U.S. patent application number 10/345866 was filed with the patent office on 2003-06-12 for cyclosporins for the treatment of respiratory diseases.
Invention is credited to Lazarova, Tsvetelina, Or, Yat Sun.
Application Number | 20030109426 10/345866 |
Document ID | / |
Family ID | 25179553 |
Filed Date | 2003-06-12 |
United States Patent
Application |
20030109426 |
Kind Code |
A1 |
Or, Yat Sun ; et
al. |
June 12, 2003 |
Cyclosporins for the treatment of respiratory diseases
Abstract
The present invention relates to novel semisynthetic cyclosporin
analogs of formula (I): 1 wherein A is 2 X is absent, --C1-C6
alkyl-, or --C3-C6 cycloalkyl- Y is selected from the group
consisting of: (i) C(O)--O--R1, where R1 is hydrogen, C1-C6-alkyl,
optionally substituted with halogen, heterocyclic, aryl,
C1-C6-alkoxy, C1-C6-alkylthio, halogen-substituted C1-C6-alkoxy, or
halogen-substituted C1-C6-alkylthio; (ii) C(O)--S--R1, where R1 is
as previously defined; (iii) C(O)--OCH.sub.2--OC(O)R2, where R2 is
C1-C6-alkyl, optionally substituted with halogen, C1-C6-alkoxy;
C1-C6-alkylthio, heterocyclic or aryl; (iv) C(S)--O--R1, where R1
is as previously defined, and (v) C(S)--S--R1, where R1 is as
previously defined; B is -.alpha.Abu-, -Val-, -Thr- or -Nva-; and U
is -(D)Ala-, -(D)Ser-, --[O-(2-hydroxyethyl)(D)Ser]-,
--[O-acyl(D)Ser]- or --[O-(2-acyloxyethyl)(D)Ser]-.
Inventors: |
Or, Yat Sun; (Cambridge,
MA) ; Lazarova, Tsvetelina; (Brookline, MA) |
Correspondence
Address: |
ENANTA PHARMACEUTICALS, INC.
ATTN: PATENT DEPT.
500 ARSENAL STREET
WATERTOWN
MA
02472
US
|
Family ID: |
25179553 |
Appl. No.: |
10/345866 |
Filed: |
January 16, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10345866 |
Jan 16, 2003 |
|
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09800856 |
Mar 5, 2001 |
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Current U.S.
Class: |
514/1.7 ;
514/1.8; 514/20.5; 530/317 |
Current CPC
Class: |
A61P 11/06 20180101;
A61P 11/02 20180101; A61K 38/00 20130101; C07K 7/645 20130101; A61P
11/00 20180101 |
Class at
Publication: |
514/11 ;
530/317 |
International
Class: |
A61K 038/13; C07K
007/64 |
Claims
What is claimed is:
1. A cyclosporin represented by the formula 7wherein A is 8X is
absent, --C1-C6 alkyl-, or --C3-C6 cycloalkyl-Y is selected from
the group consisting of: (i) C(O)--O--R1, where R1 is hydrogen,
C1-C6 alkyl, optionally substituted with halogen, heterocyclic,
aryl, C1-C6-alkoxy, C1-C6 alkylthio, halogen-substituted C1-C6
alkoxy, or halogen-substituted C1-C6 alkylthio; (ii) C(O)--S--R1,
where R1 is as previously defined; (iii) C(O)--OCH2--OC(O)R2, where
R2 is C1-C6 alkyl, optionally substituted with halogen, C1-C6
alkoxy, C1-C6 alkylthio, heterocyclic or aryl; (iv) C(S)--O--R1,
where R1 is as previously defined, and (v) C(S)--S--R1, where R1 is
as previously defined; B is -.alpha.Abu-, -Val-, -Thr- or -Nva-;
and U is -(D)Ala-, -(D)Ser-, --[O-(2-hydroxyethyl)(D)Ser]- -,
--[O-acyl(D)Ser]- or --[O-(2-acyloxyethyl)(D)Ser]-, or a
pharmaceutically acceptable salt thereof.
2. A cyclosporin according to claim 1 wherein B is -.alpha.Abu-,
and U is -(D)Ala-
3 A cyclosporin according to claim 1, wherein B is -.alpha.Abu-, U
is -(D)Ala-, X is absent, and Y is selected from a group consisting
of: C(O)--O--R1 where R1 is hydrogen, C1-C6 alkyl, optionally
substituted with halogen, heterocyclic, aryl, C1-C6-alkoxy,
C1-C6-alkylthio, halogen-substituted C1-C6 alkoxy, or
halogen-substituted C1-C6 alkylthio; C(O)--S--R1 where R1 is as
previously defined C(O)--OCH2--OC(O)R2 where R2 is C1-C6 alkyl,
optionally substituted with halogen, C1-C6-alkoxy, C1-C6-alkylthio,
heterocyclic or aryl
4. A cyclosporin according to claim 1 which is selected from the
group consisting of: Compound of Formula (I) wherein
B=-.alpha.Abu-, U=-(D)Ala-, X is absent, Y=COOCH3 Compound of
Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-, X is absent, Y=COOH
Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-, X is
absent, Y=COOEt Compound of Formula (I) wherein B=-.alpha.Abu-,
U=-(D)Ala-, X is absent, Y=COOCH2CH2CH3 Compound of Formula (I)
wherein B=-.alpha.Abu-, U=-(D)Ala-, X is absent, Y=COOCH2Ph
Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-, X is
absent, Y=COOCH2F Compound of Formula (I) wherein B=-.alpha.Abu-,
U=-(D)Ala-, X is absent, Y=COOCHF2 Compound of Formula (I) wherein
B=-.alpha.Abu-, U=-(D)Ala-, X is absent, Y=COOCF3 Compound of
Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-, X is absent,
Y=COOCH2CF3 Compound of Formula (I) wherein B=-.alpha.Abu-,
U=-(D)Ala-, X is absent, Y=COOCH2Cl Compound of Formula (I) wherein
B=-.alpha.Abu-, U=-(D)Ala-, X is absent, Y=COOCH2OCH3 Compound of
Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-, X is absent,
Y=COOCH2OCH2CH2OCH3 Compound of Formula (I) wherein B=-.alpha.Abu-,
U=-(D)Ala-, X is absent, Y=C(O)SCH2Ph Compound of Formula (I)
wherein B=-.alpha.Abu-, U=-(D)Ala-, X is --CH2CH2CH2--, Y=COOCH3
Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-, X is
absent, Y=COOFmoc.
5. A process for preparing a cyclosporin compound represented by
formula I as defined in claim 1, comprising reacting a compound of
formula 1 wherein A=-MeBmt- and B and U are as defined in claim 1
with an olefin represented by the formula CH.sub.2.dbd.CH--X--Y,
wherein X and Y are as defined in claim 1, with a catalyst in the
presence of a lithium salt in an organic solvent.
6. The process as defined in claim 5 wherein said catalyst is
Grubb's ruthenium alkylidene catalyst, Nolan's catalyst, a
benzylidene catalyst or a molybdenum catalyst.
7. The process as defined in claim 5 wherein the reaction is
carried out at from room temperature to about 100.degree. C. for 1
to 7 days.
8. A pharmaceutical composition for topical administration
comprising a cyclosporin compound of claim 1 together with a
pharmaceutically acceptable diluent or carrier therefor.
9. A method for treating inflammatory or obstructive airways
disease in a subject in need of said treatment, which comprises
topically administering to said subject a therapeutically effective
amount of a cyclosporin compound of claim 1.
10. The method of claim 9 wherein said step of topically
administering is by inhalation.
11. The method of claim 9, wherein said airways disease is asthma,
allegic rhinitis, bronchitis, COPD, chronic bronchitis or cystic
fibrosis.
Description
TECHNICAL FIELD
[0001] The present invention relates to novel semisynthetic
cyclosporin analogs for the treatment of asthma and other diseases
characterized by airflow obstruction, their use as pharmaceuticals
and pharmaceutical compositions comprising them, as well as the
processes for their production.
BACKGROUND OF THE INVENTION
[0002] Respiratory diseases are a global problem: millions of
people worldwide, both children and adults, suffer from these
medical conditions. These diseases, which include asthma, COPD, and
cystic fibrosis, as well as chronic sinusitis, reduce quality of
life, impair the ability of sufferers to perform everyday tasks
and, in some cases, cause death.
[0003] Asthma is a disease of unknown etiology in which the bronchi
are inflamed and, as a consequence, obstructed. This narrowing
results from a combination of bronchial smooth muscle contraction,
mucosal oedema, inflammatory cell infiltrate and partial or total
occlusion of the lumen with mucus, cells and cell debris. Bronchial
obstruction is either partially or totally reversible, and this
important feature distinguishes asthma from chronic bronchitis.
Asthma is an extremely common disease with a worldwide prevalence
of between 5% and 8%. In the developed world it is the most common
chronic illness and, for reasons that are unclear, the disease is
on the increase. It is now accepted that asthma is a chronic
inflammatory disorder of the airways in which many cells play a
role, in particular mast cells, eosinophils and T-lymphocytes. In
susceptible individuals this inflammation causes symptoms which are
usually associated with widespread but variable airflow
obstruction. This is often reversible, either spontaneously or with
treatment, and causes an associated increase in airway
responsiveness to a variety of stimuli.
[0004] Current drugs for the treatment for asthma are
corticosteroids, beta agonists, NSAIDS, leukotriene antagonists,
Xanthines and anticholinergics.
[0005] The illness has a wide clinical spectrum ranging from mild
episodic bronchospasm (easily controlled by the occasional use of a
bronchodilator) to a very severe, intractable asthma that sometimes
is resistant to treatment with high doses of oral corticosteroids.
Steroid resistance occurs in fewer than 5% of people with asthma.
However, these patients with severe chronic disease may have been
dependent on corticosteroids, and their disease is often so severe
that full reversibility can be difficult or impossible to
demonstrate.
[0006] Chronic obstructive airways disease, chronic obstructive
lung disease and `smoker`s chest have all been used to describe
what is now known as COPD. COPD is characterized by progressive,
irreversible airway obstruction. It can lead to death from
respiratory or cardiorespiratory failure. COPD consists of two
subsets: chronic bronchitis and emphysema. In practice, it is very
difficult to define the contribution of each of these two
conditions to the obstruction of the airway, and this has led to
the displacement of these labels by the non-specific term COPD. The
pathology of COPD is not fully elucidated, but features include
hypertrophy of mucus-secreting glands, inflammation (including
infiltration with lymphocytes) and goblet cell hyperplasia.
[0007] The treatment of COPD consists of bronchodilators,
intermittent courses of antibiotics and, in some patients, inhaled
and/or oral corticosteroids. The latter are claimed to reduce the
decline in lung function in COPD.
[0008] Cystic fibrosis is an inherited condition. Excess viscid
mucus is produced. This leads to recurrent chest infections and
progressive bronchiectasis. Approximately 50% of cystic fibrosis
sufferers have bronchial hyperresponsiveness and there is an
increased incidence of atopy. There is widespread airway narrowing
and wheezing. Most cystic fibrosis sufferers take bronchodilators;
some take inhaled corticosteroids. At least one study has reported
benefit with oral corticosteroids.
[0009] Corticosteroids are the mainstay of treatment of chronic
respiratory diseases since their introduction in the 1950's. Oral
corticosteroids have today been largely replaced by inhaled
corticosteroids, although severe asthmatics still require
medication by mouth. Inhaled corticosteroids are relatively safe
and extremely effective in most patients, and have improved the
quality of life for millions of asthma sufferers. For those with
severe asthma, however, oral therapy with corticosteroids is
required. When taken for more than a few days, oral corticosteroids
have a number of serious side effects. These include growth
retardation in children, severe osteoporosis (especially in old
age), decreased responsiveness of the pituitary adrenal axis to
stress, fluid retention, diabetes and precipitation of
psychosis.
[0010] Furthermore, an appreciable number of patients have apparent
corticosteroid resistance or unreponsiveness. Patients considered
successfully treated with inhaled or oral steroids often have to be
content with 60% of their predicted lung function. Further increase
in the dose of oral corticosteroids runs the risk of concomitant
side effects.
[0011] Although corticosteroids are effective, for the reasons
stated above, they are not ideal drugs. Over the years doctors have
occasionally used immunosuppressive agents as adjuncts to
corticosteroids in patients with extremely severe disease. Examples
of immunosuppressive drugs include azathioprine, methotrexate,
mycophenolic acid and prodrug, leflunamide, cyclosporin A,
ascomycin, FK-506 and rapamycin.
[0012] There is increasing evidence that chronic inflammation in
asthma is mediated via a network of cytokines emanating from
inflammatory and structural cells in the airways. The prominent
eosinophilic inflammation that characterizes asthma appears to be
orchestrated by cytokines derived from type 2 T-helper (Th2)-like
lymphocytes, suggesting that immunosuppressants such as cyclosporin
A might be beneficial in the control of asthma.
[0013] The cyclosporins comprise a class of structurally
distinctive, cyclic, poly-N-methylated undecapeptides, commonly
possessing pharmacological, in particular immunosuppressive,
anti-inflammatory or antiparasitic activity. The first of the
cyclosporins to be isolated was the naturally occurring fungal
metabolite Ciclosporin or Cyclosporin, also known as cyclosporin A.
3
[0014] Since the original discovery of Ciclosporin, a wide variety
of naturally occurring cyclosporins have been isolated and
identified, and many further non-natural cyclosporins have been
prepared by total- or semi-synthetic means or by the application of
modified culture techniques. The class comprised by the
cyclosporins is thus now substantial and includes, for example, the
naturally occurring cyclosporins A through Z [cf., Traber et
al.;
[0015] 1, Helv. Chim. Acta, 60,1247-1255 (1977); Traber et al.; 2,
Helv. Chim. Acta, 65, 1655-1667 (1982); Kobel et al.; Europ. J.
Applied Microbiology and Biotechnology, 14, 273-240 1982); and von
Wartburg et al.; Progress in Allergy, 38, 28-45, 1986)], as well as
various non-natural cyclosporin derivatives and artificial or
synthetic cyclosporin derivatives and artificial or synthetic
cyclosporins including dihydrocyclosporins [in which the the
-MeBmt-residue is saturated by hydrogenation]; derivatized
cyclosporins (e.g., in which the 3'-O-atom of the -MeBmt-residue is
acylated or a further substituent is introduced at the
.alpha.-carbon atom of the sarcosyl residue at the 3-position); and
cyclosporins in which variant amino acids are incorporated at
specific positions within the peptide sequence, e.g. employing the
total synthetic method for the production of cyclosporins developed
by R. Wenger--see e.g. Traber et al., 1; Traber et al., 2; and
Kobel et al., loc cit. U.S. Pat. Nos. 4,108,985, 4,220,641,
4,288,431, 4,554,351, 4,396,542 and 4,798,823; European Patent
Publication Nos. 34,567A, 56,782A, 300,784A and 300,785;
International Patent Publication No. WO 86/02080 and UK Patent
Publication Nos. 2,206,119 and 2,207,678; Wenger 1, Transpl. Proc.,
15 Suppl. 1:2230 (1983); Wenger 2, Angew. Chem. Int. Ed. 24 77
(1985) and Wenger 3, Progress in the Chemistry of Organic Natural
Products, 50,123 (1986).
[0016] The class comprised by the cyclosporins is thus now very
large and includes for example, [Thr].sup.2-,
[Val].sup.2-[Nva].sup.2 and [Nva].sup.2-[Nva].sup.5-Ciclosporin
(also known as cyclosporins C, D, G and M respectively),
[3-O-acetyl-MeBmt].sup.1-Ciclosporin (also known as
dihydro-cyclosporin D), [(D)Ser].sup.8-Ciclosporin,
[MeIle].sup.11-Ciclosporin, [(D)MeVal].sup.11-Ciclosporin (also
known as cyclosporin H), [MeAla].sup.6-Ciclosporin, [(D)
Pro].sup.3-Ciclosporin and so on.
[0017] Cyclosporin A (CsA) is active against CD4+ lymphocytes and
might, therefore, be useful for asthma. A trial of low-dose oral
CsA in patients with steroid-resistant asthma indicated that it can
improve control of symptoms in patients with severe asthma on oral
steroids.
[0018] The mechanism of CsA action in asthma is of interest. CsA
binds to the ubiquitous protein, cyclophilin, in the cytosol and
the complex binds to calcineurin, which is a calcium- and
calmodulin-dependent serine threonine phosphatase. This phosphatase
is necessary for translocation to the nucleus by the cytoplasmic
portion of the transcription factor, nuclear factor of activated
T-cells (NF-AT). Once translocated to the nucleus and bound to its
nuclear portion to become the active transcription factor, NF-AT
forms a complex with AP-1 and regulates the transcription of the
IL-2 gene, together with other genes, such as IL-5. Since CsA
prevents the cytoplasmic fraction of NF-AT from translocating, it
results in reduced transcription of IL-2. CsA has a specific
inhibitory effect in CD4+ cells through this transcription
mechanism, but may also have inhibitory effects on other cells,
including mast cells and eosinophils, through mechanisms that have
not yet been defined.
[0019] Recently, three controlled trials of CsA in asthma have been
reported. [Alexander A G, Barnes N C, Kay A B. Trial of cyclosporin
in corticosteroid-dependent chronic severe asthma. Lancet 1992;
339: 324-328; Niwanowska E, Dworski R, Domala B, Pinis G.
Cyclosporin for steroid-dependent asthma. Allergy, 1991; 46:
312-315; Lock S H, Kay A B, Barnes N C. Double-blinded,
placebo-controlled study of cyclosporin A as a
corticosteroid-sparing agent in corticosteroid-dependent asthma. Am
J Respir Crit Care Med 1996; 153: 509-14; Nizankowska E, Soja J,
Pinis G, Bochenek G, Sladek K, Domagala B, et al. Treatment of
steroid-dependent bronchial asthma with cyclosporin. Eur Respir J
1995; 8: 1091-1099.]
[0020] CsA at 5 mg/kg/day allowed a significant (about 60%)
reduction in the use of corticosteroids. Side effects with systemic
CsA were: increase in diastolic blood pressure and decrease in
renal function. Other side effects include hepatic dysfunction,
hypertrichosis, tremor, gingival hyperplasis and paraesthesia. The
systemic toxicity of CsA limits its use for the treatment of
asthma, COPD and other related lung diseases.
[0021] Therefore, it would be desirable to obtain derivatives of
CsA, which retain CsA's potential utility as a primary or adjunct
therapy for respiratory diseases, while reducing or eliminating
CsA's systemic toxicity.
SUMMARY OF THE INVENTION
[0022] The present invention relates to novel cyclosporins,
pharmaceutically acceptable salts therof, their use as
pharmaceuticals and pharmaceutical compositions comprising them, as
well as to processes for their production. The compounds of the
invention are particularly useful for topical treatment autoimmune
diseases, e.g., in the treatment of lung diseases.
[0023] More particularly, the present invention provides a
cyclosporin of the following formula (I). 4
[0024] In formula (I), amino acid residues referred to by
abbreviation, eg. -Ala-, -MeLeu-, -.alpha.Abu-, etc., are, in
accordance with conventional practice, to be understood as having
the L-configuration unless otherwise indicated. (For example,
-(D)Ala- represents a residue having the D-configuration). Residue
abbreviations preceded by "Me" as in the case of "MeLeu", represent
(.alpha.-N-methylated residues. Individual residues of the
cyclosporin molecule are numbered, as in the art, clockwise and
starting with the residue, -MeBmt- corresponding to residue 1. The
same numerical sequence is employed throughout the present
specifications and claims.
[0025] In formula (I), A is represented by 5
[0026] wherein:
[0027] X is absent, --C1-C6 alkyl-, or --C3-C6 cycloalkyl-;
[0028] Y is selected from the group consisting of;
[0029] (i) C(O)--O--R1, where R1 is hydrogen, C1-C6 alkyl,
optionally substituted with halogen, heterocyclic, aryl, C1-C6
alkoxy, C1-C6 alkylthio, halogen-substituted C1-C6 alkoxy or
halogen-substituted C1-C6 alkylthio;
[0030] (ii) C(O)--S--R1, where R1 is as previously defined;
[0031] (iii) C(O)--OCH.sub.2--OC(O)R2, where R2 is C1-C6 alkyl,
optionally substituted with halogen; C1-C6 alkoxy; C1-C6 alkylthio,
heterocyclic or aryl;
[0032] (iv) C(S)--O--R1, where R1 is hydrogen; C1-C6 alkyl,
optionally substituted with halogen, heterocyclic, aryl, C1-C6
alkoxy, C1-C6 alkylthio, halogen-substituted C1-C6 alkoxy, or
halogen-substituted C1-C6 alkylthio; and
[0033] (v) C(S)--S--R1, where R1 is as previously defined;
[0034] B is -.alpha.Abu-, -Val-, -Thr- or -Nva-; and
[0035] U is -(D)Ala-, -(D)Ser-, --[O-(2-hydroxyethyl)(D)Ser]-;
--[O-acyl(D)Ser]- or --[O-(2-acyloxyethyl)(D)Ser]-.
[0036] Accordingly, the present invention provides the use of
cyclosporin analogs for the manufacture of a preparation for the
treatment, with or without the concurrent use of other drugs, of
diseases characterized by airflow obstruction and/or of chronic
sinusitis.
DETAILED DESCRIPTION OF THE INVENTION
[0037] A first embodiment of the invention is a compound
represented by Formula I as described above, or a pharmaceutically
acceptable salt thereof.
[0038] A second embodiment of the invention is a compound
represented by Formula I as described above, wherein B is
-.alpha.Abu- and U is -(D)Ala-.
[0039] A third embodiment of the invention is a compound
represented by Formula I as described above, wherein B is
-.alpha.Abu-, U is -(D)Ala- and X is-absent.
[0040] Representative compounds of the invention include, but are
not limited to, the compounds selected from the group consisting
of:
[0041] Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-,
X is absent, Y=COOCH.sub.3
[0042] Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-,
X is absent, Y=COOH
[0043] Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-,
X is absent, Y=COOEt
[0044] Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-,
X is absent, Y=COOCH2CH2CH3
[0045] Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-,
X is absent, Y=COOCH2Ph
[0046] Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-,
X is absent, Y=COOCH2F
[0047] Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-,
X is absent, Y=COOCHF2
[0048] Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-,
X is absent, Y=COOCF3
[0049] Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-,
X is absent, Y=COOCH2CF3
[0050] Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-,
X is absent, Y=COOCH2Cl
[0051] Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-,
X is absent, Y=COOCH2OCH3
[0052] Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-,
X is absent, Y=COOCH2OCH2CH2OCH3
[0053] Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-,
X is absent, Y=C(.dbd.O)SCH2Ph
[0054] Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-,
X is --CH2CH2CH2--, Y_=COOCH3
[0055] Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-,
X is absent, Y=COOFmoc
[0056] Cyclosporins of the invention are accordingly useful for the
treatment of diseases or conditions responsive to or requiring
anti-inflammatory, immunosuppressive or related therapy, including
topical administration for the treatment of such diseases or
conditions of the eye, nasal passages, buccal cavity, skin, colon
or, especially, airways or lung. In particular cyclosporins of the
invention permit topical anti-inflammatory, immunosuppressive or
related therapy with the concomitant avoidance or reduction of
undesirable systemic side effects, for example renal toxicity or
general systemic immunosuppression.
[0057] Cyclosporins of the invention are particularly useful for
the treatment of diseases and conditions of the airways or lung, in
particular inflammatory or obstructive airways disease. They are
especially useful for the treatment of diseases or conditions of
the airways or lung associated with or characterized by
inflammatory cell infiltration or other inflammatory event
accompanied by the accumulation of inflammatory cells, e.g.
eosinophils and/or neutrophils. They are most especially useful for
the treatment of asthma.
[0058] Cyclosporins of the invention are useful in the treatment of
asthma of whatever type of genesis including both intrinsic and,
especially, extrinsic asthma. They are useful for the treatment of
atopic and non-atopic asthma, including allergic asthma, bronchitic
asthma, exercise-induced asthma, occupational asthma, asthma
induced following bacterial infection and other non-allergic
asthmas. Treatment of asthma is also to be understood as embracing
treatment of "wheezy-infant syndrome", that is treatment of
subjects, e.g., of less than 4 to 5 years of age, exhibiting
wheezing symptoms, in particular at night, and diagnosed or
diagnosable as "wheezy infants", an established patient category of
major medical concern and now more correctly indentified as
incipient or early-phase asthmatics. Cyclosporins of the invention
are in particular useful for the treatment of asthma in subjects
whose asthmatic status is either steroid-dependent or
steroid-resistant.
[0059] Cyclosporins of the invention are also useful for the
treatment of bronchitis or for the treatment of chronic or acute
airways obstruction associated therewith. Cyclosporins of the
invention may be used for the treatment of bronchitis of whatever
type or genesis, including, for example, acute bronchitis,
arachidic bronchitis, catarrhal bronchitis, chronic bronchitis,
croupous bronchitis, phthinoid bronchitis and so forth.
[0060] Cyclosporins of the invention are in addition useful for the
treatment of pneumoconiosis (an inflammatory, commonly
occupational, disease of the lungs, frequently accompanied by
airways obstruction, whether chronic or acute, and occasioned by
repeated inhalation of dusts) of whatever type or genesis,
including, for example, aluminosis, anthracosis, asbestosis,
berylliosis, chalicosis, ptilosis, siderosis, silicosis, tabacosis
and, in particular, byssinosis.
[0061] Cyclosporins of the invention may also be used for the
treatment of eosinophil-related disorders of the airways (e.g.
involving morbid eosinophilic infiltration of pulmonary tissues)
including hypereosinophilia as it affects the airways and/or lungs
as well as, for example, eosinophil-related disorders of the
airways consequential or concomitant to Loffler's Syndrome,
eosinophilic pneumonia, parasitic (in particular metazoan)
infestation (including tropical eosinophilia), bronchopulmonary
aspergillosis, polyarteritis nodosa (including Churg-Strauss
Syndrome), eosinophilic granuloma and eosinophil-related disorders
affecting the airways occasioned by drug-reaction.
[0062] The word "treatment" as used above in relation to the
treatment of diseases of the airways and lungs, in particular
asthma, is to be understood as embracing both symptomatic and
prophylactic modes, that is the immediate treatment, e.g. of acute
inflammation (symptomatic treatment) as well as advance treatment
to prevent, ameliorate or restrict long term symptomatology
(prophylactic treatment). The term "treatment" as used in the
present specification and claims in relation to such diseases is to
be interpreted accordingly as including both symptomatic and
prophylactic treatment, e.g., in the case of asthma, symptomatic
treatment to ameliorate acute inflammatory events and prophylactic
treatment to inhibit on-going inflammatory status and to ameliorate
future bronchial exacerbation associated therewith.
[0063] Cyclosporins of the invention may also be used to treat any
disease or condition of the airways or lung requiring
immunosuppressive therapy, e.g., for the treatment of autoimmune
diseases of, or as they affect, the lungs (for example, for the
treatment of sarcoidosis, alveolitis or chronic hypersensitivity
pneumonitis) or for the maintainance of allogenic lung transplant,
e.g., following lung or heart lung transplantation.
[0064] For the above purposes, some cyclosporins of the invention
preferably will be administered topically within the airways, e.g.
by the pulmonary route, by inhalation. While having potent efficacy
when administered topically, cyclosporins of the invention are
devoid of, or exhibit relatively reduced, systemic activity, e.g.
following oral administration. Cyclosporins of the invention thus
provide a means for the treatment of diseases and conditions of the
airways or lung with the avoidance of unwanted systemic side
effect, e.g., consequent to inadvertent swallowing of drug
substance during inhalation therapy. (It is estimated that during
the course of maneuvers required to effect administration by
inhalation, up to 90% or more of total drug substance administered
will inadvertently be swallowed rather than inhaled).
[0065] By the provision of cyclosporins which are topically active,
e.g. effective when inhaled but systemically inactive, the present
invention makes cyclosporin therapy available to subjects for whom
such therapy might otherwise be excluded, e.g., due to the risk of
systemic, in particular immunosuppressive, side effects.
[0066] Cyclosporins of the invention are also useful for the
treatment of other diseases or conditions, in particular diseases
or conditions having an autoimmune or inflammatory component and
for which topical therapy may be practiced, for example, treatment
of diseases and conditions of the eye such as conjunctivitis,
keratoconjunctivitis sicca, and vernal conjunctivitis and
maintenance of corneal transplant, diseases affecting the nose
including allergic rhinitis, diseases and conditions of the skin
including psoriasis, atopic dermatitis, pemphigus and contact
dermatitis, as well as diseases of the colon, for example Crohn's
disease and ulcerative collitis.
[0067] Definitions
[0068] The terms "C1-C3-alkyl" and "C1-C6-alkyl" as used herein
refer to saturated, straight- or branched-chain hydrocarbon
radicals containing between one and three or one and six carbon
atoms, respectively. Examples of C1-C3-alkyl radicals include
methyl, ethyl, propyl and isopropyl, and examples of C1-C6-alkyl
radicals include, but are not limited to, methyl, ethyl, propyl,
isopropyl, n-butyl, tert-butyl, neopentyl and n-hexyl.
[0069] The term "C1-C6-alkoxy" as used herein refers to a
C1-C6-alkyl group, as previously defined, attached to the parent
molecular moiety through an oxygen atom. Examples of C1-C6-alkoxy
groups include, but are not limited to, methoxy, ethoxy, propoxy,
isopropoxy, n-butoxy, tert-butoxy, neopentoxy and n-hexoxy.
[0070] The term "C1-C6-alkylthio" as used herein refers to a
C1-C6-alkyl group, as previously defined, attached to the parent
molecular moiety through a sulfur atom. Examples of C1-C6-alkylthio
groups include, but are not limited to, thiomethoxy, thioethoxy,
thiopropoxy, thio-isopropoxy, n-thiobutoxy, tert-thiobutoxy,
neothiopentoxy and n-thio-hexoxy.
[0071] The term "aryl" as used herein refers to unsubstituted or
substituted carbocyclic aromatic groups including, but not limited
to, phenyl, 1- or 2-naphthyl and the like.
[0072] The term "C3-C6-cycloalkyl-" as used herein refers to
carbocyclic groups of 3 to 6 carbons, respectively; for example,
cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
[0073] The term "C1-C3-alkyl-C3-C5-cycloalkyl", as used herein
refers to a C3-C5-cycloalkyl radical, as defined above, attached to
a C1-C3-alkyl radical by replacement of a hydrogen atom on the
latter.
[0074] The terms "halo" and "halogen" as used herein refer to an
atom selected from fluorine, chlorine, bromine and iodine.
[0075] The term "heterocyclic", as used herein, refers to a cyclic
aromatic radical having one or more rings, each including from five
to ten ring atoms of which at least one ring atom is selected from
S, O and N; zero, one or two ring atoms are additional heteroatoms
independently selected from S, O and N; and the remaining ring
atoms are carbon, the radical being joined to the rest of the
molecule via any of the ring atoms, such as, for example,
pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl,
thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl,
thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.
[0076] As used herein, the term "pharmaceutically acceptable salt"
refers to those salts which are, within the scope of sound medical
judgement, suitable for use in contact with the tissues of humans
and lower animals without undue toxicity, irritation, allergic
response and the like, and are commensurate with a reasonable
benefit/risk ratio. Pharmaceutically acceptable salts are well
known in the art. For example, S. M. Berge, et al. describe
pharmaceutically acceptable salts in detail in J. Pharmaceutical
Sciences, 66: 1-19 (1977), which is incorporated herein by
reference. The salts can be prepared in situ during the final
isolation and purification of the compounds of the invention, or
separately by reacting the free base function with a suitable
organic acid. Examples of pharmaceutically acceptable, nontoxic
acid addition salts are salts of an amino group formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
phosphoric acid, sulfuric acid and perchloric acid or with organic
acids such as acetic acid, oxalic acid, maleic acid, tartaric acid,
citric acid, succinic acid or malonic acid or by using other
methods used in the art such as ion exchange. Other
pharmaceutically acceptalble salts include adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,
borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.
1 Abbreviations Sar: Scarcosin MeLeu: N-Methyl-Leucine Val: Valine
Ala: Alanine MeVal: N-Methyl Valine Et: Ethyl Ph: Phenyl Fmoc:
9-Fluorenylmethoxycarbonyl- MeBmt: N-Methyl-butenyl-threoni- ne Ser
Serine Thr Threonine .alpha.Abu Nva
[0077] Synthetic Methods
[0078] The compounds and processes of the present invention will be
better understood in the following synthetic scheme which
illustrates the methods by which the compounds of the present
invention may be prepared. The groups X, Y, B and U in formula I
are as defined above. A is -MeBmt- in the starting material as
illustrated in the following reaction scheme: 6
[0079] The process for the invention for the preparation of the
compounds of formula I comprises reacting a compound of formula I
wherein A=-MeBmt- (cyclosporin A, commercially available
fermentation product) with an olefin having a terminal double bond
with Grubb's ruthenium alkylidene or benzylidene catalysts [see (a)
U.S. Pat. No. 6,111,121; (b) Reviews: Synlett, 1999, 2, 267; (c)
Reviews: Ivin, K J; Mol, J. C. Olefin Metathesis and Metathesis
Polymerization, 2.sup.nd ed, Academic Press, New York, 1997; (d) J.
Org. Chem., 1999, 64, 4798-4816; (e) Angew. Chem., Int. Ed.
English, 1997, 36, 2036-2056; (f) Tetrahedron 1998, 54, 4413-4450.]
or Nolan's ruthenium catalyst [see (a) International Patent
Application No. WO 00/15339; (b) Org. Lett., 2000, 2,1517-1519; (c)
J. Org. Chem., 2000, 65, 2204-2207] or Molybdenum catalysts [see
(a) J. Am. Chem. Soc., 1990, 112, 3875 (b), J. Am. Chem. Soc.,
1996, 118, 10926-10927] in the presence of a lithium salt in an
organic solvent such as dichloromethane, chloroform, toluene,
benzene, tetrahydrofuran, dimethylformamide, and the like at from
room temperature to about 100.degree. C. for 1-7 days to provide a
compound of formula I.
[0080] Pharmaceutical Compositions
[0081] The pharmaceutical compositions of the present invention
comprise a therapeutically effective amount of a compound of the
present invention formulated together with one or more
pharmaceutically acceptable carriers. As used herein, the term
"pharmaceutically acceptable carrier" means a non-toxic, inert
solid, semi-solid or liquid filler, diluent, encapsulating material
or formulation auxiliary of any type. Some examples of materials
which can serve as pharmaceutically acceptable carriers are sugars
such as lactose, glucose and sucrose; starches such as corn starch
and potato starch; cellulose and its derivatives such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients such as cocoa
butter and suppository waxes; oils such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such a propylene glycol; esters such as ethyl oleate
and ethyl laurate; agar; buffering agents such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol, phosphate buffer
solutions; non-toxic, compatible lubricants such as sodium lauryl
sulfate and magnesium stearate; as well as coloring agents,
releasing agents, coating agents, sweetening, flavoring and
perfuming agents. Preservatives and antioxidants can also be
present in the composition, according to the judgment of the
formulator. The pharmaceutical compositions of this invention can
be administered to humans and other animals orally, rectally,
parenterally, intracisternally, intravaginally, intraperitoneally,
topically (as by powders, ointments, or drops), buccally, or as an
oral or nasal spray.
[0082] Dosage forms for topical or transdermal administration of a
compound of this invention include ointments, pastes, creams,
lotions, gels, plasters, cataplasms, powders, solutions, sprays,
inhalants or patches. The active component is admixed under sterile
conditions with a pharmaceutically acceptable carrier and any
needed preservatives or buffers as may be required.
[0083] The ointments, pastes, creams and gels may contain, in
addition to an active compound of this invention, excipients such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0084] Powders and sprays can contain, in addition to the compounds
of this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants such as chlorofluorohydrocarbons.
[0085] For nasal administration, cyclosporins of the invention will
suitably be administered in liquid or powdered form from a nasal
applicator. Forms suitable for ophthalmic use will include lotions,
tinctures, gels, ointment and ophthalmic inserts, again as known in
the art. For rectal administration, i.e., for topical therapy of
the colon, cyclosporins of the invention may be administered in
suppository or enema form, in particular in solution, e.g., in
vegetable oil or like oily system for use as a retention enema.
[0086] It is clear that safety may be maximized by delivering the
drugs by the inhaled route either in nebuliser form or as dry
powder. Clearly the great advantage of the inhaled route, over the
systemic route, in the treatment of asthma and other diseases of
airflow obstruction and/or of chronic sinusititis, is that patients
are exposed to very small quantities of the drug and the compound
is delivered directly to the site of action.
[0087] Cyclosporins of the invention therefore are preferably
employed in any dosage form appropriate for topical administration
to the desired site. Thus, for the treatment of diseases of the
airways or lungs, cyclosporins of the invention may be administered
via the pulmonary route/by inhalation from an appropriate dispenser
device.
[0088] For this purpose, cyclosporins of the invention may be
employed in any suitable finely dispersed or finely dispersible
form, capable of administration into the airways or lungs, for
example in finely divided dry particulate form or in dispersion or
solution in any appropriate (i.e., pulmonarily administerable)
solid or liquid carrier medium. For administration in dry
particulate form, cyclosporins of the invention may, for example,
be employed as such, i.e., in micronised form without any additive
materials, in dilution with other appropriate finely divided inert
solid carrier or diluent (e.g., glucose, lactose, mannitol,
sorbitol, ribose, mannose or xylose), in coated particulate form or
in any other appropriate form as known in the art for the pulmonary
administration of finely divided solids.
[0089] Pulmonary administration may be effected using any
appropriate system as known in the art for delivering drug
substance in dry or liquid form by inhalation, e.g. an atomizer,
nebulizer, dry-powder inhaler or like device. Preferably a metered
delivery device, i.e., capable of delivering a pre-determined
amount of cyclosporin at each actuation, will be employed. Such
devices are known in the art.
[0090] Preparation of forms suitable for administration by
inhalation may be carried out by other methods known in the art. It
should be noted that several antibiotics have recently been
developed for topical inhaled usage, particularly in cystic
fibrosis, where they have been shown to be effective against
pseudomonas infections. Various inhalants are described, for
example, in DE 1491707, GB 1,392,945, GB 1,457,351, GB 1,457,352,
NL 147939, DE 1491715, GB 1,598,053, EP 5585, EP 41783, EP 45419,
EP 360463 and FR 2628638. DE 1491715, in particular, is said to be
suitable for inhalation therapy intended for bronchial or lung
diseases.
[0091] Dosages of cyclosporins of the invention employed in
practicing the method of the present invention will of course vary
depending on the site of treatment, the particular condition to be
treated, the severity of the condition, the subject to be treated
(e.g. in terms of body weight, age and so forth) as well as the
effect desired. In general, for treating diseases or conditions of
the airways or lungs, e.g., for use in treating inflammatory or
obstructive airway disease, for example asthma, cyclosporins of the
invention will suitably be administered topically to the airways or
lungs, e.g. by inhalation, at dosages of the order of from 20 to
400 mg/day, preferably from 50 or 100 to 300, e.g. from 200 to 300
mg/day. Dosages will appropriately be administered from a metered
delivery system in a series of from 1 to 5 puffs at each
administration, with administration performed once to four times
daily. Dosages at each administration will thus conveniently be on
the order of from about 5 to 100 mg, more suitably from 12.5 or 25
to 100 mg, administered with a metered delivery device capable of
delivering, e.g., 1 to 25 mg cyclosporin per actuation.
[0092] Dosage for the topical preparation will in general be one
tenth to one hundredth of the dose required for an oral
preparation.
EXAMPLES
[0093] The procedures described above for preparing the compounds
of the present invention will be better understood in connection
with the following examples, which are intended to be illustrative
only and not limiting of the scope of the invention. Various
changes and modifications of the disclosed embodiments will be
apparent to those skilled in the art. Such changes and
modifications, including without limitation, those relating to the
chemical structures, substituents, derivatives, intermediates,
syntheses, formulations and/or methods for the invention may be
made without departing from the spirit of the invention and the
scope of the appended claims.
Example 1
[0094] Compound of Formula (I): B is -.alpha.Abu-, U is -(D)Ala-, X
is absent, Y=COOCH3
[0095] Methyl acrylate (0.037 ml, 0.42 mmol), lithium bromide
(0.014 g, 0.218 mmol), and 1,3-bis(2,4,6-trimethylphenyl)
imidazol-2-ylinene(tricyc- lohexylphosphine)dichloro ruthenium(II)
bexylidene (Nolan catalyst, 0.0071 g, 0.008 mmol) were added to a
solution of cyclosporin A (0.1 g, 0.084 mmol) in methylene
chloride/tetrahydrofuran (10:1, 3 ml) at room temperature. The
reaction mixture was heated at 40.degree. C. After 24 hours, more
Nolan catalyst (0.0071 g, 0.008 mmol) and methyl acrylate (0.037
ml, 0.42 mmol) in 10:1 methylene chloride/tetrahydrofuran (3 ml)
were added and heated at 40.degree. C. for additional 24 hours.
After being cooled to room temperature, the reaction mixture was
filtered through a pre-packed solid phase extraction cartridge and
then eluted with 40:1 to 20:1, by volume, methylene
chloride/methanol. Removal of solvent in vacuo gave the title
compound as a brownish solid.
[0096] MS (ESI) m/z 1245.78 (M+H).sup.+.
Example 2
[0097] Compound of Formula (I): B is -.alpha.Abu-, U is -(D)Ala-, X
is absent, Y=COOH
[0098] The title compound of Example 2 was prepared from the title
compound of Example 1, reacted with sodium hydroxide in aqueous
methanol.
[0099] MS (ESI) m/z 1232.82 (M+H).sup.+.
Example 3
[0100] Compound of Formula (I): B is -.alpha.Abu-, U is -(D)Ala-, X
is absent, Y=COOEt
[0101] The title compound of Example 3 was prepared from
cyclosporin A, ethyl acrylate, Nolan catalyst and LiBr according to
the procedures described in Example 1.
[0102] MS (ESI) m/z 1245.78 (M+H).sup.+.
Example 4
[0103] Compound of Formula (I): B is -.alpha.Abu-, U is -(D)Ala-, X
is absent, Y=COOCH.sub.2CH.sub.2CH.sub.3
[0104] The title compound of Example 4 was prepared from
cyclosporin A, n-propyl acrylate, Nolan catalyst and LiBr according
to the procedures described in Example 1.
[0105] MS (ESI) m/z 1280.45 (M+H).sup.+.
Example 5
[0106] Compound of Formula (I): B is -.alpha.Abu-, U is -(D)Ala-, X
is absent, Y=COOCH.sub.2Ph
[0107] The title compound of Example 5 was prepared from
cyclosporin A, benzyl acrylate, Nolan catalyst and LiBr according
to the procedures described in Example 1
[0108] MS (ESI) m/z 1322.86 (M+H).sup.+.
Example 6
[0109] Compound of Formula (I): B is -.alpha.Abu-, U is -(D)Ala-, X
is absent, Y=COOCH.sub.2F
[0110] The title compound of Example 6 is prepared from cyclosporin
A, fluoromethyl acrylate, Nolan catalyst and LiBr according to the
procedures described in Example 1.
Example 7
[0111] Compound of Formula (I): B is -.alpha.Abu-, U is -(D)Ala-, X
absent, Y=COOCHF.sub.2
[0112] he title compound of Example 7 is prepared from cyclosporin
A, difluoromethyl acrylate, Nolan catalyst and LiBr according to
the procedures described in Example 1.
Example 8
[0113] Compound of Formula (I): B is -.alpha.Abu-, U is -(D)Ala-, X
is absent, Y=COOCF.sub.3
[0114] The title compound of Example 8 is prepared from cyclosporin
A, trifluoromethyl acrylate, Nolan catalyst and LiBr according to
the procedures described in Example 1.
Example 9
[0115] Compound of Formula (I), B is -.alpha.Abu-, U is -(D)Ala-, X
is absent, Y=COOCH CF.sub.3
[0116] he title compound of Example 9 is prepared from cyclosporin
A, trifluoroethyl acrylate, Nolan catalyst and LiBr according to
the procedures described in Example 1.
Example 10
[0117] Compound of Formula (I): B is -.alpha.Abu-, U is -(D)Ala-, X
is absent, Y=COOCH.sub.2Cl
[0118] The title compound of Example 10 is prepared from
cyclosporin A, chloromethyl acrylate, Nolan catalyst and LiBr
according to the procedures described in Example 1.
Example 11
[0119] Compound of Formula (I): B is -.alpha.Abu-, U is -(D)Ala-, X
is absent, Y=COOCH.sub.2OCH.sub.3
[0120] Method A. The title compound of Example 11 is prepared from
cyclosporin A, methoxymethyl acrylate, Nolan catalyst and LiBr
according to the procedures described in Example 1.
[0121] Method B. The title compound of Example 11 is prepared from
the title compound of Example 2, triethylamine and methyoxymethyl
chloride in DMF according to the procedures described in Protective
Groups in Organic Synthesis, 3 rd Ed, T. W. Greene and P. G. M.
Wuts ed., John Wiley & Sons, Inc, 1999.
Example 12
[0122] Compound of Formula (I): B is -.alpha.Abu-, U is -(D)Ala-, X
is absent, Y=COOCH.sub.2OCH.sub.2CH.sub.2OCH.sub.3
[0123] Method A. The title compound of Example 12 is prepared from
cyclosporin A, methoxyethoxymethyl acrylate, Nolan catalyst and
LiBr according to the procedures described in Example 1.
[0124] Method B. The title compound of Example 12 is prepared from
the potassium salt of the title compound of Example 2,
methoxyethoxymethyl chloride, Hunig's base in methylene chloride
according to the method described in Protective Groups in Organic
Synthesis, op. cit.
Example 13
[0125] Compound of Formula (I): B is -.alpha.Abu-, U is -(D)Ala-, X
is absent, Y=C(.dbd.O)SCH.sub.2Ph
[0126] The title compound of Example 13 is prepared from the title
compound of Example 2, benzyl mercaptan, carbodiimide and
dimethylaminopyridine in methylene chloride according to the method
described in Protective Groups in Organic Synthesis, op. cit.
Example 14
[0127] Compound of Formula (I): B is -.alpha.Abu-, U is -(D)Ala-, X
is --CH.sub.2CH.sub.2CH.sub.2--, Y=COOCH.sub.3
[0128] The title compound of Example 14 was prepared from
cyclosporin A, methyl 5-hexenoate, Nolan catalyst and LiBr
according to the procedures described in Example 1.
[0129] MS (ESI) m/z 1287.08 (M+H).sup.+.
Example 15
[0130] Compound of Formula (I): B is -.alpha.Abu-, U is -(D)Ala-, X
is absent, Y=COOFmoc
[0131] The title compound of Example 15 was prepared from
cyclosporin A, Fmoc acrylate, Nolan catalyst and LiBr according to
the procedures described in Example 1
[0132] MS (ESI) m/z 1410.89 (M+H).sup.+.
[0133] The cyclosporins of the present invention have potent
immunosuppressive anti-inflammatory activity. In particular they
inhibit antigen-induced inflammatory cell infiltration, for
example, into the airways. In vivo this activity is apparent
following topical administration, e.g., via the pulmonary route.
Some of the cyclosporins of the invention are, in contrast, found
to possess substantially reduced activity in vivo when administered
systemically, for example, following oral administration.
[0134] Anti-inflammatory properties of the cyclosporins of the
invention may be demonstrated in standard test models in vitro and
in vivo, e.g., as follows.
Example 16
[0135] Calcineurin Inhibition Assay
[0136] The immunosuppressive activity of cyclosporin is mediated
through inhibition of the phosphatase activity of the enzyme
calcineurin by a cyclophilin-cyclosporin complex. Thus, calcineurin
inhibition is widely used as an in vitro measure of the activity of
cyclosporin analogs.
[0137] Compounds were tested in an assay based on the Biomol Green
Calcineurin Assay Kit supplied by Biomol (Plymouth Meeting, Pa.),
supplemented with cyclophilin A for enzyme inhibition. The activity
of the recombinant human calcineurin was determined by release of
phosphate from a phosphopeptide representing a fragment of
camp-dependent protein kinase. Phosphate release was determined
using the colorimetric detection reagent Biomol Green.
[0138] Compounds in DMSO (2.4 .mu.l) were added to a 96-well
microplate and mixed with 50 .mu.l assay buffer (50 mM Tris, pH
7.5, 0.1 M sodium chloride, 6 mM magnesium chloride, 0.5 mM
dithiothreitol, 0.025% NP-40, 0.5 mM calcium chloride, 0.25 .mu.M
calmodulin) containing 5 .mu.M cyclophilin and 20 units of
calcineurin. After warming to 37.degree. C. for 15 min, the
enzymatic reaction was initiated by addition of phosphopeptide (7.5
.mu.l) to give a final concentration of 94 .mu.M. Phosphate release
after 60 min at 37.degree. C. was determined by addition of Biomol
Green (100 .mu.l) and measurement of the absorbance at 620 nm after
15 min at room temperature.
[0139] IC.sub.50 values were calculated from determinations of
enzyme activity at inhibitor concentrations ranging from 20 to
0.006 .mu.M.
Example 17
[0140] Immunosuppressive Activity and Applications
Murine Mixed Lymphocyte Reaction
[0141] Ca. 0.5.times.10.sup.6 lymphocytes from the spleen of female
(8-10 weeks) Balb/c mice are incubated for 5 days in 0.2 ml cell
growth medium with ca. 0.5.times.10.sup.6 lymphocytes from the
spleen of female (8-10 weeks) CBA mice. Test substance is added to
the medium at various concentrations. Activity is assessed by
ability to suppress proliferation-associated DNA synthesis as
determined by incorporation of radiolabelled thymidine.
Mishell-Dutton Test
[0142] Ca. 10.sup.7 lymphocytes from the spleen of OFI, female mice
are co-cultured with ca. 3.times.10.sup.7 sheep erythrocytes for 3
days. Test substance is added to the incubation medium in varying
concentrations. Lymphocytes are harvested and plated onto agar with
fresh sheep erythrocytes as antigen. Sensitized lymphocytes secrete
antibody that coats the erythrocytes, which lyse to form a plaque
in the presence of complement. Activity is assessed by reduction in
the number of plaque forming, i.e., antibody product, cells.
Influence on Allergen-Induced Pulmonary Eosinophilia (in vito)
[0143] Male Himalayan spotted guinea pigs (300 g, BRL) are
sensitized to ovalbumin (OA) by i.p. injection of 1 ml of a
suspension of OA (10 .mu.g/ml) with Al(OH).sub.3 (100 mg) and
B-pertussis vaccine (0.25 ml) in saline (0.9% w/v). For oral
studies the procedure is repeated 1.times. after 2 weeks and the
animals are used one week later. For inhalation studies the
procedure is repeated 2.times. at 3-week intervals and the animals
are used one week after the last injection.
[0144] Challenge is effected employing a saline solution of OA,
nebulized for discharge into an exposure chamber. Test animals are
exposed to OA by nose-only inhalation for 60 minutes. For
inhalation studies, OA solution is used at a concentration of
0.01%.
[0145] Test substance is administered (a) inhalation and/or (b)
orally. For oral studies, test substance is administered p.o. in
olive oil 1.times. daily for 3 days or in powder form in
methylcellulose once prior to OA challenge. On day 3, test animals
receive test substance 1.5 hrs. prior to and 6 hrs. after OA
challenge. For inhalation studies, test substance is micronised for
delivery to test animals restrained within a flow-past, nose-only
inhalation chamber. Administration by inhalation is effected 15
mins. prior to OA challenge.
[0146] Efficacy of administered test substance is determined by
bronchoalveolar lavage (BAL) and cell counting. For this purpose
animals are sacrificed with Na pento-barbitone (100 mg/kg i.p.) and
the trachea is exposed and cannulated. 5 successive 10 ml aliqots
of Ca.sup.2+ and Mg.sup.2+ free Hank's balanced salt solution
(HBSS), containing bovine serum albumin (BSA, 0.3%), EDTA (10 mM)
and HEPES (10 mM) is then introduced into the lung and immediately
aspirated by gentle compression of the lung tissue. Total cell
counts in pooled eluates are determined using an automatic cell
counter. Lavage fluid is centrifuged at 200 g for 10 minutes and
the cell pellet resuspended in 1 ml of supplemented HBSS. 10 .mu.l
of this cell suspension is added to 190 .mu.l of Turk's solution
(1:20) dilution). Differential cell counts are made from smears
stained by Diff-Quick. Cells are identified and counted under oil
immersion (.times.1,000). A minimum of 500 cells per smear are
counted and the total population of each cell type is
calculated.
[0147] Although the invention has been described with respect to
various preferred embodiments, it is not intended to be limited
thereto, but rather those skilled in the art will recognize that
variations and modifications may be made therein which are within
the spirit of the invention and the scope of the appended
claims.
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