studies. This original
approach allows the application of new drugs directly on human
psoriatic skin, thus creating a more clinically relevant study of
new drugs for the treatment of psoriasis. Following grafting,
psoriatic human skin is utilized to establish efficacy and timing
of various treatments in various formulations, analyzed using
morphological, histological and biochemical analysis.
Example 3
Attenuation of Scaling in PKC.alpha. Knock Out Mice
[0178] A PKC.alpha. knockout mouse model was developed and utilized
to study the effects of PKC.alpha. inactivation on skin structure
and function. As shown in FIGS. 3 and 4, attenuation of scaling was
observed in PCK.alpha. knock out mice. FIG. 3 is a histogram
showing that the average scaling severity was reduced by over 50%
in PCK.alpha. knock out mice as compared to control evidencing that
inhibition of PKC.alpha. is a key requirement in treating
psoriasis. This is also shown in FIG. 4, which is a series of
pictures comparing scaling in different mice.
Example 4
Topical PKC.alpha. Inhibitor Formulation
[0179] A topical PKC.alpha. inhibitor formulation was developed and
assessed for effectiveness in treatment of psoriasis. The peptide
PKC.alpha. inhibitor MPDY-1 (SEQ ID NO: 6) was formulated in a
cream (referred to herein as HO/02/10), the components of which are
shown in Table 2.
TABLE-US-00002 TABLE 2 MPDY-1 Cream Based Formulation INGREDIENTS
Water Glycerine Propylene Glycol Methylparaben Phenoxyethanol
Glyceryl Stearate SE Cetyl Alcohol Cosbiol PEG-40 Stearath Sucrose
Distearate Isopropyl Myristate Butylated Hydroxy Toluene Paraffin
Oil Capric/Caprylic Triglyceride Vaseline Propylparaben MPDY-1
Example 5
Effect of PKC.alpha. Inhibitors on In Vitro Epidermal
Differentiation
[0180] The formulation of Example 4 (HO/02/10) was determined to
control epidermal differentiation in vitro. Basal keratinocytes
differentiate to form the spinous layer, characterized by K1/K10
keratins, the granular layer that is characterized by
Loricrin/Filaggrin and the stratum corneum. Defects in expression
and incorporation of Loricrin and Filaggrin filaments are
associated with various immunological skin diseases including
psoriasis. Thus, the effects of HO/02/10, were assessed on skin
differentiation and proliferation. As shown in FIGS. 5 and 6,
HO/02/10 normalized skin proliferation (PCNA) (FIG. 6) and
regulated skin differentiation by reducing the expression of
Loricrin and Filaggrin, while spinous layer remained unaffected
(FIG. 5). Since psoriatic skin keratinocytes differentiate rapidly
to produce granular and mainly large amounts of corneal cells
(scales), while the spinous layer thins, HO/02/10 served to
normalize psoriatic skin by amending the skin characteristics
toward a normal phenotype.
[0181] FIG. 5 shows that HO/02/10 controls epidermal granular
differentiation in vitro. Keratinocytes derived from C57BL/6J mice
were incubated in medium containing Ca.sup.2+ to induce
keratinocytes differentiation. Cells were then incubated in the
presence of HO/02/10 (1 .mu.g/ml). Cells were harvested, run on SDS
PAGE gel and immunoblotted using anti-Filaggrin (Fil), anti
Loricrin (Lor) and anti-Keratin 1 (K1) antibody.
[0182] FIG. 6 shows that HO/02/10 reduced keratinocytes
proliferation in vitro and in vivo. Primary murine keratinocytes
from 2 day Balb/c mice were grown for 5 days to reach full
confluence in 0.05 mM Ca.sup.2+ MEM medium. HO/02/10 treatment
(10.sup.-6M and 10.sup.-5M) was applied 6 h prior to induction of
differentiation. Cells were harvested, run on SDS PAGE gel and
immunoblotted using anti-PCNA antibodies. Results are shown in FIG.
6A. C57Black mice, 8-10 weeks of age were subjected to full
thickness wounding in the upper back area to induce epidermis
remodeling and differentiation. Following the wounding, mice were
treated daily with HO/02/10 (ranged 40-4000 mg/kg/day) for 7 days.
At the termination point, mice were euthanized and upper back skin
samples were fixed in 4% paraformaldehyde solution, following
paraffin embedding and slide preparation. Skin samples were then
subjected to immunohistochemical staining utilizing PCNA antibody.
(n=18). The results are shown in FIG. 6B.
[0183] FIGS. 7 and 8 show additional expression data in
keratinocytes utilizing MPDY-1 (SEQ ID NO: 6) as well as data for
the peptide PKC.alpha. inhibitors AIP-1 (SEQ ID NO: 9), AIP-2 (SEQ
ID NO: 8), AWOT-1 (SEQ ID NO: 7) and PPDY-1 (SEQ ID NO: 10). FIG. 7
shows immunohistochemical staining utilizing anti-PCNA,
anti-Filaggrin (Fil), anti-Loricrin (Lor), anti-Keratin 1 (K1) and
anti-Keratin 14 (K14) antibody in keratinocytes treated with
various peptide PKC.alpha. inhibitors. FIG. 8 presents a summary of
expression data in keratinocytes for various peptide PKC.alpha.
inhibitors.
[0184] In order to test skin strength and elasticity, a bursting
chamber was used to measure the pressure that required for skin
samples to burst (a measurable indicator of skin elasticity and
durability). The results in FIG. 9, demonstrate that HO/02/10
treated skin exhibited enhanced skin strength. Thus, inhibition of
PKC.alpha. may be beneficial to psoriatic skin as it was shown to
enhance skin integrity and prevent bursting of psoriatic
lesions.
[0185] FIG. 9 shows that HO/02/10 dramatically enforced skin
strength. Mice skin was treated for 14 days with HO/02/10 and
subsequently was subjected to bursting pressure analysis. The
bursting chamber device consisted of a fixed volume metal cylinder
closed on one end and connected to a high-pressure CO.sup.2
container via a control valve and a manometer. On the other end of
the chamber, an adjustable frame was installed in order to mount
and hold the tested skin tissue in place. Gas was gradually
released into the chamber, and the pressure inside was continuously
monitored until bursting of the tested tissue occurs.
Example 6
Effect of Pkc Isoform Inhibitors and Activators on Skin
Inflammation
[0186] A methodology was developed to detect and quantitatively
assess inflammation in skin lesions in a wound setting which allows
one to follow cutaneous inflammatory response in the different skin
compartments and identify agents that affect this response (as a
preliminary screening). Inflammatory response was considered severe
when two of the following three conditions were evident: (1)
abscess formation; (2) excessive leukocytosis (>100 cells in a
fixed field x200); (3) high WB C/RBC ratio in blood vessels, where
>20% of WBC content within the blood vessels is shown in a fixed
field x200. Mechanistic characterization of the immunological
response is studied utilizing markers to identify infiltration and
activation of specific immunological cells. Examples for such
markers are: ICAM-1 (as a marker activated basal keratinocytes and
endothelial cells), MAC-2 (as a marker for activated macrophages)
and CD3 (T cell marker). Using this quantitative method, it was
possible to demonstrate a strong anti-inflammatory effect of
HO/02/10 and other peptide PCK.alpha. inhibitors in intact skin and
in skin lesions in different cell types and processes in several
animal models.
[0187] The representative results below demonstrate the
anti-inflammatory effect of HO/02/10 on skin wound in B57BL/6J mice
after 4 and 9 days post wounds (FIG. 10). FIG. 10 shows the dose
response of HO/02/10 effects on inflammation in C57BL/6J mice.
Skins of C57BL/6J mice were treated daily by application of
HO/02/10 (4 .mu.g/kg/day) or (40 .mu.g/kg/day) (6 mice/group).
Treatments were applied topically. Biopsies were collected 4 and 9
days post-wounding. Tissues were excised from euthanized animals
for evaluation of inflammation by histology and
immunohistochemistry.
[0188] HO/02/10 was also shown to decrease pro-inflammatory
cytokine secretion from LPS-activated splenocytes. In order to
assess general anti-inflammatory effects in vitro, mice-derived
primary splenocytes were utilized as an immunological model.
Splenocytes were derived from C57BL/6J mice, red blood cells were
lysed and cells were incubated at 500,000 per well in a 96 well
plate. LPS was added (1 .mu.g/ml for IL-1 and TNF.alpha. test, and
0.2 ng/ml for IL-6 test), and cells were treated with MPDY-1 (1
.mu.g/ml) or PBS. No LPS was added in negative control samples.
Medium was collected after 2 days and the amount of secreted
cytokines was quantified using ELISA.
[0189] FIG. 11, as well as FIGS. 17-27, 43 and 50 demonstrate the
ability of HO/02/10 to decrease dramatically the secretion of major
pro-inflammatory cytokines from activated keratinocytes, such as
TNF.alpha., IL-1 and IL-6. Specifically, IL-6 was shown to be
essential for the development of TH17 cells that are involved in
the pathogenesis of psoriasis, with enhancing effect demonstrated
for IL-1 and TNF.alpha.. TNF.alpha. and IL-6 are known targets for
psoriasis therapy. FIG. 11 demonstrates the effect of 1 .mu.g/ml
HO/02/10.
[0190] HO/02/10 was also shown to inhibit basal keratinocyte and
endothelial cell immunological activation in vivo. ICAM is an
adhesion molecule that allows leukocytes infiltration into
inflammatory lesions. Specifically in skin, basal keratinocytes
express ICAM-1 upon immunological activation which may enhance
infiltration of neutrophils and CD8-T cells into the epidermis, one
of the hallmarks of psoriasis. Thus, the effect of HO/02/10 on ICAM
expression in skin was examined by immunohistochemistry in a wound
inflammatory setting in vivo.
[0191] Down regulation of activated keratinocytes and endothelial
cells (ICAM-1 staining) in skin inflammation was observed. A two-cm
longitudinal incision was done on the upper back of a C57BL/6J
mouse. Following wounding, a sterile pad was sutured to the mouse's
skin. Animals were treated daily with HO/02/10 (n=12). Five days
post-wounding, when inflammatory phase reaches its peak, the mice
were sacrificed, skin tissues were embedded in paraffin and
immunohistochemical staining was performed utilizing anti-ICAM-1
antibodies.
[0192] As shown in FIG. 12, HO/02/10 dramatically reduces ICAM
expression on basal keratinocytes and endothelial in blood vessels
of the skin. This effect was shown to be dose dependent with
maximal effect, demonstrated at 10 .mu.g/ml.
[0193] FIG. 13 shows additional stains showing down regulation of
activated keratinocytes and endothelial cells (ICAM-1 staining) in
skin inflammation. As above, a two-cm longitudinal incision was
done on the upper back of a C57BL/6J mouse. Following wounding, a
sterile pad was sutured to the mouse's skin. Animals were treated
daily with MPDY-1 (n=6). Five days post-wounding, when inflammatory
phase reaches its peak, the mice were sacrificed, skin tissues were
embedded in paraffin and immunohistochemical staining was performed
utilizing anti-ICAM-1 antibodies.
[0194] FIG. 14 is a histogram comparing the percent of mice
exhibiting positive ICAM-1 staining at both wound edges.
[0195] The effect of MPDY-1 on macrophage infiltration was also
shown by Iba-1 staining. Iba-1 is a general marker for macrophages.
FIG. 15 is a histogram showing comparing the number of cells per
field exhibiting positive Iba-1 staining. As above, a two-cm
longitudinal incision was done on the upper back of a C57BL/6J
mouse. Following wounding, a sterile pad was sutured to the mouse's
skin. Animals were treated daily with MPDY-1 (n=6). Five days
post-wounding, when inflammatory phase reaches its peak, the mice
were sacrificed, skin tissues were embedded in paraffin and
immunohistochemical staining was performed utilizing anti-Iba-1
antibodies. A dose dependent effect of MPDY-1 on macrophage
infiltration was observed.
[0196] The effect of MPDY-1 on macrophage activation was also shown
by MAC-2 staining. MAC-2 is a specific marker for activated
macrophages. FIG. 16 shows a series of MAC-2 stains and a histogram
comparing the number of cells per field exhibiting positive MAC-2
staining. A two-cm longitudinal incision was done as described
above. Animals were treated daily with DPBS.sup.-/- (Control) or
MPDY-1 in the specified concentrations (n=6). After 5 days
immunohistochemical staining was performed utilizing anti-MAC-2
antibodies. Bar 1 .mu.m. (*p(control Vs. MPDY-1 10 .mu.g)=0.0028).
Activation of macrophages was significantly inhibited following
MPDY-1 treatment.
[0197] MPDY-1 was also shown to significantly reduce TNF.alpha.
induced IKK activation in keratinocytes in dose dependant manner as
shown in FIG. 32. Murine primary keratinocytes were grown for 4
days to full confluence in Low Ca.sup.+2 MEM. Cells were pretreated
with designated concentration of MPDY-1 as described in the figure
for 1 hour, prior to TNF.alpha. induction. Following MPDY-1
pretreatment, cells were incubated with TNF.alpha. 35 ng/ml for 15
minutes. Reaction was stopped by adding ice-cold dPBS-/- and
keratinocytes were homogenized in RIPA buffer. Samples were
subjected to SDS PAGE Western Blot analysis, utilizing
phospho-IKKa/b (Ser176/180 antibody). Pretreatment with MPDY-1
significantly reduced TNF.alpha. induced IKK activation in
keratinocytes in dose dependant manner, where lowest MPDY-1
concentration (0.1 mg/ml) exhibited the strongest inhibition thus
suppressing NFkB activation.
[0198] As discussed above, HO/02/10 was also shown to decrease
cytokine secretion from activated keratinocytes and macrophages. In
recent years it was found that both immune and skin components are
equally contributing to the cycle underlying psoriatic
pathogenesis. Resident skin cells and immunological cells (both
resident and infiltrating cells) interact in the inflammatory
psoriatic process by cell-cell interactions and cytokine secretion.
Thus, HO/02/10 was examined for its direct effect on the secretion
of pro-inflammatory, chemo-attractant and immunological pathway
related cytokines form both keratinocytes and immune cells such as
macrophages and dendritic cells. The results depicted in FIGS. 17
and 18 demonstrate that HO/02/10 down regulates secretion of immune
related cytokines such as IL-6, IL-1.alpha., GM-CSF, MIP-2 and KC
from keratinocytes and macrophages.
[0199] The results of FIG. 17 show the effect of HO/02/10 on
cytokine secretion in keratinocytes. Keratinocytes were derived
from newborn C57BL/6J mice skin. The cells were incubated for 5
days in 24 wells plates. Cells were then treated with DPBS-/-, LPS
(100 ng/ml), or HO/02/10 (1 .mu.g/ml)+LPS (100 ng/ml). Medium
containing secreted cytokines was collected after 48 hr and
analyzed using a Luminex system.
[0200] The results of FIG. 18 show that HO/02/10 down regulates
cytokine secretion in macrophages. Bone marrow cells were derived
from B6 mice. Cells were incubated for 6 days in the presence of
GM-CSF (20 ng/ml), and then were treated with DPBS-/-, LPS (100
ng/ml) or HO/02/10+LPS (1 .mu.g/ml and 100 ng/ml,
respectively).
[0201] Other peptide PKC.alpha. inhibitors were also shown to
decrease cytokine secretion from activated keratinocytes and
macrophages. FIGS. 19 to 23 show that the peptide inhibitors MPDY-1
(SEQ ID NO: 6), MPDY-1 sh (SEQ ID NO: 12) and PDY-1 (SEQ ID NO: 13)
decrease cytokine secretion from LPS and TNF.alpha. activated
keratinocytes. FIGS. 24 to 27 show that the peptide inhibitors
MPDY-1 (SEQ ID NO: 6), MPDY-1 sh (SEQ ID NO: 12) and PDY-1 (SEQ ID
NO: 13) decrease cytokine secretion from IL-17A activated
keratinocytes.
[0202] Table 3 summarizes the results according to cytokine roles
and origin for HO/02/10.
TABLE-US-00003 TABLE 3 HO/02/10 Effect On Stimulated Mice
Derived-Cells Pro- Chemo- Systemic Th1 Th17 inflammatory
attractants (% (% (% (% inhibition) (% inhibition) inhibition)
inhibition) inhibition) Keratinocytes IL-1 (80%) KC (65%) GM-CSF
IL-6 (40%) IL-6 (40%) MIP-2 (30%) (50%) G-CSF (30%) Spleen IL-1
(50%) IL-6 (40%) TNFa (50%) Bone marrow IL-1 50% KC (40%) G-CSF
IL-12 macrophages TNFa (50%) MIP-2 (30%) (40%) (40%) TNF.alpha.
(50%) Bone marrow IL-6 (30%) IP-10 DCs (20%)
[0203] Various other PKC.alpha. inhibitors were also shown to
decrease cytokine secretion in activated keratinocytes. To
determine their effects, keratinocytes were derived from newborn
BALB/C mice skin. The cells were incubated for 5 days in 24 wells
plate. Cells were then incubated with PBS -/- as control or
stimulated by LPS, TNF.alpha., or IL-17. PKC.alpha. inhibitors were
added as indicated. Medium containing secreted cytokines was
collected after 48 hr and analyzed using ELISA. FIG. 50 is a
tabular summary of cytokine secretion. The PKC.alpha. inhibitors
MPDY-1 (SEQ ID NO: 6), AIP-2 (SEQ ID NO: 8), AIP-1 (SEQ ID NO: 9),
AWOT (SEQ ID NO: 7) and PPDY-1 (SEQ ID NO: 10) were all shown to be
effective in decreasing cytokine secretion in keratinocytes.
[0204] HO/02/10 was also shown to attenuate T cell infiltration to
the skin. The effect of HO/02/10 on T cell infiltration was studied
in vivo using anti-CD3 specific staining.
[0205] As can be seen in FIG. 28, HO/02/10 down regulated T cell
infiltration to the dermis and epidermis during the inflammatory
stage. Specifically HO/02/10 inhibited T cell infiltration into the
epidermis which indicates additional anti-inflammatory properties
also characteristic of psoriasis plaques. A two-cm longitudinal
incision was done as described above. Animals were treated daily
with HO/02/10 (n=12). After nine days immunohistochemical staining
was performed utilizing anti-CD3 antibodies. FIG. 28B is a
histogram comparing the number of cells per field positively
stained for CD3. The effect was statistically significant at
concentrations of 1 .mu.g/ml and 10 .mu.g/ml, where 1 .mu.g/ml
treatment demonstrates stronger effects than 10 .mu.g/ml.
[0206] HO/02/10 was also shown to attenuate neutrophil infiltration
to the skin (FIG. 31). The effect of HO/02/10 on neutrophil
infiltration was studied in vivo using neutrophil specific
staining. A two-cm longitudinal incision was done as described
above. Animals were treated daily with DPBS-/- (Control) or
PKC.alpha. inhibitor in the specified concentrations (n=6). After
five days the mice were sacrificed, skin tissues were embedded in
paraffin and immunohistochemical staining for neutrophils was
performed. Although a dose dependent trend was observed, results
were not statistically significant.
[0207] PKC.delta. activators were also shown to have an
anti-inflammatory effect on keratinocytes and splenocytes.
Keratinocytes were derived from newborn BALB/C mice skin. The cells
were incubated for 5 days in 24 wells plate. Cells were then
incubated with PBS -/- as control or stimulated by LPS or
TNF.alpha.. The PKC.delta. inhibitor DAP-1 (SEQ ID NO: 34) was
added. Medium containing secreted cytokines was collected after 48
hr and analyzed using ELISA. FIG. 43 is a tabular summary showing
cytokine secretion in splenocytes stimulated with LPS. FIG. 44 is a
tabular summary of cytokine secretion in keratinocytes stimulated
by TNF.alpha.. DAP-1 was shown to significantly decease
inflammatory cytokine secretion in both keratinocytes and
splenocytes.
[0208] PKC.epsilon. inhibitors were also shown to have an
anti-inflammatory effect on keratinocytes. Keratinocytes were
derived from newborn BALB/C mice skin. The cells were incubated for
5 days in 24 wells plate. Cells were then incubated with PBS -/- as
control or stimulated by LPS or TNF.alpha.. The PKC.epsilon.
inhibitors EPIP-1 (SEQ ID NO: 20), EPIP-2 (SEQ ID NO: 21), or
EPIP-4 (SEQ ID NO: 23) were added. Medium containing secreted
cytokines was collected after 48 hr and analyzed using ELISA. FIGS.
45-48 show the results for secretion of specific cytokines while
FIG. 49 is a tabular summary of cytokine secretion for the various
PKC inhibitors. Several of the PKC.epsilon. inhibitors were shown
to significantly decease inflammatory cytokine secretion in
keratinocytes.
[0209] In summary, the mechanism of action of PKC isoform
inhibitors and activators was determined implicating their use as
an effective therapy for inflammation and inflammatory disease.
Such peptides were shown to 1) normalize epidermal differentiation
markers expression by reducing terminal differentiation; 2)
attenuate abnormal hyper-proliferation; 3) regulate skin structure
and augment skin strength; and 4) down-regulate inflammation by
differentially affecting different cell type recruitment and
activation in various steps of the inflammatory process.
[0210] FIG. 30 shows a schema depicting the overall effect of the
PKC isoform inhibitors and activators of the present disclosure on
the skin inflammatory and psoriatic related pathway. The scheme
summarizes the inhibitory effect of the inhibitors and activators
on various cell types and inflammatory stages in the skin. PKC
isoform inhibitors and activators inhibit secretion of
pro-inflammatory cytokines (such as, IL-1, IL-6 and TNF.alpha.) by
resident skin immune cells. Accordingly, a decrease in endothelial
cells and keratinocytes activation is achieved, resulting a
significant reduction in ICAM-1 expression, chemokines secretion
and reduce in leukocytes infiltration to the site of inflammation,
including neutrophils, macrophages, and T-cells. Cytokines involved
in the development and progression of the Th1 and Th17 pathways,
both main pathways in psoriasis, were also down regulated.
Example 7
Treatment of Multiple Sclerosis with PKC.alpha. and PKC.eta.
Inhibitors
[0211] Animal Models of Multiple Sclerosis: As CNS tissue is not
easily sampled, to gain information regarding disease mechanisms, a
number of models have been developed and are commonly used in the
literature. These models reportedly include myelin mutants,
chemically induced lesions, viral and autoimmune models that mimic
the clinical status and pathology of MS (Baker et al. (2007) ACNR
6(6):10-12). Among the models, experimental allergic
encephalomyelitis (EAE) is reportedly the most commonly used model
for MS (Baker et al. (2007) ACNR 6(6):10-12).
[0212] Autoimmune Model of multiple sclerosis: Experimental
allergic encephalomyelitis (EAE) has apparently received the most
attention as a model of the MS disease and progression, and is
routinely used in testing therapeutic strategies for MS (Baker et
al. (2007) ACNR 6(6):10-12).
[0213] This disease model exhibits many clinical and histological
features of MS and is reportedly caused by the induction of
autoimmunity to antigens that are either naturally or artificially
expressed in the CNS (Lavi et al. (2005) ISBN 0-387-25517-6; and
Owens et al. (2006) Adv Neurol 98:77-89). Following sensitization
to myelin antigen, animals appear to develop a disease, typified by
limb paralysis. This is associated with blood-brain barrier
dysfunction, mononuclear cell infiltration into the CNS and
conduction block resulting in impaired neurotransmission.
[0214] EAE is polygenic where susceptibility and the clinical
course appears to vary depending on the immunizing antigen and the
strain/species of animal being investigated. MOG, a minor myelin
protein, appears to induce chronic paralytic EAE in C57BL/6 mice.
EAE is not a single model, but a number of models that present with
varying degrees of pathology in similarity to MS (Lavi et al.
(2005) ISBN 0-387-25517-6).
[0215] To test the effects of several PKC inhibitors on development
and clinical conditions of EAE mice various studies were performed
utilizing the following experimental protocols. Specifically, the
peptide PKC.alpha. inhibitor MPDY-1 (SEQ ID NO: 6) and the peptide
PKC.eta. inhibitor MPE-1 (SEQ ID NO: 28) were assessed.
[0216] Female 8-10 week C57BL/6J mice were used for the study. The
total number of Groups was 7 (n=7 per group); total number of
animals was 49. Following anesthesia mice were immunized with
MOG35-55/CFA. Mice were immunized subcutaneously (s.c.) in the
flank with 200 .mu.g MOG35-55/CFA supplemented with 300 .mu.g
tuberculosis (Mt) H37RA (Difco). Pertussis Toxin (PTX) was injected
intravenous (i.v.) at the time of immunization and 48 h (hours)
later. The need for a boost immunization was determined based on a
preliminary calibration experiment (20 mice) with the synthesized
MOG35-55 peptide (done as a calibration procedure prior to the
treatment experiment).
[0217] Treatment was administered as follows. Starting on the day
of immunization, mice were treated by i.p (intraperitoneal)
injection three times a week (200 .mu.l/injection).
[0218] Clinical observation and scoring was performed 6 days/week
for an observation period of 49 days. Body weight was determined
prior to immunization and twice weekly thereafter. Active EAE was
scored on a scale of 0-6 depending on quantifiable clinical
presentation of mice as shown in Table 4 below.
TABLE-US-00004 TABLE 4 EAE Scoring Score Impairment 0 no impairment
1 limp tail 2 limp tail and hind limb paresis 3 .gtoreq.1 hind limb
paralysis 4 full hind limb and hind body paralysis 5 hind body
paralysis and front limb paresis 6 death
[0219] Groups of mice were treated as shown in Table 5.
TABLE-US-00005 TABLE 5 Treatment Regime Group Treatment 1
DPBS.sup.-/- (control) 2 CRAMP 2 mg/kg (CRAMP) 3 CRAMP 0.2 mg/kg
(CRAMP) 4 MPDY-1 0.1 mg/kg 5 MPDY-1 1 mg/kg 6 MPE-1 0.1 mg/kg 7
MPE-1 mg/kg
[0220] Results are presented and summarized in FIGS. 33-37.
[0221] As can be seen from FIG. 33, mice that were treated with
MPDY-1 0.1 mg/kg started to show illness signs on the 13.sup.th day
of the experiment, two days later than the control group. The mice
in the MPDY-1 0.1 mg/kg group showed a lower score during most of
the experiment days compared to the mice in the control group.
Moreover, no mouse in the MPDY-1 0.1 mg/kg group and in the MPE-1
0.1 mg/kg group died during the experiment (score 6), while, in all
the other groups mice died over the course of time (control
group--the first mice died on the 34.sup.th day of the experiment
and another one on the 35.sup.th day) A summary of all mice deaths
is provided in Table 6 below.
TABLE-US-00006 TABLE 6 Summary of Mice Deaths During Treatment
Number of dead mice Group during 49 days Details (on day) 1 2 34,
35 2 2 23, 44 3 2 27, 38 4 0 5 2 16, 38 6 0 7 4 20, 33, 37, 35
[0222] A summary of scores derived from FIG. 33-37 at specific time
points is shown in Table 7.
TABLE-US-00007 TABLE 7 Summary of Scores at Specific Time Points
Score The initial Score on day Score on day on day Group day 18 29
37 Control 11 (0.285) 3.000 2.357 2.857 CRAMP 0.2 mg/kg 10 (0.571)
3.214 2.000 2.571 CRAMP 2 mg/kg 11 (0.143) 2.643 2.643 2.143 MPDY-1
0.1 mg/kg 13 (1.000) 2.571 2.000 1.500 MPDY-1 1 mg/kg 11 (0.214)
3.143 3.286 3.071 MPE-1 0.1 mg/kg 11 (0.429) 2.857 1.929 1.429
MPE-1 1 mg/kg 10 (0.429) 3.071 4.286 4.286
[0223] Groups 4 and 6 of the treatments show resistance to
development of the clinical symptoms of the disease, including
non-mortality until the 49th day of the experiment. MPDY-1 and MPE
treatment at a concentration of 0.1 .mu.g/ml (group 4) reduced EAE
severity and protected mice from fatal EAE observed at a late stage
of the disease in control animals, thus resulting in reducing of
the average group score (MPDY-1 of 1.93; MPE-1 of 2.00) to below
the average of the control group score (2.86) at the end of the
experiment (day 42). MPDY-1 treatment at the concentration of 0.1
.mu.g/ml induced two days delay of the clinical conditions of
development of the EAE comparison as compared to control group.
Therefore, MPDY-1 and MPE appeared to be shown as effective agents
for the treatment of MS.
Example 8
In Vivo Assessment of Pruritus Treatment
[0224] A prick test model utilizing histamine to assess pruritus
was developed as shown in FIG. 38. Forearms of individual subjects
were injected with histamine solution and placebo. The formulation
of Example 4 was topically applied with MPDY-1 at various
concentrations and pruritus was assessed over a time course as
shown in FIGS. 39-42.
[0225] This test was performed by placing a drop of a solution
containing a possible allergen on the skin, and a series of
scratches or needle pricks allow the solution to enter the skin.
The extract enters into the outer layer of the skin (epidermis)
using a fine needle (such as a. 26G disposable needle). This
testing is not painful, and generally there is no bleeding involved
since the needle only scratches the surface of the skin. If the
skin develops a red, raised itchy area (called a wheal), it is as a
result of allergic reaction to that allergen. This is called a
positive reaction. A drop of extract is introduced through a fine
needle (such as a No. 26 disposable needle). The test causes no
discomfort and minimal trauma so that controls and negative test
show only the site of the prick; if anything.
[0226] 26G needles were utilized to introduce histamine stock
(Histatrol Positive Control Histamine, 1 mg/ml, code #HIST14999V,
Trupharm). The test was conducted on healthy volunteers in a
double-blind, randomized test. The formulations were applied on the
forearms. Three treatment areas were chosen and marked (the surface
of the forearm from the elbow to the wrist was divided transversely
into proximal, middle and distal thirds); the areas were pricked
prior the below treatments.
[0227] An area was treated with the active formulation in a
double-blind manner, for 10 minutes--marked as A.
[0228] An area was treated with the Placebo in a double-blind
manner, for 10 minutes--marked as C.
[0229] A color photograph was taken at time zero (T.sub.0), after
10, 20, 30 minutes.
[0230] Pruritus questionnaire was answered by the subjects 5 and 15
minutes after treatment.
[0231] In one study, three subjects were tested with treatment as
shown in Tables 8 and 9.
TABLE-US-00008 TABLE 8 Prick Test Sample Group Left Right forearm
forearm Subject 1 A5 C2 Subject 1 A4 C1 Subject 2 A2 C3 Subject 2
A5 C2 Subject 3 A4 C1 Subject 3 A1 C3
TABLE-US-00009 TABLE 9 Prick Test Treatments Treatment Group MPDY-1
1 .mu.g/ml A1 MPDY-1 10 .mu.g/ml A2 Cream 10 ppm A4 Gel 10 ppm A5
Cream W/O active material C1 Gel W/O Active material C2 DPBS C3
[0232] Subjects were provided with pruritus sensation forms and
asked to indicate the level of pruritus sensed from 0 (no response)
to 4 (uncontrollable pruritus) at different time intervals. Results
are shown in Table 10 below.
TABLE-US-00010 TABLE 10 Results Subject & Site Pruritus score 5
minutes after histamine 1-A5 2 1-A4 1 2-A2 2 2-A5 1 3-A4 1 3-A1 3
1-C2 3 1-C1 4 2-C3 4 2-C2 3 3-C1 3 3-C3 4 15 minutes after
histamine 1-A5 0 1-A4 0 2-A2 0 2-A5 0 3-A4 0 3-A1 1 1-C2 4 1-C1 4
2-C3 4 2-C2 4 3-C1 3 3-C3 4
[0233] Additionally, as is evident in FIGS. 39-42, application of
MPDY-1 significantly attenuated redness, inflammation and pruritus
as compared with control over the time course.
[0234] Although the objects of the disclosure have been described
with reference to the above example, it will be understood that
modifications and variations are encompassed within the spirit and
scope of the disclosure. Accordingly, the disclosure is limited
only by the following claims.
Example 9
Combining Insulin and PKC.alpha. Inhibitor Circumvent Adverse Side
Effects Caused by Insulin Only Treatment
[0235] Wounds were performed on the back of 8-10 week old C57BL
mice by incision and were treated daily for 7 days with either
vehicle (PBS) control or with 1 .mu.M insulin (Humulin, Eli Lilly,
USA) or a mixture of 1 .mu.M insulin combined with 1 .mu.M of the
PKC.alpha. inhibitor of SEQ ID NO: 6. Seven days after wounding all
mice were sacrificed and treated wounds were histologically
analyzed for proliferative capacity of the epidermis (proliferating
cell nuclear antigen-PCNA), angiogenesis, inflammation, epidermal
cells and the remodeling processes at the wound gap.
[0236] As shown in Table 11, the insulin-only treatment caused a
substantial increase in the incidence of abnormal angiogenesis in
the wound area, as compared with the PBS control (60% and 25%,
respectively). Since the wound healing process involves rapidly
proliferating epidermal cells, such increased angiogenesis may also
increase the risk of impaired wound closure by delaying formation
of normal granulation tissue. On the other hand, when insulin was
combined with the PKC.alpha. inhibitor of SEQ ID NO: 6 no abnormal
angiogenesis was observed in the treated wound area.
TABLE-US-00011 TABLE 11 The effect of insulin only and insulin
combined with PKC.alpha. inhibitor on the severity of angiogenesis
at the wound area Proliferative capacity of the epidermis (layers
of Treatment basal cells, PCNA) Angiogenesis PBS (control) 5 high
abnormal N = 4 7 normal 8 normal 6 normal average 6.5 Insulin only
8 high abnormal N = 5 8 normal 6 normal 5 high abnormal 5 high
abnormal average 6.4 Insulin + PKC.alpha. 6 normal inhibitor 6
normal N = 5 4 normal 2 normal 3 normal average 4.2
[0237] In addition, the insulin-only treatment resulted in
increased inflammation, hyperplasia of epidermal cells, delayed
differentiation of the spinous layer of epidermal cells and
increased scarring. None of the adverse side effects, which
resulted from the insulin-only treatment, were observed when the
PKC.alpha. inhibitor was combined with insulin.
Example 10
PKC.alpha. Inhibitor Reduces Wounds Inflammation
[0238] Late and severe inflammatory response in wounds may suppress
the process of healing, thus preventing such inflammation from
development may promote the wound healing process. Accordingly, the
effect of PKC.alpha. inhibitor HO/02 and insulin on wound
inflammation was tested in the following experiment.
[0239] Wounds were performed on the back of C57BL mice by incision
and were treated daily for 7 days with: (i) PBS, control; (ii) 1
.mu.M of the PKC.alpha. inhibitor of SEQ ID NO: 6; (iii) 1 .mu.M
insulin (Humulin, Eli Lilly, USA); or (iv) a mixture of 1 .mu.M
PKC.alpha. inhibitor and 1 .mu.M insulin. Seven days after wounding
all mice were sacrificed and the treated wounds were observed for
inflammation under a microscope. The resulting incidences of severe
inflammation observed in the wound area are summarized in Table
12.
[0240] As shown in Table 12, administering the PKC.alpha. inhibitor
to wounds caused a substantial (33.3%) decrease of severe wound
inflammation incidence, as compared to control. Insulin alone had
no anti-inflammatory effect under the experimental conditions.
TABLE-US-00012 TABLE 12 Incidence of severe inflammation Treatment
in wound (%) PBS control 60 PKC.alpha. inhibitor 40 Insulin 56
PKC.alpha. inhibitor + insulin 50
[0241] These results indicate that a PKC.alpha. inhibitor can be
used in therapy to control severe inflammation of wounds. The
demonstrated capacity of the PKC.alpha. inhibitor of SEQ ID NO:6 to
reduce inflammation, coupled with its capacity to promote epidermal
closure, dermal closure and spatial differentiation of epidermal
cells, makes it a potentially most effective therapeutic agent for
wound healing.
Example 11
Efficacy of Insulin in Combination with the PKC.alpha. Inhibitor
HO/02 in Wound Healing in STZ-Induced Diabetic Mice
[0242] Full thickness skin incisions (20 mm) were performed on the
upper back of anesthetized streptozocin (STZ)-injected (175 mg/kg
body weight) diabetic, citrate buffer injected (6 mice/group)
non-diabetic, and non-injected (6 mice/group), C57BL/6J mice.
Periodical blood glucose monitoring was performed by measuring
glucose levels from tail vein after STZ injection. Only mice
exhibiting blood glucose levels higher than 450 mg/dl were taken
into the study. Following incision, STZ-induced diabetic wounds
were treated daily by application of PBS (7/6 mice/group), insulin
(0.1 units/ml) (6 mice/group), the PKCa inhibitor of SEQ ID NO:6 (1
.mu.g/ml) (7/5 mice/group) or a formulation (herein designated
HO/03/03) containing insulin and the N-myristoylated PKC.alpha.
inhibitor of SEQ ID NO: 6 (7/6 mice/group) directly to the surgical
dressing. Biopsies were collected 9 days post-wounding. Wounds were
excised from euthanized animals for evaluation of wound-healing
parameters by histology and immunohistochemistry.
[0243] Wounds were assessed for epidermal closure by Keratin 14
staining. Wounds were considered closed when complete epidermal
staining was observed across the wound gap. Dermal contraction was
considered when both dermal edges were visible in a fixed field
.times.100 magnification utilizing H&E staining. Epidermal
differentiation was assessed by Keratin 1 staining. Wounds that
displayed positive staining across the entire wound gap were
considered as differentiated (K1 positive).
[0244] As shown in Table 13, STZ-induced diabetic animals were
wound-healing impaired. Comparison of important wound healing
parameters in diabetic treated, untreated and non-diabetic mice
revealed that the group treated with the formulation HO/03/03
exhibited a synergistic healing effect. The synergy was evident in
all critical healing stages including epidermal closure (71% vs.
17% in the diabetic control p<0.05), epidermal differentiation
(28% vs. 0%) and dermal contraction (33% vs. 0%).
[0245] The organization of hypodermis was assessed by the presence
(or absence) of hypodermis at both wound edges. Granulation tissue
formation was assessed by the presence of fibroblasts and collagen
fibers in the wound bed. The wound was considered positive for
granulation tissue formation when a continuous layer of granulation
tissue was present in the wound gap. In addition, we have defined 3
specific histological parameters characterizing severe inflammation
of diabetic wounds: (i) abscess formation at the wounded area, (ii)
excessive leukocytosis (>100 cells in a fixed field
(.times.200)) and (iii) high white blood cells (WBC)/red blood
cells (RBC) ratio within the blood vessels shown in a fixed field
(.times.200). When at least 2 of these parameters are present at
the wound gap, the wound is considered severely inflamed.
TABLE-US-00013 TABLE 13 Histological analysis of wound healing
parameters 9-days post wounding Dermal Epidermal Epidermal closure
differentiation Treatment group closure (K14) (H&E) (Kl)
Diabetic + PBS 17% 0% 0% Diabetic + Insulin 0.1 units 17% 0% 0%
Diabetic + HO/02 1 ug/ml 17% 17% 17% Diabetic + HO/03/03 71% 28%
33% Non-diabetic Buffer Citrate Injected + PBS 100% 17% 100% *
Quantitative analyses of healing parameters were made as described
above.
[0246] Results are presented as percent of wounds in each
group.
TABLE-US-00014 TABLE 14 Histological analysis of wound healing
parameters 9-days post wounding Severe Granulation Treatment group
Hypodermis inflammation tissue Diabetic + PBS 25% 67% 42% Diabetic
+ Insulin 0.1 units 10% 100% 40% Diabetic + SEQ 10% 30% 50% ID NO:
6 1 ug/ml Diabetic + HO/03/03 43% 28% 86% Non-diabetic Buffer
Citrate Injected + PBS 100% 17% 92% * Quantitative analyses of
healing parameters were made as described above.
[0247] Results are presented as percent of wounds in each
group.
[0248] As shown in Table 14, diabetic animals exhibited impairment
in other wound healing parameters such as the organization of
hypodermis at wound edges, inflammation and granulation tissue
formation. Diabetic associated impaired healing parameters were
corrected by treatment with the formulation HO/03/03 as
demonstrated by organization of the hypodermis at the wound gap
edges (43% vs. 25%) and granulation tissue formation (86% vs. 42%).
Treatment with HO/03/03 reduced inflammatory response in the wound
gap (28% vs. 67%). Healing efficacy of the formulation agents alone
(insulin or peptide of SEQ ID NO:6 exhibited only partial healing
effects.
[0249] The results summarized above demonstrate that the
formulation HO/03/03 exhibits a synergistic effect in overcoming
diabetes related healing impairments in multiple healing
parameters.
Example 12
Efficacy of Insulin in Combination with the PKC.alpha. Inhibitor
HO/02 in Wound Healing In Vivo in the Pig Skin Model
[0250] Several wound healing studies were conducted in the pig
model system for further understanding of the wound healing process
as well as the healing effects of the formulation HO/03/03.
[0251] Full thickness 35-40 mm skin incisions were performed on the
back of an anesthetized female pig (5 months old, 60-70 kg). Ten
symmetrical incisions were performed on both sides of the back area
at the same distance from the back bone (20 wounds total). The
wounds were treated daily, twice a day, by application of 1 ml PBS
(10 per group) or the formulation (herein designated HO/03/03)
containing insulin 0.1 units and the N-myristoylated PKC.alpha.
inhibitor 1 .mu.g/ml of SEQ ID NO:6 (10 per group) directly to the
wound area. Wounds were excised from sacrificed animals on 7 and 22
days post-wounding and morphological, histological and
immunohistochemical assessments were performed.
[0252] As shown in Table 15, treatment with the formulation
HO/03/03 accelerates wound healing by affecting epidermal migration
and granulation tissue formation at the early stages of healing.
Moreover, these wounds are large and prone to infections from
environmental pathogens (animals are not kept in sterile
conditions), yet HO/03/03 exhibits attenuation of the inflammatory
response at the wound gap thus promoting healing progression.
TABLE-US-00015 TABLE 15 Histological analysis of efficacy of the
formulation HO/03/03 in pig 7- days post wounding Epidermal Severe
Granulation Treatment group migration inflammation tissue formation
PBS 30% 60% 50% HO/03/03 80% 30% 85% * Results are presented as
percent of wounds in each group
TABLE-US-00016 TABLE 16 Histological analysis of efficacy of the
formulation HO/03/03 in pig 22-days post wounding Epidermal
Epidermal Dermal Treatment group closure Differentiation
Contraction PBS 88% 67% 25% HO/03/03 100% 90% 70% * Results are
presented as percent of wounds in each group
[0253] When testing advanced healing stages, HO/03/03-treated
wounds further exhibit accelerated healing. As shown in Table 16,
no difference was noted in the epidermal closure, though dermal
contraction and epidermis differentiation was significantly higher
in treated wounds. It is important to emphasize that these wounds
were performed on the same animal thus contributing to the marked
healing effects of the treatment.
[0254] From the results summarized above it is readily understood
that the formulation HO/03/03 promotes accelerated healing in the
pig skin model affecting healing parameters in early as well as
late wound healing stages.
Sequence CWU 1
1
3916PRTArtificial SequenceSynthetic peptide 1Phe Ala Arg Lys Gly
Ala 1 5 29PRTArtificial SequenceSynthetic peptide 2Phe Ala Arg Lys
Gly Ala Leu Arg Gln 1 5 37PRTArtificial SequenceSynthetic peptide
3Phe Ala Arg Lys Gly Ala Leu 1 5 48PRTArtificial SequenceSynthetic
peptide 4Phe Ala Arg Lys Gly Ala Arg Gln 1 5 58PRTArtificial
SequenceSynthetic peptide 5Thr Leu Asn Pro Gln Trp Glu Ser 1 5
69PRTArtificial SequenceSynthetic peptide 6Phe Ala Arg Lys Gly Ala
Leu Arg Gln 1 5 79PRTArtificial SequenceSynthetic peptide 7Phe Ala
Arg Lys Gly Ala Leu Arg Gln 1 5 87PRTArtificial SequenceSynthetic
peptide 8Phe Ala Arg Lys Gly Ala Leu 1 5 98PRTArtificial
SequenceSynthetic peptide 9Thr Leu Asn Pro Gln Trp Glu Ser 1 5
109PRTArtificial SequenceSynthetic peptide 10Phe Ala Arg Lys Gly
Ala Leu Arg Gln 1 5 118PRTArtificial SequenceSynthetic peptide
11Phe Ala Arg Lys Gly Ala Arg Gln 1 5 127PRTArtificial
SequenceSynthetic peptide 12Phe Ala Arg Lys Gly Ala Leu 1 5
139PRTArtificial SequenceSynthetic peptide 13Phe Ala Arg Lys Gly
Ala Leu Arg Gln 1 5 148PRTArtificial SequenceSynthetic peptide
14Glu Ala Val Ser Leu Lys Pro Thr 1 5 158PRTArtificial
SequenceSynthetic peptide 15Pro Tyr Ile Ala Leu Asn Val Asp 1 5
165PRTArtificial SequenceSynthetic peptide 16Pro Ala Trp His Asp 1
5 1712PRTArtificial SequenceSynthetic peptide 17Leu Glu Pro Glu Ala
Ala Ala Ala Ala Ala Gly Lys 1 5 10 187PRTArtificial
SequenceSynthetic peptide 18His Phe Glu Asp Trp Ile Asp 1 5
197PRTArtificial SequenceSynthetic peptide 19Val Tyr Val Ile Ile
Asp Leu 1 5 208PRTArtificial SequenceSynthetic peptide 20Glu Ala
Val Ser Leu Lys Pro Thr 1 5 218PRTArtificial SequenceSynthetic
peptide 21Pro Tyr Ile Ala Leu Asn Val Asp 1 5 225PRTArtificial
SequenceSynthetic peptide 22Pro Ala Trp His Asp 1 5
2312PRTArtificial SequenceSynthetic peptide 23Leu Glu Pro Glu Ala
Ala Ala Ala Ala Ala Gly Lys 1 5 10 247PRTArtificial
SequenceSynthetic peptide 24His Phe Glu Asp Trp Ile Asp 1 5
257PRTArtificial SequenceSynthetic Peptide 25Val Tyr Val Ile Ile
Asp Leu 1 5 2617PRTArtificial SequenceSynthetic Peptide 26Thr Arg
Lys Arg Gln Arg Ala Met Arg Arg Arg Val His Gln Ile Asn 1 5 10 15
Gly 275PRTArtificial SequenceSynthetic peptide 27Lys Arg Thr Leu
Arg 1 5 2817PRTArtificial SequenceSynthetic peptide 28Thr Arg Lys
Arg Gln Arg Ala Met Arg Arg Arg Val His Gln Ile Asn 1 5 10 15 Gly
295PRTArtificial SequenceSynthetic peptide 29Lys Arg Thr Leu Arg 1
5 307PRTArtificial SequenceSynthetic peptide 30His Phe Glu Asp Trp
Ile Asp 1 5 3114PRTArtificial SequenceSynthetic peptide 31His Phe
Glu Asp Trp Ile Asp His Phe Glu Asp Trp Ile Asp 1 5 10
3212PRTArtificial SequenceSynthetic peptide 32Met Arg Ala Ala Glu
Ala Ala Ala Ala Glu Pro Met 1 5 10 3314PRTArtificial
SequenceSynthetic peptide 33Val Tyr Val Ile Ile Asp Leu His Phe Glu
Asp Trp Ile Asp 1 5 10 3414PRTArtificial SequenceSynthetic peptide
34His Phe Glu Asp Trp Ile Asp His Phe Glu Asp Trp Ile Asp 1 5 10
3512PRTArtificial SequenceSynthetic peptide 35Met Arg Ala Ala Glu
Ala Ala Ala Ala Glu Pro Met 1 5 10 367PRTArtificial
SequenceSynthetic peptide 36His Phe Glu Asp Trp Ile Asp 1 5
3714PRTArtificial SequenceSynthetic peptide 37Val Tyr Val Ile Ile
Asp Leu His Phe Glu Asp Trp Ile Asp 1 5 10 3813PRTArtificial
SequenceSynthetic peptide 38Ser Ile Tyr Arg Arg Gly Ala Arg Arg Trp
Arg Lys Leu 1 5 10 3913PRTArtificial SequenceSynthetic peptide
39Ser Ile Tyr Arg Arg Gly Ala Arg Arg Trp Arg Lys Leu 1 5 10
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