U.S. patent application number 10/345855 was filed with the patent office on 2003-06-12 for cyclosporin analogs for the treatment of lung diseases.
Invention is credited to Lazarova, Tsvetelina Ivanova, Or, Yat Sun.
Application Number | 20030109425 10/345855 |
Document ID | / |
Family ID | 25523566 |
Filed Date | 2003-06-12 |
United States Patent
Application |
20030109425 |
Kind Code |
A1 |
Or, Yat Sun ; et
al. |
June 12, 2003 |
Cyclosporin analogs for the treatment of lung diseases
Abstract
the present invention relates to a cyclosporin analog of the
following formula (I) or a pro-drug or pharmaceutically acceptable
salt thereof: 1 In formula I, the formula for residue A is: 2 where
X is absent, --C1-C6 alkyl-, or --C3-C6 cycloalkyl-; Y is selected
from the groups: --C(O)--O--R1; --C(O)--S--R1;
--C(O)--OCH2-OC(O)R2; --C(S)--O--R1; and --C(S)--S--R1; where R1 is
hydrogen, C1-C6 alkyl optionally substituted with halogen,
heterocyclics, aryl, C1-C6 alkoxy or C1-C6 alkylthio or halogen
substituted C1-C6 alkoxy, halogen substituted C1-C6 alkylthio and
where R2 is C1-C6 alkyl optionally substituted with halogen, C1-C6
alkoxy, C1-C6 alkylthio heterocyclics or aryl; B is -.alpha.Abu-,
-Val-, -Thr- or -Nva-; and U is -(D)Ala-, -(D)Ser- or
-[O-(2-hydroxyethyl)(D)Ser]-, or -[O-acyl(D)Ser]- or
-[O-(2-acyloxyethyl)(D)Ser]-. In a second embodiment, the present
invention relates to the use of the cyclosporin analogs of the
present invention or a pro-drug or pharmaceutically acceptable salt
thereof in pharmaceutical compositions for the treatment of asthma
and other diseases characterized by airflow obstruction in a
subject. In a third embodiment, the present invention relates to
processes for the production of novel cyclosporin analogs of the
present invention. The present invention also contemplates
method(s) of treatment of asthma and other diseases characterized
by airflow obstruction in a subject by administering to the subject
therapeutically effective amounts of the cyclosporin analogs of the
present invention with or without the concurrent use of other drugs
or pharmaceutically acceptable carriers or excipients.
Inventors: |
Or, Yat Sun; (Cambridge,
MA) ; Lazarova, Tsvetelina Ivanova; (Brookline,
MA) |
Correspondence
Address: |
ENANTA PHARMACEUTICALS, INC.
ATTN: PATENT DEPT.
500 ARSENAL STREET
WATERTOWN
MA
02472
US
|
Family ID: |
25523566 |
Appl. No.: |
10/345855 |
Filed: |
January 16, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10345855 |
Jan 16, 2003 |
|
|
|
09975923 |
Oct 12, 2001 |
|
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Current U.S.
Class: |
514/1.7 ;
514/20.5; 530/317 |
Current CPC
Class: |
C07K 7/645 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
514/11 ;
530/317 |
International
Class: |
A61K 038/13; C07K
007/64 |
Claims
What is claimed is:
1. A cyclosporin analog of formula (I) or a pro-drug or a
pharmaceutically acceptable salt thereof: 10wherein, (a) A is of
the formula: 11wherein 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, heterocyclics, aryl, C1-C6 alkoxy or
C1-C6 alkylthio, halogen substituted C1-C6 alkoxy, halogen
substituted C1-C6 alkylthio; ii. --C(O)--S--R1 where R1 is
hydrogen, C1-C6 alkyl optionally substituted with halogen,
heterocyclics, aryl, C1-C6 alkoxy or C1-C6 alkylthio, halogen
substituted C1-C6 alkoxy, halogen substituted C1-C6 alkylthio; iii.
--C(O)--OCH2-OC(O)R2 where R2 is C1-C6 alkyl, optionally
substituted with halogen, C1-C6 alkoxy, C1-C6 alkylthio,
heterocyclics or aryl; iv. --C(S)--O--R1 where R1 is hydrogen,
C1-C6 alkyl optionally substituted with halogen, heterocyclics,
aryl, C1-C6 alkoxy or C1-C6 alkylthio, halogen substituted C1-C6
alkoxy, halogen substituted C1-C6 alkylthio; and v. C(S)--S--R1
where R1 is hydrogen, C1-C6 alkyl optionally substituted with
halogen, heterocyclics, aryl, C1-C6 alkoxy or C1-C6 alkylthio,
halogen substituted C1-C6 alkoxy, halogen substituted C1-C6
alkylthio. (b) B is -.alpha.Abu-, -Val-, -Thr- or -Nva-; and (c) U
is -(D)Ala-, -(D)Ser- or -[O-(2-hydroxyethyl)(D)Ser]-- ; or
-[O-acyl(D)Ser]- or -[O-(2-acyloxyethyl)(D)Ser]-.
2. A cyclosporin analog according to claim 1 or a pro-drug or a
pharmaceutically acceptable salt thereof, wherein in formula (I), B
is -.alpha.Abu-, and U is -(D)Ala-.
3. A cyclosporin analog according to claim 1 or a pro-drug or a
pharmaceutically acceptable salt thereof, wherein in formula I: (i)
A is of the formula A1 or A2, wherein: X is absent; and Y is
selected from a group consisting of: i. --C(O)--O--R1 where R1 is
hydrogen, C1-C6 alkyl optionally substituted with halogen,
heterocyclics, aryl, C1-C6 alkoxy or C1-C6 alkylthio, halogen
substituted C1-C6 alkoxy, halogen substituted C1-C6 alkylthio; ii.
--C(O)--S--R1 where R1 is hydrogen, C1-C6 alkyl optionally
substituted with halogen, heterocyclics, aryl, C1-C6 alkoxy or
C1-C6 alkylthio, halogen substituted C1-C6 alkoxy, halogen
substituted C1-C6 alkylthio; and 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, heterocyclics or aryl; (ii) B is
-.alpha.Abu-; and (iii) U is -(D)Ala-.
4. A cyclosporin analog according to claim 1 or a pro-drug or a
pharmaceutically acceptable salt thereof, selected from the group
consisting of: Compound of Formula (I) wherein B=-.alpha.Abu-,
U=-(D)Ala-, X is absent, Y=--COOCH.sub.3; 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=--COOCH.sub.2CH.sub.2CH.sub.3; Compound
of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-, X is absent,
Y=--COOCH.sub.2Ph; Compound of Formula (I) wherein B=-.alpha.Abu-,
U=-(D)Ala-, X is absent, Y=--COOCH.sub.2F; Compound of Formula (I)
wherein B=-.alpha.Abu-, U=-(D)Ala-, X is absent, Y=--COOCHF.sub.2;
Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-, X is
absent, Y=--COOCF.sub.3; Compound of Formula (I) wherein
B=-.alpha.Abu-, U=-(D)Ala-, X is absent, Y=--COOCH.sub.2CF.sub.3;
Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-, X is
absent, Y=--COOCH.sub.2Cl; Compound of Formula (I) wherein
B=-.alpha.Abu-, U=-(D)Ala-, X is absent, Y=--COOCH.sub.2OCH.sub.3-
; Compound of Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-, X is
absent, Y=--COOCH.sub.2OCH.sub.2CH.sub.2OCH.sub.3; Compound of
Formula (I) wherein B=-.alpha.Abu-, U=-(D)Ala-, X is absent,
Y=--C(.dbd.O)SCH.sub.2Ph- ; Compound of Formula (I) wherein
B=-.alpha.Abu-, U=-(D)Ala-, X is --CH.sub.2CH.sub.2CH.sub.2--,
Y=--COOCH.sub.3; and Compound of Formula (I) wherein
B=-.alpha.Abu-, U=-(D)Ala-, X is absent, Y=--COOFmoc.
5. A chemical process for preparing a cyclosporin analog of formula
I as claimed in claim 1, comprising: a. reacting a compound of
formula I, wherein A=-MeBmt- with: i. an olefin of formula
CH.sub.2.dbd.CH--X--Y, wherein X and Y are as defined in claim 1;
and ii. a catalyst; in the presence of a lithium salt in an organic
solvent; and b. hydrogenating the product of step a in an organic
solvent under hydrogen with a catalyst; and optionally converting
the product of said reaction into a pharmaceutically acceptable
salt.
6. The chemical process as claimed in claim 5, wherein the catalyst
in step (a) (ii) is Grubb's ruthenium alkylidene, Nolan's catalyst,
a benzylidene catalyst or a molybdenum catalyst.
7. The chemical process as claimed in claim 5, wherein step (b) is
performed at room temperature.
8. The chemical process as claimed in claim 7, wherein the catalyst
in step (b) is Palladium on carbon.
9. A pharmaceutical composition, said composition comprising at
least one cyclosporin analog of formula 1 as claimed in claim 1,
said cyclosporin analog being present alone or in combination with
a pharmaceutically acceptable carrier or excipient.
10. A method for treating diseases characterized by airflow
obstruction in a subject in need of treatment which comprises the
step of administering to said subject a therapeutically effective
amount of at least one cyclosporin analog of formula I as claimed
in claim 1.
11. The method of claim 10, wherein said disease is asthma.
12. The method of claim 10, wherein the step of administering the
cyclosporin analog of formula I is done by topical administration.
Description
TECHNICAL FIELD
[0001] The present invention relates to novel cyclosporin analogs
and methods for the treatment of asthma and other diseases
characterized by airflow obstruction in a subject. The present
invention further relates to pharmaceutical compositions comprising
the compounds of the present invention and processes for their
production.
BACKGROUND OF THE INVENTION
[0002] Respiratory diseases, such as asthma and other diseases
characterized by airflow obstruction, are a global problem.
Millions of people worldwide, both children and adults, suffer from
these medical conditions. These diseases reduce quality of life by
impairing the ability of sufferers to perform everyday tasks, and
in some cases, cause death. One of the major respiratory diseases
is asthma.
[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.
[0004] Asthma is an extremely common disease with a worldwide
prevalence of 5% to 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 type of airflow obstruction is often
reversible either spontaneously or with treatment and causes
associated increase in airway responsiveness to a variety of
stimuli.
[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 sometimes
resistant to treatment with high doses of oral corticosteroids.
Steroid resistance occurs in less than 5% of people with asthma.
This translates to thousands of people. These patients with severe
chronic disease may be 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 obstruction
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 cardio-respiratory 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 is 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 wheeze. Most cystic fibrosis sufferers take bronchodilators,
some take inhaled corticosteroids. And at least one study had
reported benefit with oral corticosteroids.
[0009] Current drugs for treating asthma are corticosteroids (such
as beclomethasone, triamcinolone), beta adrenergics (such as
epinephrine, albuterol, bitolterol), NSAIDS, leukotriene
antagonists, Xanthines (methyl xanthines such as theophylline,
oxtriphylline) and anticholinergics (such as atropine, ipratropium
bromide).
[0010] Corticosteroids are the mainstay of treatment of chronic
asthma and they revolutionized the treatment of this disease when
they were first introduced in the 1950's. Oral corticosteroids have
today been largely replaced by inhaled corticosteroids, although
severe asthmatics still require medication by mouth. Inhaled
cortisteroids are relatively safe and extremely effective in most
patients, and improved the quality of life for millions of
asthmatic 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.
[0011] 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
increasing the dose of oral corticosteroids runs the risk of
concomitant side effects.
[0012] Although corticosteroids are effective for asthma, 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
are azathioprine, methotrexate, mycophenolic acid and prodrug,
leflunomide, Cyclosporin A, ascomycin, FK-506 and rapamycin.
[0013] The cyclosporins comprise a class of structurally
distinctive, cyclic, poly-N-methylated undecapeptides, commonly
possessing pharmacological activity, in particular
immunosuppressive, anti-inflammatory or anti-parasitic activity.
The first of the cyclosporins to be isolated was the naturally
occurring fungal metabolite cyclosporin, Cyclosporin A represented
as follows: 3
[0014] Since the original discovery of cyclosporin, 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 comprising cyclosporins is
thus now substantial and includes, for example, the naturally
occurring Cyclosporins A through Z, for example, [Thr].sup.2,
[Val].sup.2, [Nva].sup.2 and [Nva].sup.2-, [Nva].sup.5 -Cyclosporin
(also known as Cyclosporins C, D, G and M respectively),
[(D)MeVal].sup.11-Cyclosporin (also known as Cyclosporin H), [cf.,
Traber et al.;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 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, for example,
[3-O-acetyl-MeBmt].sup.1-Cyclosporin (also known as
Dihydro-cyclosporin D), [(D)Ser].sup.8-Cyclosporin,
[Melle].sup.11-Cyclosporin, [MeAla].sup.6-Cyclosporin, [(D)
Pro].sup.3-Cyclosporin etc., 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).
[0015] 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 might be beneficial
in the control of asthma (see for example, "Pharmacokinetics,
pharmacodynamics, and safety of inhaled cyclosporin A after single
and repeated administration in ,healthy male and female subjects
and asthmatic patients," Rohatagi, S. et al., Aventis
Pharmaceutical, Collegeville, Pa., USA. J. Clin. Pharmacol. (2000),
40(11), 1211-1226). Cyclosporin A (hereinafter "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.
[0016] The mechanism of CsA action in asthma is of interest. CsA
binds to the ubiquitous protein cyclophilin, in the cytosol, and
the complex in turn binds to calcineurin, which is a calcium and
calmodulin dependent serine threonine phosphatase. Calcineurin is
necessary for the cytoplasmic portion of the transcription factor
NF-AT, a nuclear factor of activated T-cells, to translocate to the
nucleus and bind to its nuclear portion to become an active
transcription factor. NF-AT forms a complex with AP-1 and regulates
the transcription of the IL-2 gene, together with other genes, for
example, IL-5. CsA prevents the cytoplasmic portion of NF-AT from
translocating, resulting 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.
[0017] 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.]
[0018] CsA 5 mg/kg/day allowed a significant reduction in the use
of corticosteroids by 60%. 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. Therefore, it is
desirable to synthesize analogs 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
[0019] The present invention relates to novel cyclosporin analogs
and methods of treatment for the treatment of asthma and other
diseases characterized by airflow obstruction in a subject. The
present invention further relates to pharmaceutical compositions
comprising the compounds of the present invention and processes for
their production.
[0020] More particularly, the present invention relates to a
cyclosporin analog of the following formula (I) or a pro-drug or
pharmaceutically acceptable salt thereof: 4
[0021] In formula I, the formula for residue A is: 5
[0022] where X is absent, --C1-C6 alkyl-, or --C3-C6 cycloalkyl-; Y
is selected from the groups: --C(O)--O--R1; --C(O)--S--R1;
--C(O)--OCH2-OC(O)R2; --C(S)--O--R1; and --C(S)--S--R1; where R1 is
hydrogen, C1-C6 alkyl optionally substituted with halogen,
heterocyclics, aryl, C1-C6 alkoxy or C1-C6 alkylthio or halogen
substituted C1-C6 alkoxy, halogen substituted C1-C6 alkylthio and
where R2 is C1-C6 alkyl optionally substituted with halogen, C1-C6
alkoxy, C1-C6 alkylthio heterocyclics or aryl; B is -.alpha.Abu-,
-Val-, -Thr- or -Nva-; and U is -(D)Ala-, -(D)Ser- or
-[O-(2-hydroxyethyl)(D)Ser]-, or -[O-acyl(D)Ser]- or
-[O-(2-acyloxyethyl)(D)Ser]-.
[0023] In a second embodiment, the present invention relates to the
use of the cyclosporin analogs of the present invention or a
pro-drug or a pharmaceutically acceptable salt thereof in
pharmaceutical compositions for the treatment of asthma and other
diseases characterized by airflow obstruction in a subject.
[0024] In a third embodiment, the present invention relates to
processes for the production of novel cyclosporin analogs of the
present invention. In a preferred embodiment, the present invention
relates to the processes for the production of cyclosporin analogs
of formula I, with the structure of residue A as illustrated
above.
[0025] The present invention also contemplates method(s) of
treatment of asthma and other diseases characterized by airflow
obstruction in a subject by administering to the subject
therapeutically effective amounts of the cyclosporin analogs of the
present invention with or without the concurrent use of other drugs
or pharmaceutically acceptable carriers or excipients.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention relates to novel cyclosporin analogs
and methods of treatment for the treatment of asthma and other
diseases characterized by airflow obstruction in a subject. The
present invention further relates to pharmaceutical compositions
comprising the compounds of the present invention and processes for
their production. The patents and publications identified in this
specification indicate the knowledge in this field and are hereby
incorporated by reference in their entirety. In the case of
inconsistencies, the present disclosure will prevail.
[0027] More particularly, the present invention relates to a
cyclosporin analog of the following formula (I) or a pro-drug or
pharmaceutically acceptable salt thereof: 6
[0028] In formula I, the formula for residue A is: 7
[0029] where X is absent, --C1-C6 alkyl-, or --C3-C6 cycloalkyl-; Y
is selected from the groups: --C(O)--O--R1 where R1 is hydrogen,
C1-C6 alkyl optionally substituted with halogen, heterocyclics,
aryl, C1-C6 alkoxy or C1-C6 alkylthio, halogen substituted C1-C6
alkoxy or halogen substituted C1-C6 alkylthio; --C(O)--S--R1 where
R1 is hydrogen, C1-C6 alkyl optionally substituted with halogen,
heterocyclics, aryl, C1-C6 alkoxy or C1-C6 alkylthio, halogen
substituted C1-C6 alkoxy or halogen substituted C1-C6 alkylthio;
--C(O)--OCH2-OC(O)R2 where R2 is C1-C6 alkyl optionally substituted
with halogen, C1-C6 alkoxy, C1-C6 alkylthio heterocyclics or aryl;
--C(S)--O--R1 where R1 is hydrogen, C1-C6 alkyl optionally
substituted with halogen, heterocyclics, aryl, C1-C6 alkoxy or
C1-C6 alkylthio, halogen substituted C1-C6 alkoxy or halogen
substituted C1-C6 alkylthio; and --C(S)--S--R1 where R1 is
hydrogen, C1-C6 alkyl optionally substituted with halogen,
heterocyclics, aryl, C1-C6 alkoxy or C1 -C6 alkylthio, halogen
substituted C1-C6 alkoxy, halogen substituted C1-C6 alkylthio; B is
-.alpha.Abu-, -Val-, -Thr- or -Nva-; and U is -(D)Ala-, -(D)Ser- or
-[O-(2-hydroxyethyl)(D)Ser]-, or -[O-acyl(D)Ser]- or
-[O-(2-acyloxyethyl)(D)Ser]-.
[0030] In formula I, abbreviation of amino acid residues, for
example, -Ala-, -MeLeu-, -.alpha.Abu-, etc., are in accordance with
conventional practice and are to be understood as having the
L-configuration unless otherwise indicated (for example, -(D)Ala-
represents a residue having the D-configuration). Abbreviation of
residues preceded by "Me-" represents a .alpha.-N-methylated amino
acid residue, for example, "Me-Leu" is a
.alpha.-N-methylated-Leucine residue. Individual residues of a
molecule of the cyclosporin analog of the present invention 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 specification and claims.
[0031] In a most preferred embodiment, a cyclosporin analog of the
present invention is represented by formula I or a pro-drug or
pharmaceutically acceptable salt thereof, where residue B is
-.alpha.Abu- and residue U is -(D)Ala-. In another preferred
embodiment, the cyclosporin analog of the present invention is
represented by formula I or a pro-drug or pharmaceutically
acceptable salt thereof, where X is absent in residue A, residue B
is -.alpha.Abu- and residue U is -(D)Ala-.
[0032] Representative compounds of the invention include, but are
not limited to, the following compounds as illustrated below:
[0033] Compound of formula I, where in residue A, X is absent and
Y=--COOCH.sub.3; residue B=-.alpha.Abu-, and residue
U=-(D)Ala-.
[0034] Compound of formula I, where in residue A, X is absent and
Y=--COOH; residue B=-.alpha.Abu-, and residue U=-(D)Ala-.
[0035] Compound of formula I, where in residue A, X is absent and
Y=-COOEt; residue B=-.alpha.Abu-, and residue U=-(D)Ala-.
[0036] Compound of formula I, where in residue A, X is absent and
Y=--COOCH.sub.2CH.sub.2CH.sub.3; residue B=-.alpha.Abu-, and
residue U=-(D)Ala-.
[0037] Compound of formula I, where in residue A, X is absent and
Y=--COOCH.sub.2Ph; residue B=-.alpha.Abu-, and residue
U=-(D)Ala-.
[0038] Compound of formula I, where in residue A, X is absent and
Y=--COOCH.sub.2F; residue B=-.alpha.Abu-, and residue
U=-(D)Ala-.
[0039] Compound of formula I, where in residue A, X is absent and
Y=--COOCHF.sub.2; residue B=-.alpha.Abu-, and residue
U=-(D)Ala-.
[0040] Compound of formula I, where in residue A, X is absent and
Y=--COOCF.sub.3; residue B=-.alpha.Abu-, and residue
U=-(D)Ala-.
[0041] Compound of formula I, where in residue A, X is absent and
Y=--COOCH.sub.2CF.sub.3; residue B=-.alpha.Abu-, and residue
U=-(D)Ala-.
[0042] Compound of formula I, where in residue A, X is absent and
Y=--COOCH.sub.2Cl; residue B=-.alpha.Abu-, and residue
U=-(D)Ala-.
[0043] Compound of formula I, where in residue A, X is absent and
Y=--COOCH.sub.2OCH.sub.3; residue B=-.alpha.Abu-, and residue
U=-(D)Ala-.
[0044] Compound of formula I, where in residue A, X is absent and
Y=--COOCH.sub.2OCH.sub.2CH.sub.2OCH.sub.3; residue B=-.alpha.Abu-,
and residue U=-(D)Ala-.
[0045] Compound of formula I, where in residue A, X is absent and
Y=--C(.dbd.O)SCH.sub.2Ph; residue B=-.alpha.Abu-, and residue
U=-(D)Ala-.
[0046] Compound of formula I, where in residue A, X is
--CH.sub.2CH.sub.2CH.sub.2-- and Y=--COOCH.sub.3; residue
B=-.alpha.Abu-, and residue U=-(D)Ala-.
[0047] Compound of formula I, where in residue A, X is absent and
Y=--COOFmoc; residue B=-.alpha.Abu-, and residue U=-(D)Ala-.
[0048] Cyclosporin analogs of the invention are accordingly useful
for the treatment of diseases or conditions responsive to or
requiring topical anti-inflammatory, immunosuppressive or related
therapy, for example, 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,
cyclosporin analogs of the invention permit topical
anti-inflammatory, immunosuppressive or related therapy with the
concomitant avoidance or reduction of undesirable systemic side
effects, for example general systemic immunosuppression.
[0049] Cyclosporin analogs of the invention useful for the
treatment of diseases and conditions of the airways or lung, in
particular, inflammatory or obstructive airway diseases. They are
especially useful for the treatment of diseases or conditions of
the airways or lungs associated with or characterized by
inflammatory cell infiltration or other inflammatory events
accompanied by inflammatory cell accumulation, for e.g., eosinophil
and/or neutrophil. Most preferably, they are useful for the
treatment of asthma.
[0050] Cyclosporin analogs 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, for example, 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. Cyclosporin
analogs of the invention are in particular useful for the treatment
of asthma in subjects whose asthmatic status is either steroid
dependent or steroid resistant.
[0051] Cyclosporin analogs of the invention are also useful for the
treatment of bronchitis or for the treatment of chronic or acute
airways obstruction associated therewith. Cyclosporin analogs 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.
[0052] Cyclosporin analogs 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.
[0053] Cyclosporin analogs 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 effects 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.
[0054] The word "treatment" as used herein 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 for immediate treatment, for e.g., of
acute inflammation (symptomatic treatment) as well as for 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, for e.g., in the case of
asthma, symptomatic treatment to ameliorate acute inflammatory
events and prophylactic treatment to restrict on-going inflammatory
status and to ameliorate future bronchial exacerbation associated
therewith.
[0055] Cyclosporin analogs of the invention may also be used to
treat any disease or condition of the airways or lungs requiring
immunosuppressive therapy, for e.g., the treatment of autoimmune
diseases, 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,
for e.g., following lung or heart lung transplantation.
[0056] As previously indicated, for the above purposes, cyclosporin
analogs of the invention will be administered topically within the
airways, for e.g., by the pulmonary route or by inhalation. As also
previously noted, while having potent efficacy when administered
topically; cyclosporin analogs of the invention exhibit reduced
systemic toxicity. Cyclosporin analogs of the invention thus
provide a means for the treatment of diseases and conditions of the
airways or lung, for example, as hereinabove set forth, 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 manoeuvres required to
effect administration by inhalation, up to 90% or more of total
drug substance administered will normally be swallowed rather than
inhaled.
[0057] By the provision of cyclosporin analogs 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 effect.
[0058] Further uses include the treatment and prophylaxis of
inflammatory and hyperproliferative skin diseases and cutaneous
manifestations of immunologically-mediated illnesses, such as
psoriasis, atopical dermatitis, contact dermatitis and further
eczematous dermatitises, seborrhoeis dermatitis, Lichen planus,
Pemphigus, bullous pemphigoid, Epidermolysis bullosa, urticaria,
angioedemas, vasculitides, erythemas, cutaneous eosinophilias,
Lupus erythematosus, acne and Alopecia areata; various eye diseases
(autoimmune and otherwise) such as keratoconjunctivitis, vernal
conjunctivitis, keratitis, herpetic keratitis, conical cornea,
dystrophia epithelialis corneae, corneal leukoma, ocular pemphigus,
Mooren's ulcer, Scleritis, Graves' opthalmopathy,
Vogt-Koyanagi-Harada syndrome, sarcoidosis, multiple myeloma, etc.;
obstructive airway diseases, which includes conditions such as COPD
asthma (for example, bronchial asthma, allergic asthma, intrinsic
asthma, extrinsic asthma and dust asthma), particularly chronic or
inveterate asthma (for example, late asthma and airway
hyper-responsiveness), bronchitis, allergic rhinitis and the like;
inflammation of mucosa and blood vessels such as gastric ulcers,
vascular damage caused by ischemic diseases and thrombosis.
Moreover, hyperproliferative vascular diseases such as intimal
smooth muscle cell hyperplasia, restenosis and vascular occlusion,
particularly following biologically- or mechanically-mediated
vascular injury can be treated or prevented by the compounds of the
invention.
[0059] The compounds of the present invention may also find utility
in the chemosensitization of drug resistant target cells.
Cyclosporin A and FK-506 are known to be effective modulators of
P-glycoprotein, a substance which binds to and inhibits the action
of anticancer drugs; by inhibiting P-glycoprotein, they are capable
of increasing the sensitivity of multidrug resistant (MDR) cells to
chemotherapeutic agents. It is believed that the compounds of the
invention may likewise be effective at overcoming resistance
expressed to clinically useful antitumour drugs such as
5-fluorouracil, cisplatin, methotrexate, vincristine, vinblastine
and adriamycin, colchicine and vincristine.
[0060] Accordingly, the pharmaceutical compositions of the present
invention comprise a therapeutically effective amount of a
cyclosporin analog of the invention in combination with a
pharmaceutically acceptable carrier or excipient. In particular,
compositions pertaining to the present invention are useful for
treating a subject for a reversible obstructive airway disease.
[0061] The present invention also contemplates method(s) of
treatment of asthma and other diseases characterized by airflow
obstruction in a subject by administering to the subject
therapeutically effective amounts of the cyclosporin analogs of the
present invention with or without the concurrent use of other drugs
or pharmaceutically acceptable carriers or excipients, as described
throughout the present specification. Such treatment of the disease
may be done by administering a therapeutically effective amount of
a compound of the invention for such time and in such amounts as is
necessary to produce the desired result.
[0062] As used in the present invention, "therapeutically effective
amount" of one of the compounds means a sufficient amount of the
compound to treat a particular disease, at a reasonable
benefit/risk ratio. The compounds of the present invention may be
employed in pure form or, where such forms exist, in
pharmaceutically acceptable salt, ester or prodrug forms.
Alternatively, the compound may be administered as pharmaceutical
compositions containing the compound of interest in combination
with one or more drugs or pharmaceutically acceptable excipients.
It will be understood, however, that the total daily usage of the
compounds and compositions of the present invention will be decided
by the attending physician within the scope of sound medical
judgment.
[0063] The specific therapeutically-effective dose level for any
particular patient will depend upon a variety of factors including
the disorder being treated and the severity of the disorder;
activity of the specific compound employed; the specific
composition employed; the age, body weight, general health, sex and
diet of the patient; the time of administration, route of
administration, and rate of excretion of the specific compound
employed; the duration of the treatment; drugs used in combination
or coincidental with the specific compound employed; and like
factors well known in the medical arts. For example, it is well
within the skill of the art to start doses of the compound at
levels lower than required to achieve the desired therapeutic
effect and to gradually increase the dosage until the desired
effect is achieved.
[0064] Dosages of the cyclosporin analogs of the present 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 (for 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, for e.g., inflammatory or
obstructive airway disease such as asthma, cyclosporins of the
invention can be suitably administered topically to the airways or
lungs, for e.g., but not limited to, inhalation, at dosages from
about 20 to about 400 mg/day, preferably from about 50 to about 300
mg/day, most preferably from about 200 to about 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 from about 5 to 100
mg/day, more preferably from about 12.5 to about 100 mg/day, e.g.
administered with a metered delivery device capable of delivering,
for e.g., 1 to 25 mg cyclosporin per actuation. For purposes of
oral administration, more preferable doses may be in the range from
about 0.005 to about 3 mg/kg/day. If desired, the effective daily
dose may be divided into multiple doses for purposes of
administration; consequently, single dose compositions may contain
such amounts or submultiples thereof to make up the daily dose.
[0065] Definitions
[0066] The terms "C.sub.1-C.sub.3-alkyl" or "C.sub.1-C.sub.6-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 C.sub.1-C.sub.3 alkyl
radicals include methyl, ethyl, propyl and isopropyl, and examples
of C.sub.1-C.sub.6-alkyl radicals include, but are not limited to,
methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl
and n-hexyl.
[0067] The term "C.sub.1-C.sub.6alkoxy" as used herein refers to an
C.sub.1-C.sub.6-alkyl group, as previously defined, attached to the
parent molecular moiety through an oxygen atom. Examples of
C.sub.1-C.sub.6-alkoxy include, but are not limited to, methoxy,
ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy and
n-hexoxy.
[0068] The term "C.sub.1-C.sub.6-alkylthio" as used herein refers
to an C.sub.1-C.sub.6-alkyl group, as previously defined, attached
to the parent molecular moiety through a sulfur atom. Examples of
C.sub.1-C.sub.6-alkylthio include, but are not limited to,
thiomethoxy, thioethoxy, thiopropoxy, thio-isopropoxy,
n-thiobutoxy, tert-thiobutoxy, neothiopentoxy and
n-thio-hexoxy.
[0069] The term "aryl" as used herein refers to a carbocyclic ring
system having one or more aromatic rings including, but not limited
to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl and the
like. Aryl groups (including multi-cyclic aryl groups) can be
unsubstituted or substituted with one, two or three substituents
independently selected from lower alkyl, substituted loweralkyl,
haloalkyl, alkoxy, thioalkoxy, lower alkylenedioxy, lower
alkylidenedioxy, amino, alkylamino, dialkylamino, acyamino, cyano,
hydroxy, acyl, halo and/or trifluoromethyl, mercapto, nitro,
carboxylaldehyde, carboxy, alkoxycarbonyl, carbamoyl, sulfamoyl,
lower alkoxycarbonylamino, lower alkanoyl, ureido, amidino and
carboxamide. In addition, substituted aryl groups include
tetrafluorophenyl and pentafluorophenyl.
[0070] The term "C.sub.3-C.sub.6-cycloalkyl-" as used herein refers
to carbocyclic groups of 3 to 6 carbons, respectively; for example,
cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
[0071] The terms "halo" and "halogen" as used herein refer to an
atom selected from fluorine, chlorine, bromine and iodine.
[0072] The term "heterocyclics", as used herein, refers to a cyclic
aromatic radical having from five to ten ring atoms of which 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.
[0073] The term "subject" as used herein refers to a mammal or
animal. Preferably the mammal is a human. A subject refers to, for
example, dogs, cats, horses, cows, pigs, guinea pigs and the
like.
[0074] The term "pro-drug" as used herein refers to
pharmacologically acceptable derivatives, for example, but not
limited to, esters and amides, such that the resulting
biotransformation product of the derivative is the active drug.
Pro-drugs are known in the art and are described generally in,
e.g., Goodman and Gilman's "Biotransformation of Drugs," in the
Pharmacological Basis of Therapeutics, 8.sup.th Ed., McGraw Hill,
Int. Ed. 1992, page 13-15, which is hereby incorporated by
reference in its entirety.
[0075] As used herein, the term "pharmaceutically acceptable salt"
refers to those salts which are, within the scope of sound medical
judgment, 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), 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 acceptable 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,
ptoluenesulfonate, 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.
[0076] Pharmaceutical Compositions
[0077] 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, and phosphate
buffer solutions, as well as other 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 judgement 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), bucally, or as an
oral or nasal spray.
[0078] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. In addition to the active
compounds, the liquid dosage forms may contain inert diluents
commonly used in the art such as, for example, water or other
solvents, solubilizing agents and emulsifiers such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethylformamide, oils (in particular, cottonseed, groundnut,
corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid
esters of sorbitan, and mixtures thereof. Besides inert diluents,
the oral compositions can also include adjuvants such as wetting
agents, emulsifying and suspending agents, sweetening, flavoring,
and perfuming agents.
[0079] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active compound is mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
b) binders such as, for example, carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants
such as glycerol, d) disintegrating agents such as agar-agar,
calcium carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate, e) solution retarding agents such
as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, cetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof. In the case of capsules, tablets and
pills, the dosage form may also comprise buffering agents.
[0080] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like.
[0081] The solid dosage forms of tablets, dragees, capsules, pills,
and granules can be prepared with coatings and shells such as
enteric coatings and other coatings well known in the
pharmaceutical formulating art. They may optionally contain
opacifying agents and can also be of a composition that they
release the active ingredient(s) only, or preferentially, in a
certain part of the intestinal tract, optionally, in a delayed
manner. Examples of embedding compositions which can be used
include polymeric substances and waxes.
[0082] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polethylene glycols and the like.
[0083] The active compounds can also be in micro-encapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms the
active compound may be admixed with at least one inert diluent such
as sucrose, lactose or starch. Such dosage forms may also comprise,
as is normal practice, additional substances other than inert
diluents, e.g., tableting lubricants and other tableting aids such
a magnesium stearate and microcrystalline cellulose. In the case of
capsules, tablets and pills, the dosage forms may also comprise
buffering agents. They may optionally contain opacifying agents and
can also be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain part of the
intestinal tract, optionally, in a delayed manner. Examples of
embedding compositions which can be used include polymeric
substances and waxes.
[0084] Dosage forms for topical or transdermal administration of a
compound of this invention include ointments, pastes, creams,
lotions, gels, 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.
[0085] Pharmaceutically acceptable diluents or carriers may be
diluents or carriers acceptable for topical application at the
intended site of therapy, e.g. diluents or carriers acceptable for
topical administration pulmonary, dermally, nasally, ocularly or
rectaly.
[0086] 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.
[0087] 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.
[0088] Transdermal patches have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms
can be made by dissolving or dispensing the compound in the proper
medium. Absorption enhancers can also be used to increase the flux
of the compound across the skin. The rate can be controlled by
either providing a rate controlling membrane or by dispersing the
compound in a polymer matrix or gel.
[0089] Forms in topically administrable form, e.g. enabling or
facilitating topical administration, include, e.g. dry powder
preparations of the active ingredient (i.e. cyclosporin analog of
the invention) in substantially pure form, for example as employed
in the art for delivery from a dry powder inhalation device. Means
or devices enabling or facilitating topical administration include,
in particular, inhalation devices as well as containers and the
like from which the active ingredient may be delivered in a form
capable of topical application. Preferred embodiments as defined
under C will be such as permit topical administration within the
airways or lungs, e.g. by inhalation.
[0090] 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.
[0091] Preparation of forms suitable for administration by
inhalation may be carried out by methods known in the art. It
should be noted that several antibiotics have recently 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.
[0092] For this purpose cyclosporin analogs 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, cyclosporin analogs 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 know in the art for the pulmonary
administration of finely divided solids.
[0093] 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 analog at each actuation, will be employed. Such
devices are known in the art.
[0094] For nasal administration, cyclosporin analogs of the
invention will suitably be administered in liquid form from a nasal
applicator. Suitable topical forms for the treatment of diseases or
conditions of the skin will include, for example, creams, gles,
ointments, pastes, cataplasms, plasters, transdermal patches and
the like. Formulations for dermal application will appropriately
contain a skin penetration enhancer, e.g. as know in the art, for
example azone. 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, cyclosporin analogs 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.
[0095] According to the present invention, cyclosporin analogs may
be used for the manufacture of a topical preparation for the
treatment, with or without the concurrent use of other drugs. For
the above purposes, cyclosporin analogs of the invention may be
employed in any dosage form appropriate for topical administration
to the desired site. For example, for the treatment of diseases of
the airways or lungs, cyclosporin analogs of the invention may be
administered via the pulmonary route, by inhalation from an
appropriate dispenser device.
[0096] Dosage for the topical preparation will in general be one
tenth to one hundredth, of the dose required for oral
preparation.
1 Abbreviations Sar: Sarcosine MeLeu: N-Methyl-Leucine Val: Valine
Ala: Alanine MeVal: N-Methyl Valine Et: Ethyl Ph: Phenyl Fmoc:
9-Fluorenylmethoxycarbonyl- MeBmt: N-Methyl-(4R)-4-[(E)-2-b-
utenyl]-4-methyl-L-threonine .alpha.-Abu: .alpha.-Aminobutyric
acid
[0097] Synthetic Methods
[0098] The compounds and processes of the present invention will be
better understood, but are not limited to, the following synthetic
scheme which illustrates the methods by which the compounds of the
present invention (illustrated by formula I) may be prepared. The
groups X and Y, and the amino acid residues B and U in formula I
are as defined earlier in the specification. The starting material
for Scheme I, illustrated by formula I where A'=-MeBmt-, may be,
for example, but not limited to, a fermentation product or a
synthetic product made by solution phase chemistry. Preferably, the
starting material is commercially available. The starting material
as a fermentation product may be made from highly productive
strains, for example, but not limited to, Sesquicillopsis
rosariensis G. ARNOLD F605; Tolypocladium inflatum wb6-5; Fusant,
Tolypocladium inflatum KD461 etc. (in U.S. Pat. Nos. 5,256,547;
5,856,141 etc.). Alternately, the starting material may be made by
solution phase chemistry either by sequentially assembling amino
acids or by linking suitable small peptide fragments, where the
units are linked by, for example, but not limited to, amide, ester
or hydroxylamine linkages (described in, Muller, Methoden der
organischen, Chemie Vol. XV/2, pp 1 to 364, Thieme Verlag,
Stuttgart, 1974; Stewart, Young, Solid Phase Peptide Synthesis, pp
31 to 34, 71 to 82, Pierce Chemical Company, Rockford, 1984;
Bodanszky, Klausner, Ondetti, Peptide Synthesis, pp 85 to 128, John
Wiley & Sons, New York, 1976 and other standard books on
solution phase peptide chemistry). For amide linkages particular
preference is given to the azide method, the symmetric and mixed
anhydride method, in situ generated or preformed active esters and
methods using coupling reagents (e.g., dicyclohexylcarbodiimide,
N,N-dimethyl-4-aminopyridine, N-hydroxy-benzotriazole, PyBrop.RTM.
etc.). Classical solution phase chemistry using standard Z- and
Boc- methodology may be used.
[0099] Residue A, which is -MeBmt- in the starting material is
further modified, as illustrated in the following reaction scheme.
8
[0100] The process for the preparation of the compounds of formula
I comprises reacting a compound of formula I, where A'=-MeBmt- (for
example, Cyclosporin A) with an olefin having a terminal double
bond with catalysts such as Grubb's ruthenium alkylidene, Grubbs
dihydroimidazole ruthenium, Shrock-Hoveyda molybdenum catalysts 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 such as lithium bromide, lithium chloride, lithium
trifluoroacetate, lithium triflate of a lewis acid such as titanium
isopropoxide in an organic solvent. The organic solvent used may be
solvents such as, for example, dichloromethane, chloroform,
toluene, benzene, tetrahydrofuran, dimethylformamide and the like
or mixtures thereof. The reaction may be carried out from room
temperature to about 100.degree. C. for 1-7 days to provide a
compound of formula I, where residue A' is converted to residue A"
having formula (i). 9
[0101] The compounds of formula I in an organic solvent, where
residue A" has formula (i), are then subjected to standard
hydrogenation conditions using a catalyst such as, but are not
limited to, a catalytic amount of palladium on carbon in a hydrogen
atmosphere to provide the saturated compounds of formula I, where
in particular, residue A" having formula (i) is converted to
residue A, as described throughout the specification.
[0102] The organic solvents used can be solvents such as methanol,
ethanol, ethyl acetate or mixtures thereof. Other catalysts useful
to assist hydrogenation may be, for example, but not limited to,
platinum metal or its oxide [see standard books on catalytic
hydrogenation, e.g., Rylander, P. N., Hydrogenation Methods,
Academic Press: NY, 1985; Catalytic Hydrogenation in Organic
Synthesis, Academic Press: NY, 1985; {haeck over (C)}erven, L.,
Catalytic Hydrogenation, Elsevier: NY, 1986 etc.]. The reaction may
be carried out at room temperature or elevated temperature, for
example, but not limited to, 50.degree. C. or 100.degree. C.
EXAMPLES
[0103] The procedures described above for preparing the compounds
of the present invention will be better understood in connection
with the following examples, which are intentended 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
Compound of Formula I, Where in Residue A, X is Absent and
Y=--COOCH.sub.3, Residue, B=-.alpha.Abu-, and Residue,
U=-(D)Ala-
[0104] Cyclosporin methyl ester (0.030 mg, 0.024 mmol) and
palladium on carbon (0.0012 mg, 0.0012 mmol) were added to a flask
and the flask was evacuated and backfilled with hydrogen gas three
times. Anhydrous methanol (3 ml) was added and the reaction was
stirred for 18 h at ambient temperature under an atmosphere of
hydrogen. After filtration and concentration in vacuo, the product
was isolated as a white solid (0.021 mg, 70 % yield). Electrospray
mass spectrum (ESMS) M+H: 1248.91
Example 2
Compound of Formula I, Where in Residue A, X is Absent and
Y=--COOEt; Residue B=-.alpha.Abu-, and Residue U=-(D)Ala-
[0105] The title compound of example 2 was prepared from
cyclosporin ethyl ester and palladium on carbon according to the
procedures described in Example 1. ESMS M+H: 1262.3
Example 3
Compound of Formula I, Where in Residue A, X is Absent and
Y=--COOCH.sub.2CH.sub.2CH.sub.3; Residue B=-.alpha.Abu-, and
Residue U=-(D)Ala-
[0106] The title compound of example 3 was prepared from
cyclosporin propyl ester and palladium on carbon according to the
procedures described in Example 1.
Example 4
Compound of Formula I, Where in Residue A, X is Absent and
Y=--COOCH.sub.2Ph; Residue B=-.alpha.Abu-, and Residue
U=-(D)Ala-
[0107] The title compound of example 4 was prepared from
cyclosporin benzyl ester and palladium on carbon according to the
procedures described in Example 1.
Example 5
Compound of Formula I, Where in Residue A, X is absent and
Y=--COOCH.sub.2F; Residue B=-.alpha.Abu-, and Residue
U=-(D)Ala-
[0108] The title compound of example 5 was prepared from
cyclosporin fluoromethyl ester ester and palladium on carbon
according to the procedures described in Example 1
Example 6
Compound of Formula I, Where in Residue A, X is Absent and
Y=--COOCHF.sub.2; Residue B=-.alpha.Abu-, and Residue
U=-(D)Ala-
[0109] The title compound of example 6 was prepared from
cyclosporin difluoromethyl ester ester and palladium on carbon
according to the procedures described in Example1
Example 7
Compound of Formula I, Where in Residue A, X is Absent and
Y=--COOCF.sub.3; Residue B=-.alpha.Abu-, and Residue U=-(D)Ala-
[0110] The title compound of example 7 was prepared from
cyclosporin trifluoromethyl ester ester and palladium on carbon
according to the procedures described in Example 1.
Example 8
Compound of Formula I, Where in Residue A, X is Absent and
Y=--COOCH.sub.2CF.sub.3; Residue B=-.alpha.Abu-, and Residue
U=-(D)Ala-
[0111] The title compound of example 8 was prepared from
cyclosporin trifluoroethyl ester ester and palladium on carbon
according to the procedures described in Example 1.
[0112] The cyclosporin analogs of the present invention have potent
immunosuppressive and 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., pulmonary route.
[0113] The immunosuppressive and anti-inflammatory properties of
cyclosporin analogs of the invention may be demonstrated in
standard test models in vitro and in vivo for example as
follows.
Example 9
Calcineurin Inhibition Assay
[0114] 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.
[0115] 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 calorimetric detection reagent Biomol Green (Biomol
AK-111).
[0116] 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-HCl, pH
7.5; 100 mM sodium chloride; 6 mM magnesium chloride; 0.5 mM
dithiothreitol, 0.025% NP-40, 500 .mu.M calcium chloride, 0.27
.mu.M Calmodulin) containing 10 .mu.M Cyclophilin and 3 nM
Calcineurin. After warming to 37.degree. C. for 60 mins, 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 mins at room temperature.
[0117] IC.sub.50 values were calculated from determinations of
enzyme activity at inhibitor concentrations ranging from 0.1 to
0.0015 .mu.M.
Example 10
NFAT Reporter Gene Assay
[0118] NFAT activation follows precisely the activation of
calcineurin by increased free calcium levels in the cytoplasm.
Researchers from diverse fields are interested in the NFAT family
of transcription factors, which are potential targets for newer and
safer immunosuppressive drugs. In addition, the activation of NFAT
proteins involves various cellular signal transduction pathways,
including calcium mobilization and MAP kinase pathways linked to
T-cell receptors and Ras1. To assist researchers probing the
activity of NFAT proteins, Stratagene has developed a PathDetect
cis-reporter plasmid, the pNFAT-Luc reporter plasmid (Stratagene,
Inc. catalog #219094), containing the NFAT binding site from the
human IL-2 gene.2,7-9. The NFAT cis-reporting system includes the
transfection-ready pNFAT-Luc reporter plasmid and the pCIS-CK
negative control plasmid.
[0119] Construction of the pNFAT-Luc Plasmid:
[0120] The backbone of the 5749-base-pair pNFAT-Luc plasmid is the
pFR-Luc reporter plasmid of the aforementioned PathDetect
trans-reporting system. To this backbone, the GAL4 binding element
was replaced with four direct repeats of the NFAT binding sequence
(-286 to -257) from the IL-2 gene promoter, the most studied and
widely used NFAT binding sequence. For all reporter plasmids of the
PathDetect cis-reporting systems, activation of the luciferase gene
indicated interaction of uncharacterized gene products,
extracellular stimuli, growth factors, or drug candidates with
specific enhancer elements. Then a plasmid expressing the gene of
interest was cotransfected into mammalian cells along with a
cis-reporter plasmid to indicate transcription activation.
[0121] Testing the pNFAT-Luc Plasmid in Jurkat Cells:
[0122] Pharmacology studies have established that NFAT proteins can
be activated by the protein kinase C activator phorbol ester (PMA)
in combination with the calcium ionophore ionomycin, reagents that
raise free intracellular calcium. When Jurkat cells, a mature human
T-cell line, or CHO cells were transfected with the pNFAT-Luc
plasmid and treated with 60 ng/ml of PMA and 1 .mu.g/ml of
inomycin, luciferase activity increased by 13- and 16-fold,
respectively. Therefore, the enhancer element in the pNFAT-Luc
plasmid is responsive to calcium mobilization. Cells transfected
with pNFAT-Luc and then treated with either PMA or ionomycin alone
did not show a significant increase in luciferase activity.
[0123] Cyclosporin inhibits the activity of calcineurin, a protein
phosphatase regulated by intracellular calcium mobilization. All
the isoforms of NFAT protein contain a calcineurin-binding domain
and are activated by calcineurin. The inhibition of luciferase
expression from pNFAT-Luc in the present model, in both Jurkat and
CHO cells induced by PMA and ionomycin, was monitored for
cyclosporin (as a positive control) and the cyclosporin analogs of
the present invention.
[0124] In another set of experiments, rat basophilic leukemia cells
stably transfected with chemokine receptors were transtected with
pNFAT-Luc and then treated with their respective ligands (data not
shown). When both luciferase expression and calcium levels were
monitored in these cells, luciferase expression correlated very
well with calcium mobilization. Therefore, luciferase expression
from pNFAT-Luc indeed reflects the activation of endogenous NFAT
proteins by calcium immobilization.
Example 11
Immunosuppressive Activity and Applications
Murine Mixed Lymphocyte Reaction
[0125] 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
[0126] 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.
Delayed-type Hypersensitivity Resonse
[0127] On Day 0 groups of ten mice (having BALB/cByJ or any other
acceptable strain) are dosed with test compound (1 to 10%), vehicle
or the positive control, cyclophosphamide (Cyclosporin A), and
monitored from Day-2 to 7. The mice are anesthetized and their
abdomens shaved. 100 .mu.l of a 3% solution of ovalbumin are
applied to the abdomen and dried. Seven days later, the mice are
challenged by applying 5 .mu.l of ovalbumin to each side of the
right ear. After 24 hours, both the right and left ear thickness
are measured using a micrometer caliper.
Popliteal Lymph Node Assay
[0128] First, an inducer (phenytoin) is injected into the mice
footpad (having BALB/cByJ or any other acceptable strain). Then the
mice are challenged (subcutaneously or po) with ester and control
agent using graded doses, for example, 2.5, 10, 20 mg/Kg (based on
cyclosporine A data). On day 7 the popliteal lymph nodes are
excised from the dosed mice and the lymph nodes are weighed. Then
single cell suspensions of each lymph node are prepared and
enumerated. The weight index for each animal is calculated (for
example, a mean weight index<2 would indicate suppression of
immune response).
Influence on Allergen-induced Pulmonary Eosinophilia (in vitro)
[0129] 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.
[0130] 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%.
[0131] 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 substanceis 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.
[0132] 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.
[0133] In untreated animals, OA challenge induces increase of all
cell types in BAL fluid 24 hours after challenge. Prior
administration of cyclosporin analogs in accordance with the
present invention by inhalation at dosages of the order of from 1.0
to 15.0 mg/kg reduces eosinophil count in BAL in a dose dependent
manner as compared with untreated controls. Cell counts for other
leucocytes (macrophages, neutrophils etc.) are also reduced.
* * * * *