WO2003028712A2

WO2003028712A2 - Purinergic and pyrimidinergic receptor agonists for the treatment of activated cd4+ t lymphocyte-mediated immune diseases

Info

Publication number: WO2003028712A2
Application number: PCT/EP2002/010954
Authority: WO
Inventors: Liliane Schandene; Michel Goldman; Xavier Duhant; Jean-Marie Boeynaems; Didier Communi
Original assignee: Universite Libre De Bruxelles; Euroscreen S.A.
Priority date: 2001-09-28
Filing date: 2002-09-30
Publication date: 2003-04-10
Also published as: WO2003028712A3

Abstract

The present invention in particular relates to the use of purinergic and pyrimidinergic receptor agonists of formula (1) or a pharmaceutically acceptable salt thereof for inhibition of CD4+ T lymphocytes activation or as an immunosuppressive agent, wherein B1 is adenine or uracil, optionally substituted by thioalkyl, R1 and R2 represent each independently OH, H, OR7, OCOR7, OCOR7COR7, wherein R7 represents alkyl or aryl and X is of formula (2), (3) or (4), wherein R10 represents hydrogen or is of formula (7),wherein B2 is selected from the group comprising adenine, uracil, thymine, cytosine, guanine, xanthine and hypoxanthine, optionally substituted by thioalkyl, wherein n is 0, 1, 2, 3 or 4, and wherein R4, R4a and R4b represent each independently O, NH, alkylene, monohaloalkylene or dihaloalkylene and R3, R5, R6, R8, R9 represent each independently OH or SH, and wherein R11 and R12 represent each independently OH, H, OR7, OCOR7, OCOR7COR7, wherein R7 is as defined above.

Description

PURINERGIC AND PYRIMIDINERGIC RECEPTOR AGONISTS FOR THE TREATMENT OF ACTIVATED CD4⁺ T LYMPHOCYTE-MEDIATED IMMUNE DISEASES

FIELD OF THE INVENTION

The present invention relates to purinergic and pyrimidinergic receptor agonists useful for the inhibition of CD4⁺ T lymphocytes activation and to pharmaceutical compositions thereof. The present invention further relates to methods for treatment of activated CD4⁺ T lymphocyte- mediated immune diseases.

BACKGROUND TO THE INVENTION

Locally infiltrated CD4⁺ T helper cells play a crucial role in the pathogenesis of inflammatory, allergic and autoimmune diseases of inter alia airways, eye, skin and gastrointestinal tract. They orchestrate the inflammatory reaction via the secretion of various cytokines and are ultimately responsible for pathological consequences, such as bronchoconstriction in asthma or keratinocyte proliferation in psoriasis.

Conventional drugs used in the treatment of inflammatory, allergic, auto-immune diseases and graft/transplantation rejection are immunosuppressive. Cyclosporin A, tacrolimus, corticosteroids, mycophenolate, azathioprine, polyclonal anti-lymphocyte globulins are somewhat effective in electing an immunosuppressive response. However, their highly toxic profiles frequently limit their clinical benefit. These agents can be used systemically or locally. In particular inhaled corticosteroids have become the cornerstone of asthma therapy. Though safer than the systemic use, the topical use of corticosteroids can produce adverse reactions either locally (mouth and pharynx infections, skin atrophy) or systemically (adrenal suppression).

Thus, the treatment of inflammatory, allergic, autoimmune diseases and graft/transplantation rejection and other diseases requiring immunosuppression with agents having low-toxicity profiles remains a major clinical problem.

Extracellular nucleotides are a ubiquitous family of messengers that exert autocrine or paracrine actions. Their release in fluids results from cell lysis, exocytosis of nucleotide concentrating granules (synaptic vesicles, platelet dense bodies) or efflux from cytoplasm through membrane transport proteins. Several documents describe the therapeutic use of said nucleotides. WO 00/50024 relates to a method and preparation for the stimulation of mucosal hydration in a subject in need of this treatment, the method comprising administering to the mucosal surfaces of the subject uridine 5'-diphosphate (UDP), dinucleotides, cytidine 5'-diphosphate (CDP), adenosine 5'-diphosphate (ADP) in an amount effective to stimulate mucin secretion. US Pat No. 5,900,407 relates to methods for stimulation of tear secretion, for instance for treating dry eye disease, using ATP, UTP, dinucleotides and their analogs and derivatives. US Pat. No. 6,143,279 relates to the use of uridine 5'-diphosphate in a method for hydrating lung mucus secretions and treating lung disorders such as cystic fibrosis, ventilator- associated pneumonia, chronic bronchitis, chronic obstructive pulmonary disorder and primary ciliary dyskinesia.

It is an object of the present invention to provide effective agents for the treatment of inflammatory, allergic, autoimmune diseases and graft/transplantation rejection and more specifically but not limited for the treatment of activated CD4⁺ T lymphocyte-mediated immune diseases. It is another object to provide with agents for the treatment of activated CD4⁺ T lymphocyte-mediated immune diseases having an acceptable toxicity level and a sufficient activity level.

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to the use of purinergic and pyrimidinergic receptor agonists of formula (1 )

or a pharmaceutically acceptable salt thereof for the inhibition of CD4⁺ T lymphocytes activation, wherein B¹ is selected from the group comprising adenine, uracil, thymine, cytosine, guanine, xanthine and hypoxanthine, optionally substituted by thioalkyl, R¹ and R² represent each independently OH, H, OR⁷, OCOR⁷, OCOR⁷COR⁷, wherein R⁷ represents alkyl or aryl and X is of formula (2), (3) or (4),

wherein R represents hydrogen or is of formula (7),

wherein B² is selected from the group comprising adenine, uracil, thymine, cytosine, guanine, xanthine and hypoxanthine, optionally substituted by thioalkyl, wherein n is 0, 1 , 2, 3 or 4, and wherein R⁴, R^4a and R⁴ represent each independently O, NH, alkylene, monohaloalkylene or dihaloalkylene and R³, R⁵, R⁶, R⁸, R⁹ represent each independently OH or SH, and wherein R¹¹ and R¹² represent each independently OH, H, OR⁷, OCOR⁷, OCOR⁷COR⁷, wherein R⁷ is as defined above.

According to a particular embodiment, B¹ in the above formula (1) is adenine or uracil, optionally substituted by thioalkyl.

According to a particular embodiment, in the above formula (7), B² preferably is adenine, uracil, cytosine or guanine. In an interesting embodiment, B² is adenine in the formula (7) and n is 1 , 2, 3 or 4.

As a consequence of said effect on CD4⁺ T cells activation, said purinergic and pyrimidinergic receptor agonists of formula (1) are useful as immunosuppressive agents. According to a particular embodiment, said purinergic or pyrimidinergic receptor agonist is of formula (5), (6) or (8) or a pharmaceutically acceptable salt thereof,

wherein R¹, R² and X have the same meaning as defined above. Interesting compounds of the above formula are depicted in Table A.

According to a particular embodiment, said purinergic or pyrimidinergic receptor agonist is of formula (9), (10), (11) or (12) or a pharmaceutically acceptable salt thereof,

(12) wherein B¹, B², R¹, R², R^4b, R⁸, R⁹, R¹¹ and R¹² have the same meaning as that defined above, and wherein in each molecule (9), (10), (11) or (12), each R⁸ has independently from the other R⁸ substituents has an individual meaning and can change in each formula, for instance a purinergic or pyrimidinergic receptor agonist of formula (13),

Interesting compounds are those compounds of formula (9), (10), (11) or (12), wherein B¹ and B² are adenine. Non-limiting examples of such compounds are depicted in Table B. Interesting purinergic and pyrimidinergic receptor agonists of formula (1) are selected from the following groups (i) to (iv) comprising: (i) ATP and derivatives, (ATPγSBzATP2-MeSATP; (ii) UDP and UTP; (iii) ADP and derivatives (ADPβS, 2-MeSADP); (iv) dinucleotides.

More preferably said purinergic and pyrimidinergic receptor agonists of formula (1 ) are adenosine 5'-triphosphate (ATP), adenosine 5'-0-(3-thiotriphosphate) (ATPγS), 2'- and 3'-O- (4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP), uridine 5'-diphosphate (UDP) and uridine 5'-triphosphate (UTP), adenosine 5'-diphosphate (ADP), 2-methylthio-ADP (2- MeSADP), adenosine 5'-O-(2-thiodiphosphate) (ADPβS), P¹,P³-di(adenosine-5')triphosphate (Ap₃A), P¹,P⁴-di(adenosine-5')tetraphosphate (Ap₄A), P ,P⁵-di(adenosine-5')pentaphosphate (Ap₅A), P¹,P⁶-di(adenosine-5')hexaphosphate (Ap₆A), or a pharmaceutically acceptable salt of any of the aforementioned nucleotide derivatives.

In a second aspect the present invention relates to a method of inducing immunosuppression comprising administering to an individual, in need thereof, a therapeutically effective amount of a purinergic or pyrimidinergic receptor agonist of formula (1) or a pharmaceutically acceptable salt thereof.

In a third aspect, the present invention relates to a pharmaceutical composition for the treatment of activated CD4⁺ T lymphocytes-mediated immune diseases comprising a pharmaceutically acceptable carrier and a purinergic or pyrimidinergic receptor agonist of formula (1) or a pharmaceutically acceptable salt thereof.

In a fourth aspect the present invention relates to the use of a purinergic or pyrimidinergic receptor agonist of formula (1) or a pharmaceutically acceptable salt thereof for the preparation of a medicine for the treatment of an immune disease which is mediated or induced by activated CD4⁺ T lymphocytes. In preferred embodiments, said activated CD4⁺ T lymphocytes-mediated immune disease is asthma including bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma, allergic rhinitis, conjunctivitis, keratitis, keratoconjunctivitis, uveitis, eczema, atopic dermatitis, contact dermatitis, cutaneous T cell lymphoma (CTCL), Sezary syndrome, pemphigus vulgaris, bullous pemphigoid, pemphigus foliaceus, dermatomyositis, erythema nodosum, scleroderma, Bechet's disease, sarcoidosis, Sjogren's syndrome, rheumatoid arthritis, juvenile arthritis, Reiter's syndrome, lupus erythematosus, polymyositis, myocarditis, primary biliary cirrhosis, Crohn's disease, ulcerative colitis, multiple sclerosis and other demyelinating diseases, idiopathic thrombocytopenic purpura, Graves' disease and Hashimoto's disease, Addison's disease, insulin-dependent diabetes mellitus (type 1 ), transplant rejection or graft-versus-host disease.

The present invention will be further disclosed in detail hereunder. Examples are given which will further support the description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of purinergic and pyrimidinergic receptor agonists for the inhibition of CD4⁺ T lymphocytes activation, more in particular the present invention relates to the use of a purine or pyrimidine analogue or derivative of formula (1 ),

or a pharmaceutically acceptable salt thereof for the inhibition of CD4⁺ T lymphocytes activation, wherein B is B¹ is selected from the group comprising adenine, uracil, thymine, cytosine, guanine, xanthine and hypoxanthine, preferably selected from adenine or uracil, and B¹ optionally substituted by thioalkyl, R¹ and R² represent each independently OH, H, OR⁷, OCOR⁷, OCOR⁷COR⁷, wherein R⁷ represents alkyl or aryl and X is of formula (2), (3) or (4),

wherein R represents hydrogen or is of formula (7),

wherein B² is selected from the group comprising adenine, uracil, thymine, cytosine, guanine, xanthine and hypoxanthine, optionally substituted by thioalkyl, wherein n is 0, 1 , 2, 3 or 4 and wherein R⁴, R^4a and R⁴ represent each independently O, NH, alkylene, monohaloalkylene or dihaloalkylene and R³, R⁵, R⁶, R⁸, R⁹ represent each independently OH or SH, and wherein R¹¹ and R¹² represent each independently OH, H, OR⁷, OCOR⁷, OCOR⁷COR⁷, wherein R⁷ is as defined above, and wherein each R⁴ or R⁸ independently from the other R^4b or R⁸ substituents has an individual meaning and can change in each formula, as for instance in formula (7b) :

According to a particular embodiment, in the above formula (7), B² preferably is adenine, uracil, cytosine or guanine, optionally substituted by thioalkyl. In an interesting embodiment, B² is adenine, optionally substituted by thioalkyl, in the formula (7) and n is 1 , 2, 3 or 4. Although in the above formula (1), when X is of formula (2), (3) or (4) and R¹⁰ is of formula (7), the linkage between the nucleotides is from 5'->5' (e.g. P¹, P³ - Di(adenosine-5') triphosphate) , the present invention also relates to compounds wherein the linkage between the nucleotides is 2'→5' or 3'->5'. For instance, examples of such compounds which are commercially available are: adenylyl(2'^→5')cytidine, adenylyl(3'^→5')cytidine, adenylyl(2'^→5') uridine, adenylyl(3'^→5')uridine, adenylyl(3'^→5')adenosine, adenylyl(3'^→5')guanosine, cytidiyl (3'-5') cytidine, cytidylyl(2'^→5')adenosine, cytidylyl(2'^→5')guanosine, cytidylyl(3'^→5')cytidine, cytidylyl(3'^→5')guanosine, Cytidylyl(3'^→5')uridine, guanylyl(2'^→5')adenosine, guanylyl(2'^→5') cytidine, guanylyl(3'^→5')adenosine, guanylyl(3'^→5')cytidine, guanylyl(3'^→5')ιιridine, uridyly (2'^→5')uridine, uridylyl(3',5')adenosine, uridylyl (3'^→5')guanosine, uridylyl(3'^→5')uridine, 3- acetylpyridine adenine dinucleotide, and their salts.

According to a particular embodiment, said purinergic or pyrimidinergic receptor agonist is of formula (5), (6) or (8) or a pharmaceutically acceptable salt thereof,

wherein R¹, R² and X have the same meaning as that defined above.

(12) wherein B¹, B², R¹, R², R^4b, R⁸, R⁹, R¹¹ and R¹² have the same meaning as that defined above. Interesting compounds are those compounds of formula (9), (10), (11) or (12), wherein B¹ and B² are adenine. Non-limiting examples of such compounds are depicted in Table B. Other interesting compounds are those compounds of formula (9), (10), (11) or (12), wherein B¹ and B² are uracil. Non-limiting examples of such compounds are depicted in Table B. Other interesting compounds are those compounds of formula (9), (10), (11) or (12), wherein B¹ and B² are different. Non-limiting examples of such compounds are depicted in Table C.

The term "alkyl" as used herein refers to saturated monovalent hydrocarbon radicals having straight, branched or cyclic moieties or combinations thereof and contains 1-20 carbon atoms, preferably 1-10 carbon atoms, more preferably 1-8 carbon atoms, still more preferably 1-6 carbon atoms, yet more preferably 1-4 carbon atoms. Alkyl radicals of interest are methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, isoamyl, hexyl, cyclohexyl. The term "aryl" as used herein, includes a monovalent organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl. The term "alkylene" as used herein refers to saturated bivalent hydrocarbon radicals having straight, branched or cyclic moieties or combinations thereof and contains 1-20 carbon atoms, preferably 1-10 carbon atoms, more preferably 1-8 carbon atoms, still more preferably 1-6 carbon atoms, yet more preferably 1-4 carbon atoms. Examples of alkylene radicals are methylene, ethylene, and propylene. The term "halo" or "halogen" as used herein is generic for fluoro, chloro, bromo or iodo. The terms "monohaloalkylene" and "dihaloalkylene" as used herein refers to alkylene radical as defined above substituted by respectively one and two halogen radicals.

Interesting purinergic and pyrimidinergic receptor agonists advantageous for said use are selected from the group comprising adenosine 5'-triphosphate (ATP), adenosine 5'-0-(3- thiotriphosphate) (ATPγS), 2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP), uridine 5'-diphosphate (UDP) and uridine 5'-triphosphate (UTP), adenosine 5'- diphosphate (ADP), 2-methylthio-ADP (2-MeSADP), adenosine 5'-0-(2-thiodiphosphate) (ADPβS), P¹,P³-di(adenosine-5')triphosphate (ApaA), P¹,P⁴-di(adenosine-5')tetraphosphate (Ap₄A), P¹,P⁵-di(adenosine-5')pentaphosphate (Ap₅A), P¹,P⁶-di(adenosine-5')hexaphosphate (Ap₆A), or a pharmaceutically acceptable salt of any of the aforementioned nucleotide derivatives.

It should be noted that when the compound "2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'- triphosphate (BzATP)" is mentioned, a mixture of 2'-0-(4-benzoyl-benzoyl) adenosine 5'- triphosphate and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate can be meant. The compound BzATP is a mixture of positional isomers 2'-0-(4-benzoyl-benzoyl) adenosine 5'- triphosphate and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate.

The cited nucleotides are potent agonists of the metabotropic P2Y receptors, which are parts of the superfamily of G-protein-coupled receptors. At this stage, 8 genuine human P2Y receptors have been identified and characterized: P2Y_{1 f} P2Y₂, P2Y₄, P2Y₆, P2Yn, P2Y₁₂ and P2Y₁₃, as well as the uridine 5'-diphosphoglucose (UDP-glucose) receptor which is structurally related to P2Y₁₂. According to a preferred embodiment said purinergic or pyrimidinergic receptor useful for the present invention is selected from the group comprising P2Y_{1 f} P2Y₂, P2Y₄, P2Y₆, P2Yn, P2Y₁₂ and P2Y₁₃ receptors and UDP-glucose receptor. In one embodiment, said P2Y receptor is the P2Yn receptor. In another embodiment said P2Y receptor is the P2Y₁₃ receptor.

Extracellular nucleotides exert numerous actions and induce a wide spectrum of biological effects, mediated by several P2Y and P2X receptors, on monocytes and macrophages, dendritic cells, lymphocytes and granulocytes. Various nucleotides were found to induce an inhibition of the CD4⁺ T cell activation. The release of four cytokines (IL-2, IL-5, IL-10 and IFN-γ) involved in both Th1 and Th2 responses was significantly inhibited in the presence of nucleotides. Simultaneously to inhibition of IL-2 secretion, the level of the IL-2 receptor (CD25) expression was down-regulated: these two effects contribute to the inhibition of proliferation, assessed with the classical [³H]thymidine uptake test. In both activated and non- activated CD4⁺ T cell, cAMP accumulation was observed in response to ATP and ATP derivatives, ATPγS and BzATP. The rank order of potency was ATPγS≡.BzATP>ATP>2MeSATP»UTP=UDP=0. The immunosuppressive effects on both Th1 and Th2 responses, , are mediated by cAMP. An increase in cAMP, and inter alia the resulting activation of protein kinase A, inhibit T cell activation. UTP and UDP also inhibited the activation of CD4⁺ T lymphocytes as reflected by the inhibition of four cytokines (IL-2, IL- 5, IL-10 and IFN-γ), but this effect was not mediated by an increase in cAMP. Purinergic and pyrimidinergic receptor agonists of formula (1 ) or a pharmaceutically acceptable salt are therefore useful as immunosuppressive agent. Activation of CD4+ lymphocytes, as assessed by inhibition of cell proliferation and cytokine secretion, was also inhibited by ADP, ADPβS and 2-MeSADP and the dinucleotides Ap₃A, Ap₄A, Ap₅A and Ap₆A , independently of cAMP increase. The purinergic and pyrimidinergic receptor agonists useful according to the present invention may have asymmetric centers, occur as racemates, racemic mixtures, and as individual diastereoisomers, with all possible stereochemical isomers including optical isomers, being included in the present invention.

The present invention when referring to purinergic or pyrimidinergic receptor agonist, as cited herein, also includes within its scope not only the specific compound(s) listed or described, but also alternative forms of the compound such as pharmaceutically acceptable salts, solvates, hydrates, and the like. The pharmaceutically acceptable salts of the compounds of this invention include the conventional non-toxic salts of the compounds as formed, e.g., from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like: and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like.

The invention further relates to the use of use of a purine or pyrimidine analogue or derivative of formula (1) or a pharmaceutically acceptable salt of any of the aforementioned nucleotide derivatives, as an immunosuppressive agent.

In a further aspect, the present invention includes a method that is specifically intended to reduce or suppress an immune response or reduce inflammation in an individual in need of such a treatment. Inflammation can be suppressed inter alia by suppressing the immune reaction. According to one embodiment, the present invention relates to methods for suppressing inflammation in an individual by suppressing the immune reaction. According to a preferred embodiment, immunosuppression and inflammation are both suppressed in said individual.

More in particular, the method is useful for the suppression or reduction of T lymphocyte mediated immune response and more preferably CD4⁺ T lymphocytes mediated immune response in an individual. The term "T cell mediated immune response " as used herein refers to the increased release by T cells of factors such as cytokines in response to antigen. Examples of cytokines released from T cells in response to antigen include IL-2, IL-5, IL-10 and IFN-γ. The term "individual" as used herein refers to a mammal. The individual will preferably be a human, but may also be a domestic livestock, laboratory or pet animals.

Specifically, the present invention relates to a method of inducing immunosuppression comprising administering to an individual, in need thereof, a therapeutically effective amount of a purinergic or pyrimidinergic receptor agonist of formula (1 ) or a pharmaceutically acceptable salt thereof. Preferably said immunosuppression is being induced in an individual suffering from asthma including bronchial asthma, allergic asthma, intrinsic asthma and dust asthma. Preferably said immunosuppression is being induced in an individual suffering from allergic rhinitis. Preferably said immunosuppression is being induced in an individual suffering from conjunctivitis. Preferably said immunosuppression is being induced in an individual suffering from keratitis. Preferably said immunosuppression is being induced in an individual suffering from keratoconjunctivitis. Preferably said immunosuppression is being induced in an individual suffering from uveitis. Preferably said immunosuppression is being induced in an individual suffering from psoriasis. Preferably said immunosuppression is being induced in an individual suffering from eczema. Preferably said immunosuppression is being induced in an individual suffering from atopic dermatitis. Preferably said immunosuppression is being induced in an individual suffering from contact dermatitis. Preferably said immunosuppression is being induced in an individual suffering from cutaneous T cell lymphoma (CTCL). Preferably said immunosuppression is being induced in an individual suffering from Sezary syndrome. Preferably said immunosuppression is being induced in an individual suffering from pemphigus vulgaris. Preferably said immunosuppression is being induced in an individual suffering from bullous pemphigoid. Preferably said immunosuppression is being induced in an individual suffering from pemphigus foliaceus. Preferably said immunosuppression is being induced in an individual suffering from dermatomyositis. Preferably said immunosuppression is being induced in an individual suffering from erythema nodosum. Preferably said immunosuppression is being induced in an individual suffering from scleroderma. Preferably said immunosuppression is being induced in an individual suffering from Bechet's disease. Preferably said immunosuppression is being induced in an individual suffering from sarcoidosis. Preferably said immunosuppression is being induced in an individual suffering from Sjogren's syndrome. Preferably said immunosuppression is being induced in an individual suffering from rheumatoid arthritis. Preferably said immunosuppression is being induced in an individual suffering from juvenile arthritis. Preferably said immunosuppression is being induced in an individual suffering from Reiter's syndrome. Preferably said immunosuppression is being induced in an individual suffering from lupus erythematosus. Preferably said immunosuppression is being induced in an individual suffering from polymyositis. Preferably said immunosuppression is being induced in an individual suffering from myocarditis. Preferably said immunosuppression is being induced in an individual suffering from primary biliary cirrhosis. Preferably said immunosuppression is being induced in an individual suffering from Crohn's disease. Preferably said immunosuppression is being induced in an individual suffering from ulcerative colitis. Preferably said immunosuppression is being induced in an individual suffering from multiple sclerosis and other demyelinating diseases. Preferably said immunosuppression is being induced in an individual suffering from idiopathic thrombocytopenic purpura. Preferably said immunosuppression is being induced in an individual suffering from Graves' disease and Hashimoto's disease. Preferably said immunosuppression is being induced in an individual suffering from Addison's disease. Preferably said immunosuppression is being induced in an individual suffering from insulin-dependent diabetes mellitus (type 1 ). Preferably said immunosuppression is being induced in an individual suffering from transplant rejection or graft-versus-host disease.

The "therapeutically effective amount" of said above-described purinergic or pyrimidinergic receptor agonist relates to the amount or quantity of said agonist required to achieve the desired therapeutic and/or prophylactic effect, for example the dosage of said agonist which results in suppression of an immune response in the individual.

A particular purinergic or pyrimidinergic receptor agonist advantageous for said method is of formula (5), (6), (8), (9), (10), (11) or (12), or a pharmaceutically acceptable salt thereof.

According to another embodiment, said purinergic or pyrimidinergic receptor agonist of formula (1) is a P2Yn or a P2Y₁₃ receptor agonist, and is selected from the group comprising ATP, adenosine 5'-0-(3-thiotriphosphate) (ATPγS), 2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP) and 2-methylthio-ATP (2-MeSATP). More preferably, said purinergic and pyrimidinergic receptor agonist is ATP, adenosine 5'-0-(3-thiotriphosphate) (ATPγS) or 2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP). According to another embodiment, said purinergic or pyrimidinergic receptor agonist is uridine 5'- diphosphate (UDP), uridine 5'-triphosphate (UTP), ADP, 2-methylthio-ADP (2-MeSADP), adenosine 5'-0-(2-thiodiphosphate) (ADPβS), P¹,P³-di(adenosine-5')triphosphate (Ap₃A), P ,P⁴-di(adenosine-5')tetraphosphate (Ap₄A), P¹,P⁵-di(adenosine-5')pentaphosphate (Ap₅A), P¹,P⁶-di(adenosine-5')hexaphosphate (Ap₆A), or a pharmaceutically acceptable salt of any of the aforementioned nucleotide derivatives.

In a further aspect, the present invention relates to a pharmaceutical composition for the treatment of activated CD4⁺ T lymphocytes-mediated immune diseases comprising a pharmaceutically acceptable carrier and a purinergic or pyrimidinergic receptor agonist of formula (1) or a pharmaceutically acceptable salt thereof.

A particular purinergic or pyrimidinergic receptor agonist advantageous for said pharmaceutical composition is of formula (5), (6), (8), (9), (10), (11) or (12) or a pharmaceutically acceptable salt thereof.

An interesting purinergic and pyrimidinergic receptor agonist of formula (1) according to the invention is preferably a P2Yn or P2Y₁₃ receptor agonist, and is selected from the group comprising ATP, adenosine 5'-0-(3-thiotriphosphate) (ATPγS), 2'- and 3'-0-(4-benzoyl- benzoyl) adenosine 5'-triphosphate (BzATP), 2-methylthio-ATP (2-MeSATP) or a pharmaceutically acceptable salt thereof. More preferably said P2Y receptor agonist is ATP, adenosine 5'-0-(3-thiotriphosphate) (ATPγS), 2'- or 3'-0-(4-benzoyl-benzoyl) adenosine 5'- triphosphate (BzATP). Other interesting purinergic and pyrimidinergic receptor agonists of formula (1 ) are uridine 5'-diphosphate (UDP), uridine 5'-triphosphate (UTP), adenosine 5'- diphosphate (ADP), 2-methylthio-ADP (2-MeSADP), adenosine 5'-0-(2-thiodiphosphate) (ADPβS), P¹,P³-di(adenosine-5')triphosphate (Ap₃A), P¹,P⁴-di(adenosine-5')tetraphosphate (Ap₄A), P ,P⁵-di(adenosine-5')pentaphosphate (ApsA), P¹,P⁶-di(adenosine-5')hexaphosphate (Ap₆A), or a pharmaceutically acceptable salt of any of the aforementioned nucleotide derivatives..

Examples of activated CD4+ T-lymphocyte-mediated immune diseases include but are not limited to: asthma including bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma, allergic rhinitis, conjunctivitis, keratitis, keratoconjunctivitis, uveitis, psoriasis, eczema, atopic dermatitis, contact dermatitis, cutaneous T cell lymphoma (CTCL), Sezary syndrome, pemphigus vulgaris, bullous pemphigoid, pemphigus foliaceus, dermatomyositis, erythema nodosum, scleroderma, Bechet's disease, sarcoidosis, Sjogren's syndrome, rheumatoid arthritis, juvenile arthritis, Reiter's syndrome, lupus erythematosus, polymyositis, myocarditis, primary biliary cirrhosis, Crohn's disease, ulcerative colitis, multiple sclerosis and other demyelinating diseases, idiopathic thrombocytopenic purpura, Graves' disease and Hashimoto's disease, Addison's disease, insulin-dependent diabetes mellitus (type 1 ), transplant rejection and graft-versus-host disease.

According to a preferred embodiment, the present invention relates to a pharmaceutical composition for the treatment of activated CD4⁺ T lymphocytes-mediated immune diseases wherein said activated CD4⁺ T lymphocytes-mediated immune disease is selected from the group comprising asthma, allergic rhinitis, eczema, atopic dermatitis, contact dermatitis, bullous pemphigoid, pemphigus foliaceus, dermatomyositis, lupus erythematosus, Sjogren's syndrome, Crohn's disease and ulcerative colitis.

The pharmaceutical composition of the present invention may further include thickeners, diluents, buffers, preservatives, surface active agents, liposomes, or lipid formulations, and the like. Pharmaceutically acceptable carriers may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions suitable for ingestion, inhalation, intranasal administration, ocular application, skin application or administration as a suppository to the rectum or vagina. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and certain organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.

Said pharmaceutical composition may be administered to an individual in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topical including on the skin, ophthalmic, vaginal, rectal, intranasal, oral, by inhalation, or parenteral, for example by intravenous drip, subcutaneous, intratumor, intraperitoneal, intralymphatic or intramuscular injection. According to a preferred embodiment topical administration is preferred for topical immunosuppression in airway, eye and skin diseases.

The pharmaceutical composition according to the invention may suitably be provided in the form of a spray, an aerosol, tablets (entero-or not-enterocoated), capsule (hard or soft, entero-or not-enterocoated, controlled ileal release or not), a suspension, a dispersion, granules, a powder, a solution, an emulsion, chewable tablets, tablets for dissolution, drops, a gel, a paste, a syrup, a cream, a lozenge (powder, granulate, tablets), a lotion, a foam, an instillation fluid, a gas, a vapor, an ointment, a patch, a stick, implants (ear, eye, skin, nose, rectal, or vaginal), vagitories, suppositories, enema, foam, or uteritories suitable for administration via the oral, nasal, vaginal, sublingual, ocular, rectal, urinary, intramammary, pulmonary, otolar, or buccal route.

In a further aspect the present invention relates to the use of purinergic or pyrimidinergic receptor agonist of formula (1) as described in detail above, or a pharmaceutically acceptable salt thereof for treating allergic airway diseases or for the preparation of a medicine for treatment of an allergic airway disease. More preferably, said allergic airway disease is asthma or allergic rhinitis.

The present invention also relates to the use of purinergic and pyrimidinergic receptor agonist of formula (1 )

or a pharmaceutically acceptable salt thereof for treating skin diseases or for the preparation of a medicine for the treatment of skin disease, wherein B¹ is adenine or uracil, optionally substituted by thioalkyl, R¹ and R² represent each independently OH, H, OR⁷, OCOR⁷, OCOR⁷COR⁷, wherein R⁷ represents alkyl or aryl and X is of formula (2), (3) or (4),

9 9 9

R^ηu-0-P— R^ιυ-0-P- -R⁴-P— 9

R^1u- O-l -R - AP-R 4^4a-P- (2) R³ (3) k R³ (4) R 5⁶6 R⁵ R³

wherein R¹⁰ is of formula (7),

wherein B² is selected from the group comprising adenine, uracil, thymine, cytosine, guanine, xanthine and hypoxanthine, optionally mono- or di-substituted by thioalkyl, wherein n is 0, 1 , 2, 3 or 4, and wherein R⁴, R^4a and R⁴ represent each independently O, NH, alkylene, monohaloalkylene or dihaloalkylene and R³, R⁵, R⁶, R⁸, R⁹ represent each independently OH or SH. wherein R⁴, R^4a and R^4b represent each independently O, NH, alkylene, monohaloalkylene or dihaloalkylene and R³, R⁵, R⁶, R⁸, R⁹ represent each independently OH or SH, and wherein R¹¹ and R¹² represent each independently OH, H, OR⁷, OCOR⁷, OCOR⁷COR⁷, wherein R⁷ is as defined above, and wherein each R4b or R8 independently from the other R^4b or R⁸ substituents has an individual meaning and can change in each formula, for instance as in formula (7b). Preferably said skin disease is psoriasis. Preferably said skin disease is eczema. Preferably said skin disease is atopic dermatitis. Preferably said skin disease is contact dermatitis. Preferably said skin disease is bullous pemphigoid. Preferably said skin disease is pemphigus foliaceus. Preferably said skin disease is dermatomyositis. Preferably said skin disease is lupus erythematosus.

Interesting examples of compounds for treating skin diseases are depicted in tables B and C.

According to a particular embodiment, B¹ in the above formula (1) of the receptor agonist for treating skin disease or for the preparation of the medicine for treating skin diseases, is adenine or uracil, optionally substituted by thioalkyl.

According to a particular embodiment, in the above formula (7) of the receptor agonist for treating skin disease or for the preparation of a medicine for treating skin diseases, B² preferably is adenine, uracil, cytosine or guanine, optionally substituted with thioalkyl. In an interesting embodiment, B² is adenine in the formula (7) and n is 1 , 2, 3 or 4.

The present invention further relates to the use of purinergic or pyrimidinergic receptor agonist of formula (1 ) as described in detail above, or a pharmaceutically acceptable salt thereof for treating inflammatory bowel diseases or for the preparation of a medicine for treatment of inflammatory bowel diseases. According to a preferred embodiment said inflammatory bowel disease is Crohn's disease. According to another embodiment said inflammatory bowel disease is ulcerative colitis.

A particular purinergic or pyrimidinergic receptor agonist advantageous for use in the treatment of immune diseases which are mediated or induced by activated CD4⁺ T lymphocytes or in the preparation of a medicine for treatment an activated CD4⁺ T lymphocytes-mediated immune disease is of formula (5), (6), (8), (9), (10), (11 ) or (12), or is a pharmaceutically acceptable salt of any of the purinergic or pyrimidinergic receptor agonists of formula (5), (6), (8), (9), (10), (11) or (12). An interesting purinergic or pyrimidinergic receptor agonist of formula (1 ) is a P2Yn or P2Y₁₃ receptor agonist, and is selected from the group comprising ATP, adenosine 5'-O-(3- thiotriphosphate) (ATPγS), 2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP), 2-methylthio-ATP (2-MeSATP) or a pharmaceutically acceptable salt thereof. According to a preferred embodiment said purinergic and pyrimidinergic receptor agonist is ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPγS), 2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP) or a pharmaceutically acceptable salt thereof. According to another preferred embodiment, said purinergic or pyrimidinergic receptor agonist is uridine 5'-diphosphate (UDP), uridine 5'-triphosphate (UTP), adenosine 5'-diphosphate (ADP), 2- ethylthio-ADP (2-MeSADP), adenosine 5'-0-(2-thiodiphosphate) (ADPβS), P¹,P³- di(adenosine-5')triphosphate (Ap₃A), P¹,P⁴-di(adenosine-5')tetraphosphate (Ap₄A), P¹,P⁵- di(adenosine-5')pentaphosphate (ApsA), P¹,P⁶-di(adenosine-5')hexaphosphate (Ap₆A), or a pharmaceutically acceptable salt thereof.

Non limiting examples of purinergic and pyrimidinergic receptor agonists of formula (1) useful according to the invention are listed Tables A, B and C.

Table A

61-

fξβOl/ZOdΑ/ΣJd Z 8Z0/C0 OΛV

vz

fξβOl/ZOdΑ/ΣJd Z 8Σ0/C0 OΛV

22

fξβOl/ZOdΑ/ΣJd ZUHZOItO OΛV

ε2

fςβoi/zodsi/iDd zuazmo OΛV

vZ

fξβOl/ZOdΑ/ΣJd Z 8Z0/C0 OΛV

92

PSβOl/ZOdΑ/lDd Z 8Z0/C0 OΛV

LZ

82

fξβOl/ZOdΑ/ΣJd Z 8Z0/C0 OΛV

62

PSβOl/ZOdΑ/lDd Z 8Z0/C0 OΛV

ι.ε

tξβOl/ZOdΑ/lDd Z 8Z0/C0 OΛV

2ε

fξβOl/ZOdΑ/ΣJd Z 8Z0/C0 OΛV

εε

tξβoi/zodΑ/∑jd Z 8Z0/C0 OΛV

gε

ζ60l/Z0dΑ/13d Z 8Z0/C0 OΛV

9ε

ζ60l/Z0dΑ/13d Z 8Z0/C0 OΛV

zε

fξβOl/ZOdΑ/ΣJd Z 8Z0/C0 OΛV

ζ60l/Z0dΑ/13d Z 8Z0/C0 OΛV

0 θ|qe

fξβOl/ZOdΑ/ΣJd Z 8Z0/C0 OΛV

IV

ζ60l/Z0dΑ/13d Z 8Z0/C0 OΛV

ZP

ζόOl/ZOdΑ/lDd Z 8Z0/C0 OΛV

zv

fξβOl/ZOdΑ/ΣJd Z 8Z0/C0 OΛV

rt

PSβOl/ZOdΑ/lDd Z 8Z0/C0 OΛV

9t?

fξβOl/ZOdΑ/ΣJd Z 8Z0/C0 OΛV

ζόOl/ZOdΑ/ΣJd Z 8Z0/C0 OΛV

The invention will be more readily understood by reference to the following examples and figures, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1a: shows the effect of adenine and uridine nucleotides on cAMP accumulation in freshly purified (FP) or activated (Act) human CD4⁺ T cells.

Figure 1b: represents concentration-action curves of ATP and ATP derivatives on the cAMP accumulation in human CD4⁺ T cells activated with pre-coated anti-CD3 and soluble anti- CD28 mAb during 72 h.

Figure 1c: shows the effect of 8-p-SPT and indomethacin (Indo) on the cAMP production in human CD4⁺ T cell.

Figure 2: represents concentration-inhibition curves for ATPγS and BzATP effect on IL-2 (a), IFN-γ(b), IL-5 (c) and IL-10 (d) secretion from human CD4⁺ T cells.

Figure 3: shows the inhibition of CD4+ T lymphocytes proliferation by ATP derivatives.

Figure 4: is a graph indicating the effect of ATP derivatives on CD25 expression in human CD4⁺ T cells after 72 hours of activation with pre-coated anti-CD3 and soluble anti-CD28 mAb.

Figure 5: is a graph showing the concentration-dependent inhibition by UTP and UDP of IL-2 (a), IL-5 (b), IL-10 (c) and IFN-γ (d) secretion from activated human CD4⁺T cells

Figure 6: is a graph that compares the concentration-dependent inhibition by Ap₄A and ATPγS of IFN-γ (a) and IL-10 (b) secretion from activated CD4⁺ T cells.

Figure 7: is a graph showing the inhibition of CD4⁺ T lymphocytes proliferation by nucleotide diphosphates and dinucleotides (concentrations expressed in μM). EXAMPLES

Materials and Methods

ATP, adenosine 5'-0-(3-thiotriphosphate) (ATPγS), 2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP), uridine 5'-diphosphate (UDP), uridine 5'-triphosphate (UTP), ADP, 2-methylthio-ADP (2-MeSADP), adenosine 5'-0-(2-thiodiphosphate) (ADPβS), P¹,P³-di(adenosine-5')triphosphate (Ap₃A), P¹,P⁴-di(adenosine-5')tetraphosphate (Ap₄A), P¹,P⁵-di(adenosine-5')pentaphosphate (ApsA), P¹,P⁶-di(adenosine-5')hexaphosphate (Ap₆A), and indomethacin (Indo) were obtained from Sigma Chemicals (St. Louis, MO, USA). 2- methylthio-ATP (2-MeSATP), CGS-21680 (a potent agonist of A_2A receptors) and 8-(p- sulphophenyl) theophylline (8-p-SPT) were purchased from Research Biochemicals International (Natick, MA, USA). The anti-CD3 monoclonal antibody (mAb) OKT3 (Orthoclone OKT3®) was provided by Janssen-Cilag (Berchem, Belgium) and the anti-CD28 mAb (clone CD28.2) by BD Pharmingen (San Diego, CA, USA). [³H]thymidine (25 Ci mmol^'1) was from Moravek Biochemicals (Brea, CA, USA). Rolipram was a gift from the Laboratoires Jacques Logeais (Trappes, France).

Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy blood donors by density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway). After three washes in Hank's balanced salt solution (HBSS) (Gibco Life Technologies, Paisley, UK) CD4⁺ T cells were isolated from PBMC using the MACS negative depletion system (Miltenyi Biotec, Auburn, CA). No contaminating CD8⁺ T cells, B cells, monocytes, or natural killer cells were detected.

Purified CD4⁺ T cells were cultured in RPMI 1640 medium (Gibco Life Technologies, Paisley, UK) supplemented with 10 % heat-inactivated fetal bovine serum (FCS) from Hyclone (Logan, Utah), 25 mM Hepes buffer, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 μg ml -1 gentamicin and 50 μM 2-β -mercaptoethanol at 37 °C in 5 % C02. The CD4⁺ T cells (1 X 10⁵ /well) were activated in flat-bottomed 96-well plates pre-coated with the anti-CD3 mAb (10 μg ml^"1) in the presence of soluble anti-CD28 mAb (1 μg ml^"1) and presence or absence of different concentrations of nucleotides. Culture supernatants were harvested after 24, 72 or 96 hours for measurement of cytokine concentration and the remaining cells were resuspended in PBS to determine CD25 surface expression as well as apoptosis and necrosis by flow cytometry. After 56 hours of culture, proliferation was assessed by [³H]thymidine (0.5 μCi/well) uptake during the following 16 hours. Each experimental condition was tested in triplicate.

Flow cytometric analysis of surface phenotype of the CD4⁺ T cells was performed by two- or three-color staining using fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, and piridinin-chlorophyll-a-protein (PerCP)-conjugated mouse anti-human mab. Purified CD4⁺ T cells were stained with mabs against: CD3, CD4, CD8, CD14, CD16, CD19, CD25, CD45, CD56, CD69, all from BD Pharmingen (San Diego, CA). The percentage of apoptotic and necrotic CD4⁺ T cells was determined using FITC-conjugated Annexin-V and propidium iodide (PI), both from BD Pharmingen (San Diego, CA). Samples were assayed in duplicate and analyzed using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and the Cell Quest® software

Commercially available kits were used for quantification of various cytokine levels: IL-2 (R&D Systems, Oxon, UK), IL-10 and IFN- from Biosource International (Camarillo, CA). IL-5 levels were measured by two-site sandwich ELISA using antibodies from BD Pharmingen (San Diego, CA). Each experimental condition was tested in triplicate.

Cells were preincubated for 30 min in complete culture medium with 25 μM rolipram and then incubated in the same medium for 12 min in the presence of the agonists. The incubation was stopped by the addition of 1 ml HCI 0.1 M. The incubation medium was dried up and the samples were resuspended in water and diluted as required. Cyclic AMP was quantified by radioimmunoassay after acetylation as previously described (Brooker G. et al. Adv. Cyclic. Nucleotide Res. 1979, 10:1). Each experimental condition was tested in triplicate.

Triplicates were obtained for each measurement (i.e. for each agonist, each donor and each concentration). Analysis of variance for repeated measures (concentration at three levels) with two inter-subjects factors (donor and agonist) was performed. Adjustment for multiple comparisons between the three concentrations was performed using the Sidak correction (SPSS 10.0 from SPSS Inc., Chicago, USA). Example 1: Effect of nucleotides on camp in freshly purified and activated human CD4⁺ T cells.

The effect of nucleotides on cAMP was determined. A comparison was made between CD4⁺ T cells freshly purified and activated during 72 h with the association of immobilized anti-CD3 mAb and soluble anti-CD28 mAb, used as an antigen-presenting-cell (APC)-independent and polyclonal T cell stimulus. Freshly purified or activated cells were pre-incubated for 30 min with rolipram (25 μM) and then incubated in the same medium in the presence of various concentrations of nucleotides for 12 min. cAMP was quantified by radioimmunoassay after acetylation. The data represent the mean DS.D. of triplicate experimental points obtained in one representative experiment of three.

The effects of adenine and uridine nucleotides on cAMP accumulation in freshly purified (FP) or activated (Act) human CD4⁺ T cells are shown in Figure 1a At 100 μM concentration, ATPDS and BzATP, stimulated significantly the cAMP production in both populations. This stimulation was observed in all the 6 preparations of CD4⁺ T cells that were tested. ATPγS (100 μM) produced a 20 ± 11 fold cAMP increase in freshly isolated cells and a 28 ± 15 fold cAMP increase 72 hours following activation (mean ± S.D. of 6 experiments).

Concentration-action curves of ATP and ATP derivatives on the cAMP accumulation in activated human CD4⁺ T cells showed that the rank order of agonist potency in activated CD4⁺ T cells was ATPγS ≡ BzATP > ATP (Figure 1 b).

Freshly purified or activated cells were pre-incubated with 8-p-SPT (300 μM) or Indo (5 μg ml^"1) for 15 min, both with rolipram (25μM), then incubated with ATPγS (100 μM), adenosine (100 μM) or CGS-21680 (100 nM) for 12 min. cAMP was quantified by RIA after acetylation. In 3 experiments out of 6, a stimulation of cAMP accumulation could also be detected in response to adenosine (100 μM) or the A_2A receptor agonist CGS-21680 (100 nM), but only in activated cells (Figure. 1c). That cAMP response was sensitive to inhibition by 8-p-SPT (300 μM), an A2 receptor antagonist, while the effect of ATPγS was not (Figure. 1c). On the other hand, indomethacin, an inhibitor of prostaglandin release, had a minimal effect on the cAMP response to ATPγS : 25 ± 14 % inhibition (mean of 3 experiments ± S.D. ; range : 11-38 %). ADP, ADPβS, 2-MeSADP, Ap₃A, Ap₄A, Ap₅A and Ap₆A did not increase the cAMP level in CD4⁺ T cells. Example 2: Effects of nucleotides on cytokine production by CD4⁺ T cells.

The freshly purified cells were incubated with pre-coated anti-CD3 and soluble anti-CD28 mAbs (10⁶ cells/ml), and various concentrations of nucleotides. After 24 h (IL-2), 72 h (IFN- γ and IL-10) or 96 h (IL-5) of incubation, the supernatants were harvested for ELISA. IFN- (Figure 2b), IL-2 (Figure 2a), IL-5 (Figure 2c) and IL-10 (Figure 2d) production by CD4⁺ T cells was measured in absence or presence of adenine nucleotide derivatives added at the beginning of the culture. Results of similar experiments performed with UTP and UDP on one hand, Ap₄A on the other hand, are shown respectively in Figure 5 and Figure 6. Data represent the mean ± S.D. of triplicate experimental points obtained in one representative experiment of three. The results obtained are shown in Figure 2a, 2b, 2c, 2d, 5a, 5b, 5c, 5d, 6a and 6b and are listed in Table I, Table II and Table III.

As shown in Figure 2a, 2b, 2c, 2d, Tables I and II, both ATPγS and BzATP significantly inhibited in a concentration-dependent manner the secretion of the four cytokines tested. The inhibition increases significantly with the concentration (P < 0.001 for each cytokine), but no significant difference could be detected between the agonists, except for IL-10. These effects were insensitive to 8-p-SPT (data not shown), indicating that they are not mediated by adenosine and A2 receptors. Moreover, the inhibition of IL-2 and IL-10 production by CD4⁺ T cells was not modified by the addition of indomethacin (5 μg ml^"1) (data not shown). No significant amounts of cytokines were detectable in culture supernatants of non activated CD4⁺ T cell (<10 pg ml^"1).

As shown in Figure 5a, 5b, 5c and 5d, UDP and UTP also inhibited the release of the four cytokines in a concentration-dependent way.

Other nucleotides inhibited the release of cytokines by activated CD4⁺ T cells. Results are shown in Table III and Figure 6a and 6b.

Table I: Effect of ATγS and BzATP on cytokine secretion by activated CD4⁺ T cells.

The data listed above represent the mean (pg/ml) ± standard deviation (SD) of triplicates for each donor and the mean percentage of inhibition of each cytokine secretion from activated CD4⁺ T cells in presence of ATPγS or BzATP (100 μM). Control represents the normal level of secretion of activated cells (no nucleotide).

Table II: P-values after statistical analysis of inhibition of cytokine secretion data.

Analysis of variance for repeated measures (concentration at three levels) with two inter-subjects factors (donor and agonist) was performed. Adjustment for multiple comparisons between the three concentrations was performed using the Sidak correction (SPSS 10.0 from SPSS Inc., Chicago, USA). Table III: Comparison between the effects of various nucleotides on the release of IFN-γ by CD4⁺ T cells

The table shows the mean inhibitory effect (in %) of various nucleotides on the secretion of IFN-γ by CD4⁺T cells activated by the combination of pre-coated anti-CD3 and soluble anti-CD28 mAbs. SD is the standard deviation (or range in the case where n=2) and n the number of individual experiments with cells from different donors.

Example 3: Effects of ATP analogues on CD4⁺ T cell proliferation

Human CD4⁺ T cells were purified and activated with pre-coated anti-CD3 and soluble anti- CD28 mAb. Nucleotides were added as the same time as the two antibodies. The cells were incubated during 56 h then 16 h in presence of [³H]thymidine (0.5 μCi/well). Data are given as the mean percentage of control [³H]thymidine incorporation. Data represent the mean ± S.D. of triplicate experimental points obtained in one representative experiment of three.

A shown in figure 3, ATPγS and BzATP added at concentrations ranging from 1 to 100 μM, inhibited the proliferation of CD4⁺ T cells activated by immobilized anti-CD3 and soluble anti- CD28 mAb as assessed by [³H]thymidine incorporation. An average of 41 ± 13 and 46 ± 7 (mean ± S.D.) percentage inhibition of CD4⁺ T cell proliferation were observed with ATPγS and BzATP (both 100 μM) respectively. Also, the decrease in proliferation was not reversed by the simultaneous addition of 8-p-SPT (data not shown). Labeling of the CD4⁺ T cells with Annexin-FITC and propidium iodide showed that nucleotides did not increase the number of apoptotic and necrotic cells (data not shown). Other nucleotides inhibited the proliferation ofCD4+ T lymphocytes. Results are shown in Figure 7.

Example 4: ATP analogues down-regulate the expression of CD25 during CD4⁺ T cell activation

The mechanism of action of the ATP derivatives on CD4⁺ T cell proliferation was studied, CD25 expression induced on these cells during activation by immobilized anti-CD3 and soluble anti-CD28 mab was evaluated by flow cytometry in presence or absence of the nucleotides. The nucleotides were added as the same time as the two antibodies. CD25 expression was analysed by flow cytometry after 72 h of incubation. Data are given as the mean ± range of duplicate points obtained in one representative experiment of three.

A shown in figure 4, addition of ATPγS (100 μM) or BzATP (100 μM) at the beginning of the culture inhibited CD25 expression on the CD4⁺ T cells after 72 hours activation by more than 40 %.

In both activated and non-activated CD4⁺ T cell, cAMP accumulation was observed in response to ATP and ATP derivatives, ATPγS and BzATP. The rank order of potency was ATPγS=BzATP>ATP>2MeSATP»UTP=UDP=0. The above-described experiments demonstrate that ATP derivatives induce an inhibition of the CD4⁺ T cell activation. The release of four cytokines (IL-2, IL-5, IL-10 and IFN-γ) involved in both Th1 and Th2 responses was significantly inhibited in the presence of adenine nucleotides. Simultaneously to inhibition of IL-2 secretion, the level of the IL-2 receptor (CD25) expression was down-regulated: these two effects contribute to the inhibition of proliferation, assessed with the classical [³H]thymidine uptake test. Although they did not increase cAMP, UTP and UDP also inhibited the release of the four cytokines (IL-2, IL-5, IL-10, IFN-γ). Other nucleotides were also able to inhibit the release of cytokines (IFN-γ and 11-10) by CD4+ T cells, as well as their proliferation: ADP, ADPβS, 2-MeSADP, Ap₃A, Ap₄A, ApsA and Ap₆A. They did not act via an increase in camp.

These results demonstrated the effectiveness of the immunosuppressive action of the purinergic and pyrimidinergic receptor agonists.

Example 5 : In vitro and in vivo testing of active compounds

The compounds are tested in animal models, both topically and systemically : skin graft rejection, herpes simplex keratitis, cardiac graft rejection, islet graft rejection, experimental allergic encephalomyelitis. The in vitro responsiveness of T lymphocytes from patients with autoimmune diseases (diabetes 1 , multiple sclerosis, coeliac disease, is evaluated. The effect of topical administration in human patients (aerosol, ophthalmic drops...) is monitored by measuring locally, in biopsies and/or fluids, the number of T cells, the proportion of various subsets (CD4 versus CD8...), the expression of various surface markers of activation (HLA- DR...), the cell expression and local secretion of cytokines (IL-2, IFN-γ, IL-4, IL-5...).

Claims

1. Use of purinergic and pyrimidinergic receptor agonists of formula (1 )

or a pharmaceutically acceptable salt thereof for the inhibition of CD4⁺ T lymphocytes activation, wherein B¹ is adenine or uracil, optionally substituted by thioalkyl, R¹ and R² represent each independently OH, H, OR⁷, OCOR⁷, OCOR⁷COR⁷, wherein R⁷ represents alkyl or aryl and X is of formula (2), (3) or (4),

wherein R represents hydrogen or is of formula (7),

wherein B² is selected from the group comprising adenine, uracil, thymine, cytosine, guanine, xanthine and hypoxanthine, optionally substituted by thioalkyl, wherein n is 0, 1 , 2, 3 or 4, and wherein R⁴, R^4a and R^4b represent each independently O, NH, alkylene, monohaloalkylene or dihaloalkylene and R³, R⁵, R⁶, R⁸, R⁹ represent each independently OH or SH, and wherein R¹¹ and R¹² represent each independently OH, H, OR⁷, OCOR⁷, OCOR⁷COR⁷, wherein R⁷ is as defined above.

2. Use of purinergic or pyrimidinergic receptor agonist of formula (1 )

or a pharmaceutically acceptable salt thereof as an immunosuppressive agent, wherein B¹ is adenine or uracil, optionally substituted by thioalkyl, R¹ and R² represent each independently OH, H, OR⁷, OCOR⁷, OCOR⁷COR⁷, wherein R⁷ represents alkyl or aryl and X is of formula (2), (3) or (4),

wherein R represents hydrogen or is of formula (7),

wherein B² is selected from the group comprising adenine, uracil, thymine, cytosine, guanine, xanthine and hypoxanthine, optionally substituted by thioalkyl, wherein n is 0, 1,

2, 3 or 4, and wherein R⁴, R^4a and R^4b represent each independently O, NH, alkylene, monohaloalkylene or dihaloalkylene and R³, R⁵, R⁶, R⁸, R⁹ represent each independently OH or SH.

3. Use according to claim 1 or 2, wherein said purinergic or pyrimidinergic receptor is selected from the group comprising P2Y_L P2Y₂, P2Y₄, P2Y₆, P2Yn, P2Y₁₂ and P2Y₁₃ receptors and the UDP-glucose receptor.

4. Use according to any of claims 1 to 3, wherein said purinergic or pyrimidinergic receptor is the P2Yn receptor.

5. Use according to any of claims 1 to 3, wherein said purinergic or pyrimidinergic receptor is the P2Y₁₃ receptor.

6. Use according to any of claims 1 to 5, wherein said purinergic or pyrimidinergic receptor agonist of formula (1) is selected from the group comprising ATP, adenosine 5'-0-(3- thiotriphosphate) (ATPγS), 2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP), 2-methylthio-ATP (2-MeSATP) or a pharmaceutically acceptable salt thereof.

7. Use according to any of claims 1 to 5, wherein said purinergic or pyrimidinergic receptor agonist of formula (1 ) is uridine 5'-diphosphate (UDP), uridine 5'-triphosphate (UTP), ADP, 2-methylthio-ADP (2-MeSADP), adenosine 5'-0-(2-thiodiphosphate) (ADPβS), P¹,P³-di(adenosine-5')triphosphate (Ap₃A), P¹,P⁴-di(adenosine-5')tetraphosphate (Ap₄A), P ,P⁵-di(adenosine-5')pentaphosphate (ApsA), P ,P⁶-di(adenosine-5')hexaphosphate (Ap₆A) or a pharmaceutically acceptable salt thereof.

8. Use according to any of claims 1 to 7, wherein said purinergic or pyrimidinergic receptor agonist of formula (1) is selected from the group as depicted in tables A, B or C.

9. Method of inducing immunosuppression comprising administering to an individual in need thereof, a therapeutically effective amount of a purinergic or pyrimidinergic receptor agonist of formula (1),

or a pharmaceutically acceptable salt thereof, wherein B¹ is adenine or uracil, optionally substituted by thioalkyl, R and R² represent each independently OH, H, OR⁷, OCOR⁷, OCOR⁷COR⁷, wherein R⁷ represents alkyl or aryl and X is of formula (2), (3) or (4),

wherein R represents hydrogen or is of formula (7),

wherein B² is selected from the group comprising adenine, uracil, thymine, cytosine, guanine, xanthine and hypoxanthine, optionally substituted by thioalkyl, wherein n is 0, 1 , 2, 3 or 4, and wherein R⁴, R^4a and R^4b represent each independently O, NH, alkylene, monohaloalkylene or dihaloalkylene and R³, R⁵, R⁶, R⁸, R⁹ represent each independently OH or SH.

10. Method according to claim 9, wherein said immunosuppression is being induced in an individual suffering from asthma, including bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma, allergic rhinitis, eczema, atopic dermatitis, contact dermatitis, cutaneous T cell lymphoma (CTCL), Sezary syndrome, pemphigus vulgaris, bullous pemphigoid, pemphigus foliaceus, dermatomyositis, erythema nodosum, scleroderma, Bechet's disease, sarcoidosis, Sjogren's syndrome, rheumatoid arthritis, juvenile arthritis, Reiter's syndrome, lupus erythematosus, polymyositis, myocarditis, primary biliary cirrhosis, Crohn's disease, ulcerative colitis, multiple sclerosis or other demyelinating diseases, idiopathic thrombocytopenic purpura, Graves' disease or

Hashimoto's disease, Addison's disease, insulin-dependent diabetes mellitus (type 1 ), transplant rejection or graft-versus-host disease.

11. Method according to claim 9 or 10, wherein said purinergic or pyrimidinergic receptor agonist of formula (1 ) is selected from the group comprising ATP, adenosine 5'-0-(3- thiotriphosphate) (ATPγS), 2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP), 2-methylthio-ATP (2-MeSATP), uridine 5'-diphosphate (UDP) and uridine 5'- triphosphate (UTP), ADP, 2-methylthio-ADP (2-MeSADP), adenosine 5'-0-(2- thiodiphosphate) (ADPβS), P¹,P³-di(adenosine-5')triphosphate (ApsA), P¹,P⁴- di(adenosine-5')tetraphosphate (Ap₄A), P¹,P⁵-di(adenosine-5')pentaphosphate (Ap₅A),

P¹,P⁶-di(adenosine-5')hexaphosphate (Ap₆A) or a pharmaceutically acceptable salt thereof.

12. Pharmaceutical composition for the treatment of activated CD4⁺ T lymphocytes induced immune diseases comprising a pharmaceutically acceptable carrier and a purinergic or pyrimidinergic receptor agonist of formula (1)

or a pharmaceutically acceptable salt thereof, wherein B¹ is adenine or uracil, optionally substituted by thioalkyl, R¹ and R² represent each independently OH, H, OR⁷, OCOR⁷, OCOR⁷COR⁷, wherein R⁷ represents alkyl or aryl and X is of formula (2), (3) or (4),

wherein R represents hydrogen or is of formula (7),

13. Pharmaceutical composition according to claim 12, wherein said purinergic or pyrimidinergic receptor agonist of formula (1) is selected from the group comprising ATP, adenosine 5'-0-(3-thiotriphosphate) (ATPγS); 2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP), 2-methylthio-ATP (2-MeSATP), uridine 5'-diphosphate (UDP) and uridine 5'-triphosphate (UTP), ADP, 2-methylthio-ADP (2-MeSADP), adenosine 5'-0- (2-thiodiphosphate) (ADPβS), P¹,P³-di(adenosine-5')triphosphate (Ap₃A), P¹,P⁴- di(adenosine-5')tetraphosphate (Ap₄A), P¹,P⁵-di(adenosine-5')pentaphosphate (ApsA), P¹,P⁶-di(adenosine-5')hexaphosphate (Ap₆A) or a pharmaceutically acceptable salt thereof.

14. Use of a purinergic or pyrimidinergic receptor agonist of formula (1)

or a pharmaceutically acceptable salt thereof for the preparation of a medicine for the treatment of allergic airway disease, wherein B¹ is adenine or uracil, optionally substituted by thioalkyl, R¹ and R² represent each independently OH, H, OR⁷, OCOR⁷, OCOR⁷COR⁷, wherein R⁷ represents alkyl or aryl and X is of formula (2), (3) or (4),

wherein R¹⁰ represents hydrogen or is of formula (7),

wherein B² is selected from the group comprising adenine, uracil, thymine, cytosine, guanine, xanthine and hypoxanthine, optionally mono- or di-substituted by thioalkyl, wherein n is 0, 1 , 2, 3 or 4, and wherein R⁴, R^4a and R^4b represent each independently O,

NH, alkylene, monohaloalkylene or dihaloalkylene and R³, R⁵, R⁶, R⁸, R⁹ represent each independently OH or SH.

15. Use according to claim 14, wherein said allergic airway disease is asthma or allergic rhinitis.

16. Use of a purinergic or pyrimidinergic receptor agonist of formula (1)

or a pharmaceutically acceptable salt thereof for the preparation of a medicine for the treatment of skin disease, wherein B¹ is adenine or uracil, optionally substituted by thioalkyl, R¹ and R² represent each independently OH, H, OR⁷, OCOR⁷, OCOR⁷COR⁷, wherein R⁷ represents alkyl or aryl and X is of formula (2), (3) or (4),

wherein R is of formula (7),

wherein B² is selected from the group comprising adenine, uracil, thymine, cytosine, guanine, xanthine and hypoxanthine, optionally mono- or di-substituted by thioalkyl, wherein n is 0, 1 , 2, 3 or 4, and wherein R⁴, R^4a and R^4b represent each independently O, NH, alkylene, monohaloalkylene or dihaloalkylene and R³, R⁵, R⁶, R⁸, R⁹ represent each independently OH or SH.

17. Use according to claim 16, wherein said skin disease is selected from the group of diseases comprising psoriasis, eczema, atopic dermatitis, contact dermatitis, bullous pemphigoid, pemphigus foliaceus, dermatomyositis, lupus erythematosus.

18. Use of a purinergic or pyrimidinergic receptor agonist of formula (1)

or a pharmaceutically acceptable salt thereof for the preparation of a medicine for treatment of inflammatory bowel disease, wherein B¹ is adenine or uracil, optionally substituted by thioalkyl, R¹ and R² represent each independently OH, H, OR⁷, OCOR⁷, OCOR⁷COR⁷, wherein R⁷ represents alkyl or aryl and X is of formula (2), (3) or (4),

R¹⁰-O- 9P— R ₁ ¹ ₀ ⁰-O- AP-R⁴- AP— Rⁱ° θ- 9lj>-R⁴- 9P-R^4a- 9P-

(2) R³ (³) R⁵ R³ (4) R⁶ R⁵ R³

wherein R¹⁰ represents hydrogen or is of formula (7),

19. Use according to claim 17, wherein said inflammatory bowel disease is Crohn's disease or ulcerative colitis.

20. Use according to any of claims 13 to 18, wherein said purinergic or pyrimidinergic receptor agonist of formula (1 ) is selected from the group comprising ATP, adenosine 5'-

0-(3-thiotriphosphate) (ATPγS), 2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'- triphosphate (BzATP), 2-methylthio-ATP (2-MeSATP), uridine 5'-diphosphate (UDP), uridine 5'-triphosphate (UTP), ADP, 2-methylthio-ADP (2-MeSADP), adenosine 5'-0-(2- thiodiphosphate) (ADPβS), P ,P³-di(adenosine-5')triphosphate (Ap₃A), P ,P⁴- di(adenosine-5')tetraphosphate (Ap₄A), P¹,P⁵-di(adenosine-5')pentaphosphate (Ap₅A),