WO2003028712A2 - Agonistes de recepteur purinergique et pyrimidinergique pour le traitement de maladies immunitaires mediees par le lymphocyte t cd4+ active - Google Patents

Agonistes de recepteur purinergique et pyrimidinergique pour le traitement de maladies immunitaires mediees par le lymphocyte t cd4+ active Download PDF

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WO2003028712A2
WO2003028712A2 PCT/EP2002/010954 EP0210954W WO03028712A2 WO 2003028712 A2 WO2003028712 A2 WO 2003028712A2 EP 0210954 W EP0210954 W EP 0210954W WO 03028712 A2 WO03028712 A2 WO 03028712A2
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adenosine
formula
represent
independently
purinergic
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PCT/EP2002/010954
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WO2003028712A3 (fr
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Liliane Schandene
Michel Goldman
Xavier Duhant
Jean-Marie Boeynaems
Didier Communi
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Universite Libre De Bruxelles
Euroscreen S.A.
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Publication of WO2003028712A2 publication Critical patent/WO2003028712A2/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/06—Anti-spasmodics, e.g. drugs for colics, esophagic dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • 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.
  • 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.
  • 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.
  • nucleotide concentrating granules synaptic vesicles, platelet dense bodies
  • efflux from cytoplasm through membrane transport proteins.
  • 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.
  • UDP uridine 5'-diphosphate
  • CDP cytidine 5'-diphosphate
  • ADP adenosine 5'-diphosphate
  • 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.
  • 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.
  • the present invention relates to the use of purinergic and pyrimidinergic receptor agonists of formula (1 )
  • B 1 is selected from the group comprising adenine, uracil, thymine, cytosine, guanine, xanthine and hypoxanthine, optionally substituted by thioalkyl
  • R 1 and R 2 represent each independently OH, H, OR 7 , OCOR 7 , OCOR 7 COR 7 , wherein R 7 represents alkyl or aryl and X is of formula (2), (3) or (4),
  • R represents hydrogen or is of formula (7)
  • B 2 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 R 4 , R 4a and R 4 represent each independently O, NH, alkylene, monohaloalkylene or dihaloalkylene and R 3 , R 5 , R 6 , R 8 , R 9 represent each independently OH or SH, and wherein R 11 and R 12 represent each independently OH, H, OR 7 , OCOR 7 , OCOR 7 COR 7 , wherein R 7 is as defined above.
  • B 1 in the above formula (1) is adenine or uracil, optionally substituted by thioalkyl.
  • B 2 preferably is adenine, uracil, cytosine or guanine.
  • B 2 is adenine in the formula (7) and n is 1 , 2, 3 or 4.
  • said purinergic and pyrimidinergic receptor agonists of formula (1) are useful as immunosuppressive agents.
  • said purinergic or pyrimidinergic receptor agonist is of formula (5), (6) or (8) or a pharmaceutically acceptable salt thereof,
  • said purinergic or pyrimidinergic receptor agonist is of formula (9), (10), (11) or (12) or a pharmaceutically acceptable salt thereof,
  • each R 8 has independently from the other R 8 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 B 1 and B 2 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, (ATP ⁇ SBzATP2-MeSATP; (ii) UDP and UTP; (iii) ADP and derivatives (ADP ⁇ S, 2-MeSADP); (iv) dinucleotides.
  • said purinergic and pyrimidinergic receptor agonists of formula (1 ) are adenosine 5'-triphosphate (ATP), adenosine 5'-0-(3-thiotriphosphate) (ATP ⁇ S), 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) (ADP ⁇ S), P 1 ,P 3 -di(adenosine-5')triphosphate (Ap 3 A), P 1 ,P 4 -di(adenosine-5')tetraphosphate (Ap 4 A), P ,P 5 -di(adeno),
  • 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.
  • 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.
  • 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.
  • 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,
  • 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 ),
  • B is B 1 is selected from the group comprising adenine, uracil, thymine, cytosine, guanine, xanthine and hypoxanthine, preferably selected from adenine or uracil, and B 1 optionally substituted by thioalkyl
  • R 1 and R 2 represent each independently OH, H, OR 7 , OCOR 7 , OCOR 7 COR 7 , wherein R 7 represents alkyl or aryl and X is of formula (2), (3) or (4),
  • R represents hydrogen or is of formula (7)
  • B 2 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 R 4 , R 4a and R 4 represent each independently O, NH, alkylene, monohaloalkylene or dihaloalkylene and R 3 , R 5 , R 6 , R 8 , R 9 represent each independently OH or SH, and wherein R 11 and R 12 represent each independently OH, H, OR 7 , OCOR 7 , OCOR 7 COR 7 , wherein R 7 is as defined above, and wherein each R 4 or R 8 independently from the other R 4b or R 8 substituents has an individual meaning and can change in each formula, as for instance in formula (7b) :
  • B 1 in the above formula (1) is adenine or uracil, optionally substituted by thioalkyl.
  • B 2 preferably is adenine, uracil, cytosine or guanine, optionally substituted by thioalkyl.
  • B 2 is adenine, optionally substituted by thioalkyl, in the formula (7) and n is 1 , 2, 3 or 4.
  • X is of formula (2), (3) or (4) and R 10 is of formula (7), the linkage between the nucleotides is from 5'->5' (e.g.
  • the present invention also relates to compounds wherein the linkage between the nucleotides is 2' ⁇ 5' or 3'->5'.
  • 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, cytidyl (3'-5') cytidine, cytidylyl(2'
  • said purinergic or pyrimidinergic receptor agonist is of formula (5), (6) or (8) or a pharmaceutically acceptable salt thereof,
  • R 1 , R 2 and X have the same meaning as that defined above.
  • said purinergic or pyrimidinergic receptor agonist is of formula (9), (10), (11) or (12) or a pharmaceutically acceptable salt thereof,
  • B 1 , B 2 , R 1 , R 2 , R 4b , R 8 , R 9 , R 11 and R 12 have the same meaning as that defined above.
  • interesting compounds are those compounds of formula (9), (10), (11) or (12), wherein B 1 and B 2 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 B 1 and B 2 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 B 1 and B 2 are different. Non-limiting examples of such compounds are depicted in Table C.
  • alkyl 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.
  • aryl as used herein, includes a monovalent organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl.
  • alkylene 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.
  • 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.
  • adenosine 5'-triphosphate ATP
  • adenosine 5'-0-(3- thiotriphosphate) ATP ⁇ S
  • 2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate BzATP
  • uridine 5'-diphosphate UDP
  • uridine 5'-triphosphate UDP
  • UDP uridine 5'-triphosphate
  • UDP uridine 5'-triphosphate
  • UDP uridine 5'-triphosphate
  • UDP uridine 5'-triphosphate
  • UDP uridine 5'-triphosphate
  • UDP uridine 5'-triphosphate
  • UDP uridine 5'-triphosphate
  • UDP uridine 5'-triphosphate
  • UDP uridine 5'-triphosphate
  • UDP uridine 5'-triphosphate
  • UDP uridine 5'
  • 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.
  • 8 genuine human P2Y receptors have been identified and characterized: P2Y 1 f P2Y 2 , P2Y 4 , P2Y 6 , P2Yn, P2Y 12 and P2Y 13 , as well as the uridine 5'-diphosphoglucose (UDP-glucose) receptor which is structurally related to P2Y 12 .
  • UDP-glucose uridine 5'-diphosphoglucose
  • said purinergic or pyrimidinergic receptor useful for the present invention is selected from the group comprising P2Y 1 f P2Y 2 , P2Y 4 , P2Y 6 , P2Yn, P2Y 12 and P2Y 13 receptors and UDP-glucose receptor.
  • said P2Y receptor is the P2Yn receptor.
  • said P2Y receptor is the P2Y 13 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- ⁇ ) involved in both Th1 and Th2 responses was significantly inhibited in the presence of nucleotides.
  • 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 [ 3 H]thymidine uptake test.
  • cAMP accumulation was observed in response to ATP and ATP derivatives, ATP ⁇ S and BzATP.
  • the immunosuppressive effects on both Th1 and Th2 responses, are mediated by cAMP.
  • 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- ⁇ ), 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, ADP ⁇ S and 2-MeSADP and the dinucleotides Ap 3 A, Ap 4 A, Ap 5 A and Ap 6 A , 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.
  • 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.
  • inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like
  • organic acids such as acetic, propionic, succinic, glycolic, stea
  • 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.
  • 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.
  • the present invention relates to methods for suppressing inflammation in an individual by suppressing the immune reaction.
  • immunosuppression and inflammation are both suppressed in said individual.
  • T cell mediated immune response 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- ⁇ .
  • individual refers to a mammal. The individual will preferably be a human, but may also be a domestic livestock, laboratory or pet animals.
  • 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.
  • said immunosuppression is being induced in an individual suffering from asthma including bronchial asthma, allergic asthma, intrinsic asthma and dust asthma.
  • said immunosuppression is being induced in an individual suffering from allergic rhinitis.
  • said immunosuppression is being induced in an individual suffering from conjunctivitis.
  • said immunosuppression is being induced in an individual suffering from keratitis.
  • said immunosuppression is being induced in an individual suffering from keratoconjunctivitis.
  • said immunosuppression is being induced in an individual suffering from uveitis.
  • said immunosuppression is being induced in an individual suffering from psoriasis.
  • said immunosuppression is being induced in an individual suffering from eczema.
  • said immunosuppression is being induced in an individual suffering from atopic dermatitis.
  • said immunosuppression is being induced in an individual suffering from contact dermatitis.
  • said immunosuppression is being induced in an individual suffering from cutaneous T cell lymphoma (CTCL).
  • CTCL cutaneous T cell lymphoma
  • said immunosuppression is being induced in an individual suffering from Sezary syndrome.
  • said immunosuppression is being induced in an individual suffering from pemphigus vulgaris.
  • said immunosuppression is being induced in an individual suffering from bullous pemphigoid.
  • said immunosuppression is being induced in an individual suffering from pemphigus foliaceus.
  • said immunosuppression is being induced in an individual suffering from dermatomyositis.
  • said immunosuppression is being induced in an individual suffering from erythema nodosum.
  • said immunosuppression is being induced in an individual suffering from scleroderma.
  • said immunosuppression is being induced in an individual suffering from Bechet's disease.
  • said immunosuppression is being induced in an individual suffering from sarcoidosis.
  • said immunosuppression is being induced in an individual suffering from Sjogren's syndrome.
  • said immunosuppression is being induced in an individual suffering from rheumatoid arthritis.
  • said immunosuppression is being induced in an individual suffering from juvenile arthritis.
  • said immunosuppression is being induced in an individual suffering from Reiter's syndrome.
  • said immunosuppression is being induced in an individual suffering from lupus erythematosus.
  • said immunosuppression is being induced in an individual suffering from polymyositis.
  • said immunosuppression is being induced in an individual suffering from myocarditis.
  • said immunosuppression is being induced in an individual suffering from primary biliary cirrhosis.
  • said immunosuppression is being induced in an individual suffering from Crohn's disease.
  • said immunosuppression is being induced in an individual suffering from ulcerative colitis.
  • said immunosuppression is being induced in an individual suffering from multiple sclerosis and other demyelinating diseases.
  • said immunosuppression is being induced in an individual suffering from idiopathic thrombocytopenic purpura.
  • said immunosuppression is being induced in an individual suffering from Graves' disease and Hashimoto's disease.
  • said immunosuppression is being induced in an individual suffering from Addison's disease.
  • said immunosuppression is being induced in an individual suffering from insulin-dependent diabetes mellitus (type 1 ).
  • 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.
  • said purinergic or pyrimidinergic receptor agonist of formula (1) is a P2Yn or a P2Y 13 receptor agonist, and is selected from the group comprising ATP, adenosine 5'-0-(3-thiotriphosphate) (ATP ⁇ S), 2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP) and 2-methylthio-ATP (2-MeSATP).
  • said purinergic and pyrimidinergic receptor agonist is ATP, adenosine 5'-0-(3-thiotriphosphate) (ATP ⁇ S) or 2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP).
  • 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) (ADP ⁇ S), P 1 ,P 3 -di(adenosine-5')triphosphate (Ap 3 A), P ,P 4 -di(adenosine-5')tetraphosphate (Ap 4 A), P 1 ,P 5 -di(adenosine-5')pentaphosphate (Ap 5 A), P 1 ,P 6 -di(adenosine-5')hexaphosphate (Ap 6 A), or a pharmaceutically acceptable salt of any of the aforementioned nucleotide derivatives.
  • UDP uridine 5'- diphosphate
  • UDP uridine 5'-triphosphate
  • ADP 2-methylthi
  • 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 P2Y 13 receptor agonist, and is selected from the group comprising ATP, adenosine 5'-0-(3-thiotriphosphate) (ATP ⁇ S), 2'- and 3'-0-(4-benzoyl- benzoyl) adenosine 5'-triphosphate (BzATP), 2-methylthio-ATP (2-MeSATP) or a pharmaceutically acceptable salt thereof.
  • said P2Y receptor agonist is ATP, adenosine 5'-0-(3-thiotriphosphate) (ATP ⁇ S), 2'- or 3'-0-(4-benzoyl-benzoyl) adenosine 5'- triphosphate (BzATP).
  • uridine 5'-diphosphate UDP
  • uridine 5'-triphosphate UDP
  • adenosine 5'- diphosphate ADP
  • 2-methylthio-ADP 2-MeSADP
  • adenosine 5'-0-(2-thiodiphosphate) ADP ⁇ S
  • P 1 ,P 3 -di(adenosine-5')triphosphate Ap 3 A
  • P 1 ,P 4 -di(adenosine-5')tetraphosphate Ap 4 A
  • P ,P 5 -di(adenosine-5')pentaphosphate ApsA
  • P 1 ,P 6 -di(adenosine-5')hexaphosphate Ap 6 A
  • a pharmaceutically acceptable salt of any of the aforementioned nucleotide derivatives A.
  • 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
  • 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.
  • 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.
  • 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,
  • 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 )
  • B 1 is adenine or uracil, optionally substituted by thioalkyl
  • R 1 and R 2 represent each independently OH, H, OR 7 , OCOR 7 , OCOR 7 COR 7 , wherein R 7 represents alkyl or aryl and X is of formula (2), (3) or (4),
  • R 10 is of formula (7)
  • B 2 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 R 4 , R 4a and R 4 represent each independently O, NH, alkylene, monohaloalkylene or dihaloalkylene and R 3 , R 5 , R 6 , R 8 , R 9 represent each independently OH or SH.
  • R 4 , R 4a and R 4b represent each independently O, NH, alkylene, monohaloalkylene or dihaloalkylene and R 3 , R 5 , R 6 , R 8 , R 9 represent each independently OH or SH, and wherein R 11 and R 12 represent each independently OH, H, OR 7 , OCOR 7 , OCOR 7 COR 7 , wherein R 7 is as defined above, and wherein each R4b or R8 independently from the other R 4b or R 8 substituents has an individual meaning and can change in each formula, for instance as in formula (7b).
  • said skin disease is psoriasis.
  • said skin disease is eczema.
  • said skin disease is atopic dermatitis.
  • said skin disease is contact dermatitis.
  • said skin disease is bullous pemphigoid.
  • said skin disease is pemphigus foliaceus.
  • said skin disease is dermatomyositis.
  • said skin disease is lupus erythematosus.
  • B 1 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.
  • B 2 in the above formula (7) of the receptor agonist for treating skin disease or for the preparation of a medicine for treating skin diseases, B 2 preferably is adenine, uracil, cytosine or guanine, optionally substituted with thioalkyl.
  • B 2 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.
  • said inflammatory bowel disease is Crohn's disease.
  • 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 P2Y 13 receptor agonist, and is selected from the group comprising ATP, adenosine 5'-O-(3- thiotriphosphate) (ATP ⁇ S), 2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP), 2-methylthio-ATP (2-MeSATP) or a pharmaceutically acceptable salt thereof.
  • said purinergic and pyrimidinergic receptor agonist is ATP, adenosine 5'-O-(3-thiotriphosphate) (ATP ⁇ S), 2'- and 3'-0-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP) or a pharmaceutically acceptable salt thereof.
  • 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) (ADP ⁇ S), P 1 ,P 3 - di(adenosine-5')triphosphate (Ap 3 A), P 1 ,P 4 -di(adenosine-5')tetraphosphate (Ap 4 A), P 1 ,P 5 - di(adenosine-5')pentaphosphate (ApsA), P 1 ,P 6 -di(adenosine-5')hexaphosphate (Ap 6 A), or a pharmaceutically acceptable salt thereof.
  • UDP uridine 5'-diphosphate
  • UDP uridine 5'-triphosphate
  • Non limiting examples of purinergic and pyrimidinergic receptor agonists of formula (1) useful according to the invention are listed Tables A, B and C.
  • 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 ATP ⁇ S and BzATP effect on IL-2 (a), IFN- ⁇ (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- ⁇ (d) secretion from activated human CD4 + T cells
  • Figure 6 is a graph that compares the concentration-dependent inhibition by Ap 4 A and ATP ⁇ S of IFN- ⁇ (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 ⁇ M).
  • ATP adenosine 5'-0-(3-thiotriphosphate) (ATP ⁇ S), 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) (ADP ⁇ S), P 1 ,P 3 -di(adenosine-5')triphosphate (Ap 3 A), P 1 ,P 4 -di(adenosine-5')tetraphosphate (Ap 4 A), P 1 ,P 5 -di(adenosine-5')pentaphosphate (ApsA), P 1 ,P 6 -di(adenosine-5')hexaphosphate (Ap 6 A), and indom
  • PBMC Peripheral blood mononuclear cells
  • 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 ⁇ g ml -1 gentamicin and 50 ⁇ M 2- ⁇ -mercaptoethanol at 37 °C in 5 % C02.
  • FCS heat-inactivated fetal bovine serum
  • the CD4 + T cells (1 X 10 5 /well) were activated in flat-bottomed 96-well plates pre-coated with the anti-CD3 mAb (10 ⁇ g ml "1 ) in the presence of soluble anti-CD28 mAb (1 ⁇ g 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 [ 3 H]thymidine (0.5 ⁇ Ci/well) uptake during the following 16 hours. Each experimental condition was tested in triplicate.
  • CD4 + T cells 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.
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • PerCP piridinin-chlorophyll-a-protein
  • 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
  • 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.
  • nucleotides on cAMP were 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 ⁇ M) 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.
  • Freshly purified or activated cells were pre-incubated with 8-p-SPT (300 ⁇ M) or Indo (5 ⁇ g ml "1 ) for 15 min, both with rolipram (25 ⁇ M), then incubated with ATP ⁇ S (100 ⁇ M), adenosine (100 ⁇ M) 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 ⁇ M) or the A 2A receptor agonist CGS-21680 (100 nM), but only in activated cells (Figure. 1c).
  • the freshly purified cells were incubated with pre-coated anti-CD3 and soluble anti-CD28 mAbs (10 6 cells/ml), and various concentrations of nucleotides. After 24 h (IL-2), 72 h (IFN- ⁇ 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.
  • both ATP ⁇ S 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.
  • the inhibition of IL-2 and IL-10 production by CD4 + T cells was not modified by the addition of indomethacin (5 ⁇ g 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 ).
  • UDP and UTP also inhibited the release of the four cytokines in a concentration-dependent way.
  • 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 AT ⁇ S 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 ATP ⁇ S or BzATP (100 ⁇ M). 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.
  • the table shows the mean inhibitory effect (in %) of various nucleotides on the secretion of IFN- ⁇ by CD4 + T cells activated by the combination of pre-coated anti-CD3 and soluble anti-CD28 mAbs.
  • 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 [ 3 H]thymidine (0.5 ⁇ Ci/well). Data are given as the mean percentage of control [ 3 H]thymidine incorporation. Data represent the mean ⁇ S.D. of triplicate experimental points obtained in one representative experiment of three.
  • ATP ⁇ S and BzATP added at concentrations ranging from 1 to 100 ⁇ M, inhibited the proliferation of CD4 + T cells activated by immobilized anti-CD3 and soluble anti- CD28 mAb as assessed by [ 3 H]thymidine incorporation.
  • An average of 41 ⁇ 13 and 46 ⁇ 7 (mean ⁇ S.D.) percentage inhibition of CD4 + T cell proliferation were observed with ATP ⁇ S and BzATP (both 100 ⁇ M) respectively.
  • the decrease in proliferation was not reversed by the simultaneous addition of 8-p-SPT (data not shown).
  • Example 4 ATP analogues down-regulate the expression of CD25 during CD4 + T cell activation
  • 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.
  • IL-2 receptor (CD25) expression was down-regulated: these two effects contribute to the inhibition of proliferation, assessed with the classical [ 3 H]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- ⁇ ). Other nucleotides were also able to inhibit the release of cytokines (IFN- ⁇ and 11-10) by CD4+ T cells, as well as their proliferation: ADP, ADP ⁇ S, 2-MeSADP, Ap 3 A, Ap 4 A, ApsA and Ap 6 A. They did not act via an increase in camp.
  • 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.
  • skin graft rejection herpes simplex keratitis
  • cardiac graft rejection 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 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- ⁇ , IL-4, IL-5).

Abstract

L'invention concerne l'utilisation d'agonistes de récepteur purinergique et pyrimidinergique de formule (I) ou un sel pharmaceutiquement acceptable de ceux-ci pour l'inhibition de l'activation de lymphocytes T CD4+, ou en tant qu'agent immunosuppresseur. B1 représente adénine ou uracile, éventuellement substituée par thioalkyle, R1 et R2 représentent chacun indépendamment OH, H, OR7, OCOR7, OCOR7COR7, R7 représente alkyle ou aryle et X est représenté par la formule (2), (3) ou (4). R10 représente hydrogène ou est de formule (7). B2 est sélectionné dans le groupe comprenant adénine, uracile, thymine, cytosine, guanine, xanthine et hypoxanthine, éventuellement substituées par thioalkyle, n représente 0, 1, 2, 3 ou 4, et R4, R4a et R4b représentent chacun indépendamment O, NH, alkylène, monohaloalkylène ou dihaloalkylène et R3, R5, R6, R8, R9 représentent chacun indépendamment OH ou SH, et R11 et R12 représentent chacun indépendamment OH, H, OR7, OCOR7, OCOR7COR7. R7 est défini comme ci-dessus.
PCT/EP2002/010954 2001-09-28 2002-09-30 Agonistes de recepteur purinergique et pyrimidinergique pour le traitement de maladies immunitaires mediees par le lymphocyte t cd4+ active WO2003028712A2 (fr)

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WO2006097547A1 (fr) * 2005-03-17 2006-09-21 Proyecto De Biomedicina Cima S.L. Emploi de 5'-methylthioadenosine (mta) dans la prevention et/ou le traitement de maladies auto-immunes et/ou du rejet de greffons
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WO2006038865A1 (fr) * 2004-10-01 2006-04-13 Betagenon Ab Derives de nucleotides servant a traiter le diabete de type 2 ou d'autres maladies
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