WO1995011681A1 - Antagonistes du recepteur de l'adenosine humaine - Google Patents

Antagonistes du recepteur de l'adenosine humaine Download PDF

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WO1995011681A1
WO1995011681A1 PCT/US1994/012272 US9412272W WO9511681A1 WO 1995011681 A1 WO1995011681 A1 WO 1995011681A1 US 9412272 W US9412272 W US 9412272W WO 9511681 A1 WO9511681 A1 WO 9511681A1
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adenosine
receptor
adenosine receptor
human
xanthine
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PCT/US1994/012272
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Michael P. Doyle
Marlene A. Jacobson
Brian R. Duling
Robert G. Johnson
Joel M. Linden
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Merck & Co., Inc.
The University Of Virginia Patents Foundation
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Publication of WO1995011681A1 publication Critical patent/WO1995011681A1/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
    • 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
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate

Definitions

  • the present invention concerns the use of compounds identified as specific modulators of adenosine's physiological actions.
  • the pharmacology of these compounds is characterized through the use of cloned human adenosine receptors of the Al, A2a, A2b and A3 class and their subtypes.
  • Compounds identified as antagonists of the A3 adenosine receptor subtype are useful in preventing mast cell degranulation and are therefore useful in the treatment or prevention of disease states induced by activation of the A3 receptor and mast cell activation.
  • These disease states include but are not limited to asthma, myocardial reperfusion injury, allergic reactions including but not limited to rhinitis, poison ivy induced responses, urticaria, scleroderma, arthritis, other autoimmune diseases and inflammatory bowel diseases.
  • Adenosine is a naturally occurring nucleoside which exhibits diverse and potent physiological actions in the cardiovascular, nervous, pulmonary, renal and immune systems. Adenosine has been demonstrated to terminate superventricular tachycardia through blockage of atrioventricular nodal conduction (J.P. DiMarco, et al., (1985) J. Am. Col. Cardiol. 6:417-425, A. Munoz, et al., (1984) Eur. Heart J. 5:735-738). Adenosine is a potent vasodilator except in the kidney and placenta (R.A. Olsson, (1981 ) Ann. Rev. Physiol. 43:385- 395).
  • Adenosine produces bronchoconstriction in asthmatics but not in nonasthmatics (Cushly et al., 1984, Am. Rev. Respir. Dis. 129:380- 384). Adenosine has been implicated as a preventative agent and in treatment of ventricular dysfunction following episodes of regional or global ischemia (M.B. Forman and C.E. Velasco (1991) Cardiovasc. Drugs and Therapy 5:901-908) and in cerebral ischemia(M.C. Evans, et al., (1987) Neurosci. Lett. 83:287, D.KJ.E.,Von Lubitz, et al., (1988) Stroke 19: 1133).
  • Dog Al and A2a adenosine receptors were the first adenosine receptors to be cloned. See F. Libert, et al., (1989) Science 244:569-572, C. Maennant, et al., Biochem. Biophys. Res. Comm.,
  • the human Al adenosine receptor differs by 18 amino acids from the dog A 1 sequence and 16 amino acids from the rat Al sequence.
  • the human A2a adenosine receptor differs by 28 and 71 amino acids, respectively from the dog and rat A2a sequences.
  • the amino acid sequence for the human A3 receptor is 72% identical with the rat A3 receptor and 85% identical with the sheep A3 receptor sequences.
  • adenosine The actions of adenosine are mediated through G-protein coupled receptors, the Al , A2a, A2b and A3 adenosine receptors.
  • the adenosine receptors were initially classified into Al and A2 subtypes on the basis of pharmacological criteria and coupling to adenylate cyclase (Van Caulker. D.. Muller, M. and Hamprecht, B. (1979) J. Neurochem. 33, 999- 1003.).
  • a fourth subtype, A3, had remained pharmacologically undetected until its recent identification by molecular cloning.
  • the rat A3 sequence, tgpcrl was first cloned from rat testis by Meyerhoff et al. (see above). Subsequently, a cDN A encoding the identical receptor was cloned from striatum and functionally expressed by Zhou et al. (see above). When compared to the other members of the G-protein coupled receptor family, the rat sequence had the highest homology with the adenosine receptors (> 40% overall identity, 58% within the transmembrane regions).
  • the rat A3 receptor exhibited a unique pharmacology relative to the Al and A2 adenosine receptor subtypes and was reported not to bind the xanthine antagonists 1 ,3-dipropyl-8-phenylxanthine (DPCPX) and xanthine amine congener (XAC).
  • DPCPX ,3-dipropyl-8-phenylxanthine
  • XAC xanthine amine congener
  • the sheep homolog of the A3 receptor was cloned from hypophysial pars tuberalis (see Linden et al. above).
  • the sheep receptor is 72% identical to the rat receptor, binds the radioligand 125l-ABA and is also coupled to inhibition of cyclic AMP.
  • the agonist affinity order of the sheep receptor is I-ABA > APNEA> NECA > R-PIA » CPA.
  • the pharmacology of xanthine antagonists was extensively studied and the sheep receptor was found to exhibit high affinity for 8- phenylxanthines with para-acidic substitutions.
  • the expression of the sheep A3 adenosine receptor transcript is widespread throughout the brain and is most abundant in the lung and spleen.
  • Adenosine receptor agonists, antagonists and binding enhancers have been identified and implicated for usage in the treatment of physiological complications resulting from cardiovascular, pulmonary, renal and neurological disorders.
  • Adenosine receptor agonists have been identified for use as vasodilators ((1989) FASEB. J. 3(4) Abs 4770 and 4773, (19910 J. Med. Chem. (1988) 34:2570), antihypertensive agents (D.G. Taylor et al., FASEB J. (1988) 2:1799), and anti -psychotic agents (T.G. Heffner et al., (1989) Psychopharmacology 98:31 -38).
  • Adenosine receptor agonists have been identified for use in improving renal function (R.D. Murray and P.C. Churchill,(1985) J. Pharmacol. Exp. Therap. 232:189-193) .
  • Adenosine receptor allosteric or binding enhancers have shown utility in the treatment of ischemia, seizures or hypoxia of the brain (R.F. Bruns, et al. (1990) Mol. Pharmacol. 38:939-949; CA. Janusz. et al., (1991) Brain Research 567:181-187).
  • the cardioprotective agent, 5-amino-4- imidazole carboxamide (AICA) ribose has utility in the treatment of ischemic heart conditions, including unstable angina and acute myocardial infarction (H.E. Gruber, et al. (1989) Circulation 80: 1400- 1414).
  • 8-phenylxanthines methods of their synthesis and their use in human and veterinary therapy for conditions associated with the cell surface effects of adenosine have been described (EP 0 203 721 , published 12/3/86). However, this publication is silent as to adenosine receptor subtypes and subtype specificity of disclosed compounds. In WO 90/00056, a group of 1,3 -unsymmetrical straight chain alkyl- substituted 8-phenylxanthines were described as being potent bronchodilators. This disclosure is likewise silent as to the subtype specificity of disclosed compounds.
  • Figure 1 Full length amino acid sequence of human Al adenosine receptor.
  • Figure 2 A-B Full length nucleotide sequence of the cloned human Al adenosine receptor complementary DNA depicted from the 5' to 3' terminus.
  • Figure 3 Full length amino acid sequence of human A2a adenosine receptor.
  • Figure 4A-B Full length nucleotide sequence of cloned human A2a adenosine receptor complementary DNA depicted from the 5' to 3' terminus.
  • Figure 5 Full length amino acid sequence of human A2b receptor.
  • Figure 6 A-B Full length nucleotide sequence of cloned human A2b adenosine receptor complementary DNA depicted from the 5' to 3' terminus.
  • Figure 7 Saturation binding of [ ⁇ H]-cyclohexyladenosine (CHA) to human A 1 adenosine receptor in COS7 assay.
  • Figure 8 Saturation binding of [ 3 H]-CGS21680 to human A2a adenosine receptor in COS7 assay.
  • Figure 9 Full length amino acid sequence of human A3 adenosine receptor.
  • Figure 10A-B Full length nucleotide sequence of the cloned human A3 adenosine receptor complementary DNA depicted from the 5' to 3' terminus.
  • Figure 11 (A) Equilibrium binding of 125 I-ABA to membranes prepared from A3 stable transfected CHO cells shows specific (•) and nonspecific (o) binding. Nonspecific binding was measured in the presence of 1 ⁇ M I- ABA.
  • Figure 12A-B Competition by agonists and antagonists for 125 I-ABA binding to membranes prepared from stably transfected CHO cells expressing the human A3 adenosine receptor.
  • Agonists top panel
  • ( • ) NECA, (o) R-PIA, ( • ) CPA, ( ) S-PIA; antagonists bottom panel
  • Figure 13A-F Competition by antagonists of NECA-inhibited cyclic AMP accumulation in CHO cells stably expressing the human A3 adenosine receptor.
  • Dose response curves to NECA measured in the absence or presence of two concentrations of BW-A1433, XAC and I-ABOPX.
  • Figure 14 A-B Northern blot analysis of the four human adenosine receptor subtypes.
  • A 5 ⁇ g poly(A)+ RNA from various human tissues probed with HS-21a. The two blots shown were transferred, hybridized and exposed separately.
  • B 7.5 ⁇ g poly(A) + RNA from various human tissues probed with either A 1 , A2a, or A2b. Each blot was transferred and exposed separately.
  • Figure 16 An image showing the abluminal surface of an arteriole
  • SUBSTITUTE SHEET (RULE 2fl after staining with methylene blue, clearly labeling adherent mast cells, some of which are degranulated from prior exposure to adenosine.
  • Figure 17 Change in microvessel diameter with A3 agonist.
  • Figure 20 Abolition of agonist induced contractile response by pre- treatment with an adenosine A3 receptor specific antagonist.
  • the invention concerns the use of compounds, identified through the use of recombinant human adenosine receptors Al , A2a, A2b and A3, and functional assays, to specifically modulate the physiologic role of adenosine activation of its various receptors.
  • the human A3 adenosine receptor was cloned from a striatal cDNA library using a probe derived from the homologous rat sequence.
  • the cDNA encodes for a protein of 318 amino acids and exhibits 72% and 85% overall identity with the rat and sheep A3 adenosine receptor sequences, respectively.
  • adenosine receptor agonists were determined to be N- ethylcarboxamidoadenosine (NECA) > R-phenylisopropyladenosine (R- PIA) > N ⁇ -cyclopentyladenosine (CPA) > S-phenylisopropyladenosine (S-PIA).
  • NECA N- ethylcarboxamidoadenosine
  • R- PIA R-phenylisopropyladenosine
  • CCA N ⁇ -cyclopentyladenosine
  • S-PIA S-phenylisopropyladenosine
  • a partial listing of the pharmacology is that the potency order of antagonists is I-ABOPX > 1 ,3-dipropyl-8-(4-acrylate) phenylxanthine (BW-A1433) > xanthine amino cogener (XAC) » 1,3- dipropyl-8-cyclopentylxanthine (DPCPX).
  • Adenosine, NECA, R- and S- PIA and CPA inhibited forskolin-stimulated cAMP accumulation by 30- 40% in the stably transfected CHO cells; I- ABA is a partial agonist.
  • the dose response curves of NECA-induced inhibition of forskolin-stimulated cAMP accumulation were right-shifted.
  • Antagonist potencies determined by Schild analyses correlated well with those established by competition for radioligand binding.
  • the tissue distribution of transcripts for all of the human adenosine receptor subtypes was compared.
  • the A3 adenosine receptor transcript is widespread, and in contrast to the Al , A2a and A2b transcripts, the most abundant expression is found in the lung and liver.
  • the rat A3 adenosine receptor transcript is primarily expressed in testis and the sheep transcript is most abundant in the lung, spleen and pineal.
  • the human tissue distribution of A3 mRNA is more similar to the widespread profile found in sheep than to the restricted profile found in the rat. Numerous physiological effects of adenosine may be mediated by A3 adenosine receptors in man.
  • This invention provides a method for achieving specific blockade of the A3 subtype of the adenosine receptor.
  • adenosine, adenosine metabolites and other A3 adenosine receptor agonists induce mast cell degranulation in an animal model and that this can be prevented by selective antagonists of the A3 receptor.
  • the release of enzymes, bioactive amines and arachidonic acid metabolites following mast cell activation causes vasoconstriction, edema, leukocyte accumulation, and ultimately, tissue damage.
  • Mast cell degranulation is a component of: myocardial reperfusion injuryu, hypersensitivity reactions (asthma, allergic rhinitis, and urticaria), ischemic bowel disease, autoimmune inflammationa, and atopic dermatitis.
  • the invention consists of the use of any of a series of highly specific A3 adenosine receptor antagonists to treat or prevent these diseases and pathologic effects that result from mast cell degranulation.
  • Other physiologic effects induced through activation of the A3 adenosine receptor are also amenable to modulation through blockade of A3 adenosine mediated responses in basophiles, eosinophiles and other immune cells.
  • mast cell degranulation is clearly involved in the pathophysiology of allergies such as asthma. Autoimmune diseases are also characterized by immune reactions which attack targets, including self-proteins in the body such as collagen, mistaking them for invading antigens. The resulting damage, caused at least in part by mast cell degranulation, is amenable to treatment by the method of this invention which comprises administration of selective A3 adenosine receptor antagonists effective to inhibit mast cell degranulation.
  • Addison's disease (adrenal), autoimmune hemolytic anemia (red cells), Crohn's disease (gut), Goodpasture's syndrome (kidney and lungs), Grave's disease (thyroid), Hashimoto's thyroiditis (thyroid), idiopathic thrombocytopinic purpura (platelets), Insulin-dependent diabetes militus (pancreatic beta cells), multiple sclerosis (brain and spinal cord), myasthenia gravis (nerve/muscle synapses), Pemphigus vulgaris (skin), pernicious anemia (gastric parietal cells), poststreptococcal glomerulonephritis (kidney), psoriasis (skin), rheumatoid arthritis (connective tissue), scleroderma (heart, lung, gut, kidney), Sjogren's syndrome (liver,
  • the method of this invention provides a means for preventing or treating disease states associated with vascular constriction induced through activation of the A3 subtype of the adenosine receptor.
  • the method comprises contacting said receptor in the vasculature with an amount of a compound which selectively blockades activation of the A3 adenosine receptor subytpe.
  • this blockade occurs on granulocytes, including mast cells, exhibiting the A3 adenosine receptor.
  • xanthine or a xanthine derivative is used to effect a reduction in vasoconstriction in the vasculature without any substantial effect (binding or blockade) of the Al , A2a or A2b subtypes of the adenosine receptor.
  • the method extends to the treatment or prevention of disease states mediated through activation of the A3 subtype of the adenosine receptor on mast cells.
  • Prevention of mast cell degranulation through blockade of the A3 subtype of the adenosine receptor by contacting mast cells with an inhibitory effective amount of a xanthine or xanthine derivative specific for the A3 receptor subtype therefore also forms part of this invention.
  • Disease states associated with A3 adenosine receptor activation and mast cell degranulation include, but are not limited to asthma, myocardial reperfusion injury, allergic reactions including but not limited to rhinitis, asthma, poison ivy induced responses, urticaria, scleroderma, arthritis, and inflammatory bowel diseases.
  • A3 adenosine receptor subtype Compounds having specific affinity for the A3 adenosine receptor subtype are identified through the pharmacology displayed in binding to isolated receptors of the various subtypes. Many compounds have been identified broadly as antagonists of the adenosine receptor. However, the subtype specificity of these compounds, in particular the pharmacology of the primate A3 receptor subtype, was not known prior to the instant patent disclosure. This disclosure defines xanthines and xanthine derivatives displaying potent and specific A3 subtype specificity. This disclosure also defines the unexpectedly similar tissue distribution and pharmacology of the human and sheep A3 receptors. Because of this discovered similarity, it is now possible to utilize known sheep A3 pharmacology to predict primate pharmacology.
  • this disclosure demonstrates the functional effects of using A3 specific compounds in blocking adenosine induced effects. Microvasculatrue contraction is inhibited, intracellular cAMP reduction, which is normally induced by A3 receptor agonism, is blocked and mast cell degranulation is inhibited.
  • a cDNA from human striatum designated HS-21a that encodes a human A3 adenosine receptor has been cloned.
  • the cDNA is homologous with rat (5,6) and sheep clones (7), and all three sequences encode receptors that couple adenosine induced inhibition of cAMP accumulation when stably expressed in CHO cells.
  • 125 I-ABA previously used as a radioligand for Al adenosine receptors (12), was found to be suitable for detecting recombinant CHO cells expressing human A3 adenosine receptors.
  • the sheep A3 adenosine receptor transcript is widely distributed, with high levels found in lung and spleen and moderate levels found in brain, pineal and testis. In marked contrast, the rat A3 adenosine receptor transcript is found primarily in testis. Therefore, it was of great interest to determine the tissue distribution of the human A3 adenosine receptor transcript.
  • the rat A3 adenosine receptor differs from the human and sheep receptors in that it was reported not to bind the xanthine antagonists, XAC and DPCPX (6).
  • the sheep and now the human A3 adenosine receptors have been found to bind both antagonists and also have high affinity for 8-substituted xanthines having acidic substitutions.
  • the acidic substitutions increase the binding affinity of these compounds for the A3 receptor, they decrease the affinity for the Al , A2a and A2b subtypes.
  • a limited number of xanthine analogs were evaluated in the pharmacological characterization of the rat A3 receptor and it was reported that these compounds do not significantly bind to the rat A3 receptor. The opposite is true in the case with the sheep A3 homolog.
  • the human A3 receptor has a high affinity for this class of compounds.
  • I- ABA appears to be a full and partial agonist, respectively, for lowering cyclic AMP in CHO cells transfected with sheep and human receptors.
  • the human receptor has higher affinity for CPA in comparison to the sheep receptor.
  • An agonist affinity order of I-ABA > NECA > R-PIA > S-PIA » CPA was established for the sheep receptor (7).
  • the human receptor has a generally higher affinity for all of the agonists and a preference for CPA over S-PIA, resulting in an agonist profile of I-ABA > NECA > R-PIA > CPA > S-PIA.
  • the antagonist affinity order profiles are similar between human and sheep receptors, however, the human homolog exhibits a higher affinity for XAC. Since 8-phenylxanthines with para-acidic residues were found to bind with high affinity to sheep A3 adenosine receptors (7), we evaluated acidic 8-phenylxanthines as human A3 adenosine receptor antagonists. Included among these are compounds with various 3-substitutions that were evaluated previously as potent antagonists of Al adenosine receptors (13). One of these compounds, I- ABOPX, was found to have the highest affinity as an A3 adenosine receptor antagonist, with a Kj of 18 nM for the human receptor and 2 nM for the sheep receptor.
  • the potency order profiles of agonist and antagonist binding to the A3 receptor differ substantially from the profiles established for the other cloned human adenosine receptor subtypes (4). Pharmacologically, the A3 receptors appear to more closely resemble Al than the A2 adenosine receptors. This is consistent with the fact that the human A3 sequence is more similar to the A 1 subtype (identity score 49%) than to the A2a and A2b subtypes. All subtypes of human adenosine receptors are blocked by xanthine antagonists such as BW- A1433, XAC and DPCPX, but differ in their affinities and potency order profiles for these ligands.
  • the Al subtype has high affinity for agonists with saturated rings in the N6 position of the adenine ring, and xanthine antagonists with saturated rings in the C ⁇ position.
  • the human A3 receptor has a slightly higher affinity for ligands with unsaturated than saturated rings in the N ⁇ position of agonists (R-PIA > CPA) and in the C ⁇ position of xanthines (BW- A1433 and XAC > DPCPX).
  • R-PIA CPA
  • BW- A1433 and XAC > DPCPX C ⁇ position of xanthines
  • a model has been proposed in which the N ⁇ -substituents of the adenine ring can be superimposed upon the C- ⁇ -regions of xanthines (27,28 ).
  • a similar relationship has been suggested to exist for the sheep A3 receptor since parallel changes in potency for agonists and antagonists were observed when the N ⁇ and C ⁇ positions, respectively were substituted with unsaturated or saturated rings (7).
  • the human A3 receptor was found to also exhibit corresponding changes in agonist and antagonist affinities when saturated substitutions were introduced at the N ⁇ and C-- positions.
  • an A3 adenosine receptor antagonist will have a pKi for the A3 sutype of 7 or greater, and a pKi for other adenosine receptor subtype of 6 or less.
  • xanthine compounds having the following characteristics are preferred: An acidic, aromatic substitution at the 8 position of the xanthine (the acidity decreases affinity to Al subtype to below a pKi of about 6, and the aromatic substitution reduces the affinity for the A2 subtype to below about 6).
  • Rl , R2, and R3, independently, are as defined below:
  • alkyl, alkenyl, cycloalkyl is substituted or unsubstituted aryl is benzyl, phenyl; substituted aryl is an aryl substituted with an alkyl, amino or halogen; and acidic aryl is an aryl substituted with a carboxylate, oxyacetate, acrylate, sulphonate, phosphonate, or tetrazol.
  • R 2 wherein Rl , R2, and R3, independently, are as defined below:
  • the xanthine is:
  • Rl , R2, and R3, independently, are as defined below:
  • the xanthine is selected from the group consisting of IABOPX, BW-A1433, BW-934, BW-A215.
  • a comparison of the distribution among tissues of human adenosine receptor transcripts suggests that the Al , A2a, A2b and A3 subtypes are all expressed in a number of tissues, but the pattern of transcript distribution is variable. Within the four tissues analyzed, Al and A2a adenosine receptor transcripts are highly expressed in the brain. In contrast, the human A3 adenosine receptor transcript is most abundant in the lung and liver.
  • the physiological role of the A3 adenosine receptor was not defined. Recently, a receptor exhibiting an agonist pharmacological profile thought to be characteristic of the rat A3 subtype and insensitive to blockade by 8- (para-sulfophenyl)theophylline, was suggested to mediate in vivo hypotension in the angiotensin ⁇ -supported circulation of the pithed rat (30). The effect of 8-(para-sulfophenyl)theophylline on the cloned rat A3 receptor has not been evaluated and further pharmacological characterization is required to determine if the rat receptor binds this antagonist.
  • 8-Para-sulfophenyltheophylline has broad action on Al , A2a and A2b adenosine receptor subtypes (2), and also blocks both the human and sheep A3 receptors.
  • Another possible physiological role for the A3 adenosine receptor subtype in reproduction and spermatogenesis has been proposed on the basis of the abundant transcript found in rat testis and the in situ localization of mRNA within the central luminal regions of seminiferous tubules where sperm maturation occurs (5). It is possible that A3 adenosine receptors also are involved in the maturation of sperm in human and ovine testis, since low to moderate levels of receptor transcript are found in these species, respectively.
  • A3 adenosine receptor subtype mediates a physiological action in the pulmonary system.
  • Adenosine has been shown to mediate both vasodilation and vasoconstriction (31,32) in the pulmonary vasculature.
  • vasodilation and vasoconstriction 31,32
  • adenosine produces bronchoconstriction which can be antagonized by theophylline (33).
  • the establishment of the pharmacological profile for the A3 receptor in both the human and the sheep, and the availability of subtype selective ligands facilitates the identification of the physiological functions mediated by the A3 adenosine receptor subtype and the treatment of disease states mediated through agonism of this receptor subtype.
  • Adenosine has been shown to produce bronchoconstriction in asthmatics but not in nonasthmatics, demonstrating that adenosine plays a role in the etiology of this disease state (Cushly et al., 1984, AM. Rev. Respir. Dis. 129:380-384). Adenosine mediated bronchoconstriction in asthmatics is blocked by a combination of histamine and leukotriene antagonists (Bjorck et al., Am. Rev. Resp. Dis. 1992, 145:1087-1091). This indicates that adenosine acts by releasing histamine, leukotriene and other agents from mast cells or other cells that contain these allergic mediators.
  • the instant patent disclosure provides evidence that the blockade of A3 adenosine receptor mediated action in the vasculature is useful to treat and prevent disease states in humans. reports that adenosine potentiates the release of granule contents from mast cells isolated from rat peritoneum (Lohse et al., N.-S. Arch. Pharmacol. 335:555-560. 1987; Marquardt et al., J. Immunol. 120:871 -878, 1978), and that mast cell degranulation causes constriction in some vascular beds resulting in C5a-induced myocardial ischemia (Ito et al., Am. J. Physiol. 264 (Heart Circ. Physiol.
  • the trigger for mast cell degranulation is usually thought to be an allergen. Allergens are endocytosed by marcrophages and degraded. The resulting fragments are displayed on T lymphocytes. B lymphocytes are stimulated to mature into plasma cells which are able to secrete allergen-specific molecules known as immunoglobulin E, (IgE). These antibodies attach to receptors on mast cells in tissue and on basophils circulating in blood - to trigger degranulation (see L. Lichtenstein, Sci. Am. 269:1 16-125, 1993). As described below, our data shows that activation of A3 adenosine receptors can produce mast cell degranulation and enhance the effect of allergens.
  • IgE immunoglobulin E
  • Adenosine and antigens trigger an influx of calcium to induce mast cell granules to release their contents an promote synthesis and release of cytokines, prostaglandins and leukotrienes.
  • the various chemicals released by mast cells are responsible for many allergic symptoms. Long term release of these chemicals can induce basophils, eosinophils, and other cells flowing through blood vessels to migrate into the tissue. Migration is promoted due to the expression and activation of adhesion molecules on the circulating cells and on vascular endotheilial cells. The circulating cells adhere to the endothelial cells, roll among them, and eventually cross into the surrounding matrix. These recruited cells secrete chemicals of their own that damage tissue. Thus, there are long term secondary effects which may also be prevented by specific blockade of mast cell degranulation.
  • the method is directed to prevention or treatment of myocardial ischemia/reperfusion.
  • the basis of this application of the method is that a period of myocardial ischemia followed by reperfusion produces damage to the myocardium. Part of this damage may be secondary to mast cell degranulation triggered by adenosine during ischemia.
  • A3 adenosine receptor antagonists may be useful for the treatment of patients prone to reperfusion injury. This includes patients with coronary artery diseases in general, and patients about to have occluded arteries opened (reperfused) by various interventions (coronary artery bypass grafts, angioplasty or thrombolytic therapy).
  • Adenosine-induced mast cell degranulation during a period of transient ischemia may be responsible for the phenomenon of preconditioning (i.e. a transient ischemic episode reduces myocardial damage resulting from a subsequent prolonged ischemic episode). Accordingly, mast cells are temporarily depleted of damaging mediators during the preconditioning period.
  • Lodoxamide a drug that acts to inhibit mast cells degranulation, reduces myocardial ischemic injury; Ito, B.R., Engler, R.L., Del Balzo, U., Role of cardiac mast cells in complement C5a-induced myocardial ischemia. Am. J. Physiol. 33 :H1346-H 1354, 1992. conclusion: Cardiac mast cells are involved in complement-induced release of vasoactive eicosanoids, including TxA2.).
  • the human Al, A2a, A2b and A3 receptor subtype cDNAs were subcloned into the expression vectors pSVL (PHARMACIA), CMV5 (Mumby, et al. 1990, PNAS, 87:728-732) or pREP (INVITROGEN).
  • pSVL human kidney cell line, ATCC CRL 1651 , ATCC, Rockville, MD
  • pREP pREP
  • Membranes prepared from the transfected cells were utilized for the determination of binding affinity, selectivity and specificity of the human adenosine receptors for various ligands.
  • Stable expression of the human adenosine receptors in mammalian cells was achieved after integration of the transfected cDNA into the chromosomes of the host cells.
  • These stable cell lines constituently express the cloned human adenosine receptors and can be propagated infinitely.
  • Stable cell lines expressing the human adenosine subtype cDNAs individually can be used in the binding assay to measure the affinity and selectivity of the receptors for adenosine agonists, antagonists and enhancers.
  • Membranes prepared from transfected COS7 cells were utilized in a binding assay to measure the affinity of the human adenosine receptors for the radiolabeled adenosine agonists, [3H]-cyclo- hexyladenosine (CHA), [ 3 H]-CGS21680 (2-(p-(2-carboxyethyl)- phenylamino)-5'-N-ethyl-carboxamidoadenosine), [3H]-5'-N- ethylcarboxamido adenosine ([3H]-NECA), or adenosine (125I-ABA).
  • Monolayer cell culture of transfected COS7 cells were dissociated with 1 mM EDTA in phosphate buffered saline and resuspended in 5 mM Tris, pH7.6/10 mM MgC ⁇ .
  • the cells were subjected to freeze-thaw lysis and the suspension was homogenized in a glass dounce homogenizer.
  • the membranes were pelleted, resuspended in binding buffer, 50 mM Tris pH 7.6/10 mM MgCl2 and incubated with adenosine deaminase before the binding assay.
  • the binding assay was performed by incubating 50-100 ⁇ g of membranes with increasing concentrations of radiolabeled adenosine agonists.
  • Bound ligand was separated from free ligand by filtration on a SKATRON CELL HARVESTER equipped with a receptor binding filtermat. Bound radioactivity was measured by scintillation counting. Substances which bind to or enhance binding to expressed human adenosine receptors in COS and CHO cells can be identified in competition binding assays with radiolabeled adenosine or xanthine analogs. For the competition binding assay, membranes were incubated with 5nM [3H]-CHA 5nM [ 3 H]-CGS21680 or lOnM [3H]-NECA and various concentrations of adenosine agonists or antagonists.
  • a transient expression system in Xenopus oocytes was established by microinjection of in vitro transcribed mRNA from the cloned adenosine receptor cDNAs.
  • the expression system allows the measurement of the biological effects (i.e.. changes in cAMP levels) upon activation of the expressed adenosine receptors with ligand binding.
  • the cAMP levels are measured by a radioimmunoassay (RIANEN kit, DuPont/NEN) using the acetylation protocol.
  • Activation of the expressed receptors by ligand binding are coupled to either increases or decreases in the intracellular cAMP levels dependent upon the subtype of adenosine receptor (Van Calker et al., (1979) J.
  • the activity of any potential adenosine receptor agonist can be evaluated by measuring the changes in cAMP levels in oocytes injected with adenosine receptor mRNA but not in uninjected or negative control injected oocytes.
  • the activity of any potential adenosine receptor antagonist can be evaluated by determining the inhibition of the cAMP response induced by adenosine in oocytes injected with adenosine receptor transcripts but not negative control or uninjected oocytes.
  • the changes in cAMP accumulation can alternatively be measured in stably transfected CHO cells expressing the human adenosine receptor subtypes.
  • the cAMP accumulation assay has a number of advantages over the binding assay established in the mammalian cell expression system as a screen for adenosine receptor modulating agents.
  • the assay allows the measurement of a biological effect (i.e., changes in cAMP levels) resulting from the activation of the expressed receptors by ligand binding.
  • the native agonist adenosine is utilized in the assay to activate the expressed receptors.
  • the functionality of additional adenosine receptor subtypes identified by molecular cloning which may not have defined ligands for binding analysis can be evaluated with the natural agonist and without prior identification of a selective, high affinity, radiolabeled ligand.
  • an adenosine A3 specific antagonist is administered in an amount effective to induce blockade of the receptor.
  • Compounds having a pKi of greater than about 7 for the A3 receptor and below about 6 for other adenosine receptor subtypes may be administered by any effective means to achieve either localized or systemic contact of the antagonist with target A3 adenosine receptors. This might include intraveous, intramuscular, intrasynovial, intranasal, nebulized intrapulmanory, intraperitoneal or other common means for administration of therapeutic compounds. Dosages of between about 1 ⁇ g/kg and 10 mg/kg are envisioned, as necessary, to achieve the desired effect of A3 adenosine receptor blockade.
  • EXAMPLE 1 STEP A In the first step of obtaining the partial cDNAs encoding the human Al and A2a adenosine receptors, total RNA was extracted by homogenizing 2.3g human ventricle in 20 ml 5M guanidine isothiocyanate, 0.1M sodium citrate, pH 6.3, lmM EDTA, pH 7.0, 5% beta-mercaptoethanol, and 0.5% sodium lauryl sarcosinate. The homogenate was centrifuged for 10 min. at 10,000 rpm and the resulting supernatant was layered onto a cushion of 5.7M CsCl/O.lM EDTA, pH 7.0. After 20 hrs. of centrifugation at 24,000 rpm, the resulting pellet was precipitated one time and then passed over an oligo(dT)-cellulose (PHARMACIA, Piscataway, NJ) column to isolate poly(A)+ RNA.
  • PARMACIA oligo(dT)-cellulose
  • oligo(dT) primed library was synthesized from 5 ⁇ g of the poly(A) + human ventricle RNA using the YOU-PRIME cDNA SYNTHESIS KIT (PHARMACIA, Piscataway, NJ). See Gubler and Hoffman Gene 25:263 (1983). The resulting double-stranded cDNA was ligated into ⁇ gtlO EcoRI arms (PROMEGA, Madison, WI) and packaged according to the GIGAPACK II GOLD PACKAGING EXTRACT protocol (STRATAGENE, La Jolla, CA). See Huynh et al. (1985) DNA Cloning Techniques: A Practical Approach. IRL Press, Oxford, p.49 and Kretz et al. Res.
  • the E. coli strain C600HA (PROMEGA, Madison, WI) was infected with library phage, plated on agar plates, and incubated at 37°C The phage DNA was transferred to HYBOND-N nylon membranes (AMERSHAM, Arlington Heights, IL) according to the manufacturer's specifications.
  • Synthetic probes were constructed from overlapping oligonucleotides (Al probe: 62+63, A2 probe: 52+53; see Table I for their sequences) based on the published dog Al (RDC7 ) and A2a(RDC8) sequences (F Libert, et al,(1989) Science 244:569-572).
  • the oligonucleotides were annealed and filled-in with c ⁇ 2p_dc ⁇ p (NEN, Wilmington, DE) and Klenow enzyme.
  • the filters were hybridized with the appropriate probe in 5XSSC, 30% formamide, 5XDenhardt's solution, 0.1 % SDS, and O.lmg/ml sonicated salmon sperm DNA at 42°C, overnight.
  • the human ventricle Al cDNA (hval-3a) and human ventricle A2a cDNA (hva2-13) contain portions of coding sequences for proteins homologous to the reported dog Al and A2a cDNAs, respectively.
  • the coding region of the human Al clone corresponds to nucleotides 482 through 981 ( Figures 2A and 2B) and is 92% identical to the dog Al sequence at the nucleotide level.
  • the coding region of the human A2a clone corresponds to nucleotides 497 through 1239 ( Figure 4 A and 4B), and is 90% identical to the dog A2a sequence at the nucleotide level.
  • (hvAl-3a) is a 543 bp NotI fragment containing 23 bp 3' untranslated sequence and is 460 bp short of the initiation methionine based on sequence homology to the dog Al cDNA.
  • RACE rapid amplification of cDNA ends
  • MA Frohman et al,(1988), Proc. Natl. Acad. Sci. USA, 85:8998-9002 was used to generate the 5' coding region of the cDNA.
  • First strand cDNA was synthesized from l ⁇ g of the human ventricle poly(A) + RNA in a total volume of 40 ⁇ l containing 50mM Tris, pH 8.0, 140mM KC1, lOmM MgCl 2 , 10mM DTT, 15mM each dNTP, 20 units RNasin (PROMEGA,
  • AMV reverse transcriptase at 37°C for 2 hrs.
  • the reaction was then diluted to 120 ml with 0.5 mM Tris, pH 7.6/0.05 mM EDTA and passed through a SEPHACRYL S-300 SPUN COLUMN (PHARMACIA,
  • primer 71 25pmol primer 71 , and 25pmol human primer 80 (see table I) in a total volume of 50 ⁇ l.
  • Primer 70 is 5'-gactcgagtcgacatcga(t)i7
  • primer 71 is
  • the gel slice was melted and 1 ⁇ l was used as template in a secondary PCR amplification reaction containing lOOpmol primer 71 and human primer 81 (see Table I) for 30 cycles of 1 min at 94°C, 2 min at 56°C, 3 min at 72°C
  • the secondary PCR amplification product was digested with EcoRI and Sail and electrophoresed on a 1.4% agarose gel. An area corresponding to 500-600bp was excised and ligated into EcoRI/Sall digested pBLUESCRIPT II KS+ (STRATAGENE, La Jolla, CA).
  • the sequence of the 515 bp PCR product (5 ⁇ VA1-9) was determined by the SEQUENASE protocol (USBC, Cleveland, OH).
  • the partial human ventricle Al cDNA and the PCR product contain overlapping sequence and represent the complete coding region for the human Al receptor, including 14 and 23 bp of 5' and 3' untranslated sequences, respectively.
  • the sequence of the human Al adenosine receptor cDNA so identified, is shown in Figures 2 A and 2B.
  • a probe was generated by Klenow enzyme extension, including ⁇ 32p_dCTP, of annealed oligonucleotides 62 and 63, and used to screen a human kidney cDNA library (CLONTECH, Palo Alto, CA).
  • E. coli strain C600hfl PROMEGA, Madison, WI
  • the probe was incubated with the filters in 750mM NaCl, 75mM sodium citrate, 30% formamide, 0.1 % sodium dodecyl sulfate, 0.5mg/mL polyvinylpyrrolidone, 0.5mg/mL bovine serum albumin, 0.5mg/mL Ficoll 400, and O.lmg/mL salmon sperm DNA, at 42°C overnight.
  • the filters were washed in 0.9M NaCl and 90mM sodium citrate at 50°C A positively hybridizing phage (hkAl-14), was identified and purified by replating and screening with the probe twice more.
  • the final phage plaque was transferred to 0.5 mL 50mM Tris, pH 7.5, 8mM MgS04 85 mM NaCl, lmg/mL gelatin, and 1 ⁇ L of a 1 :50 dilution in water of the phage stock was used as template for PCR amplification. 50 pmol each of lamL and lamR (Table I) oligonucleotide primers were included, then a final 15 min at 72°, according to the GENE AMP protocol (PERKIN ELMER CETUS, Norwalk, CT).
  • the human ventricle A2a adenosine receptor partial cDNA (hvA2-13) is a 1.6 kb NotI fragment containing approximately 900 bp 3' untranslated sequence and is 496 bp short of the initiation methionine based on sequence homology to the dog A2a cDNA clone. Two consecutive rounds of 5' RACE were utilized to generate the 5' coding region of the cDNA.
  • First strand cDNA was synthesized from 1 ⁇ g of the human ventricle poly(A) + RNA in a total volume of 40 ⁇ l containing 50mM Tris, pH 8.0, 140mM KC1, lOmM MgCl 2 , 10mM
  • the products in the column effluents were polyadenylated in lOOmM potassium cacodylate, pH 7.2, 2 mM CoCl 2 , 0.2 mM DTT, 0.15 mM dATP, and 14 units terminal deoxynucleotidyl transferase in a total volume of 31 ⁇ l for 10 min. at 37°C
  • the poly(A) tailing reaction was terminated by heating at 65°C for 15 min. and then diluted to 500 ⁇ l with TE.
  • PCR amplification products were digested with EcoRI and Sail and electrophoresed on a 1.4% agarose gel. Areas corresponding to 200-400 bp were excised and ligated into EcoRI/Sall digested pBLUESCRIPT II KS+ (STRATAGENE, La Jolla, CA).
  • sequences of the two A2a PCR products, the 332 bp 1st round RACE product (5'hvA2-14) and the 275 bp 2nd round RACE product (5'hvA2-29) were determined by the SEQUENASE (USBC, Cleveland, OH) protocol.
  • SEQUENASE USBC, Cleveland, OH
  • sequence homology comparisons with the dog A2a adenosine receptor cDNA sequence the 1st round RACE product (5'hvA2-14) was 258 bp short of the initiation methionine and the second round RACE product (5 ⁇ VA2-29) was determined to extend lbp upstream of the initiation methionine.
  • the human ventricle A2a partial cDNA clone (hvA2-13) and the human A2a PCR products (5'hvA2-14 and 5'hva2-29) contain overlapping sequence and together represent the complete coding sequence for the human adenosine A2a receptor, and include 1 bp and 0.8 kb of 5' and 3' untranslated sequence, respectively.
  • the sequence of the human A2a adenosine receptor is shown in Figures 4 A and 4B.
  • a double-stranded DNA probe was generated by Klenow enzyme extension, including p_dCTP, of annealed oligonucleotides 66 and 67, and used to screen a human striata cDNA library (STRATAGENE, La Jolla, CA).
  • the oligonucleotide sequence was based on a region of the human ventricle A2a cDNA sequence.
  • E. coli strain XLl-blue (STRATAGENE, La Jolla, CA) cells were infected with library phage and grown overnight on agar plates at 37 °C. Phage DNA was transferred to HYBOND-N nylon filters according to the manufacturer's protocol (AMERSHAM, Arlington Heights, BL).
  • the probe was incubated with the filters in 750 mM NaCl, 75 mM sodium citrate, 10% formamide, 0.5% sodium dodecyl sulfate, 0.5 mg/mL polyvinylpyrrolidone, 0.5 mg/mL bovine serum albumin, 0.5 mg/mL Ficoll 400, and 0.02 mg/mL salmon sperm DNA, at 42°C overnight.
  • the filters were washed in 0.9 M NaCl and 90 mM sodium citrate at 50°C A positively hybridizing phage (hbA2-22A) was identified and purified by replating and screening with the probe twice more, and subcloned into the plasmid pBLUESCRIPT SK- by the manufacturer's protocol (STRATAGENE, La Jolla, CA). See Short et al. (1988) Nucl. Acids Res. 16:7583-7600; Sorge (1988) Stratagies 1:3-7.
  • the human brain A2a adenosine receptor cDNA spans bp 43 of the A2 coding sequence ( Figures 4 A and 4B) through the translation STOP codon, and includes about 900 bp of 3' untranslated sequence.
  • the sequence of this human brain A2a cDNA is identical to the human ventricle A2a adenosine receptor cDNA (hvA2-13, 5'hvA2-14 and 5'hvA2-29).
  • a double-stranded DNA probe was generated by Klenow enzyme extension of annealed oligonucleotides 129 and 130, including o.32p-clCTP, and used to screen a human frontal cortex cDNA library (STRATAGENE, La Jolla, CA).
  • the oligonucleotide sequence was based on a region of the human A2a and Al cDNA sequence.
  • E. coli strain XL-1 blue (STRATAGENE, La Jolla, CA) cells were infected transf erred to HYBOND-N nylon filters according to the manufacturer's protocol (AMERSHAM, Arlington Heights, IL).
  • the probe was incubated with the filters in 750 mM NaCl, 75 mM sodium citrate, 10% formamide, 0.5% sodium dodecyl sulfate, 0.5 mg/mL polyvinyl-pyrrolidone, 0.5 mg/mL bovine serum albumin, 0.5 mg/mL Ficoll 400, and 0.02 mg/mL salmon sperm DNA, at 42°C overnight.
  • the filters were washed in 0.9 M NaCl and 90 mM sodium citrate at 50°C
  • a positively hybridizing phage (hb-32c) was identified and purified by replating and screening with the probe twice more.
  • the insert was subcloned to the plasmid pBLUESCRIPT SK- according to the manufacturer's protocol (STRATAGENE, La Jolla, CA). Sequence analysis by the SEQUENASE protocol (USBC, Cleveland, OH) demonstrated a complete open reading frame coding for amino acid sequence homologous to both of the previously isolated human Al and A2a clones.
  • This homologous adenosine receptor subtype cDNA is the A2b subtype having the sequences in Figures 5 and 6.
  • a 1.3 kb Smal- Xmnl fragment was ligated into the Smal site of pSVL (PHARMACIA, Piscataway, NJ), giving the full length coding sequence of the A2b adenosine receptor in a plasmid suitable for its expression in COS and CHO cells. See Sprague et al. (1983) J. Virology 45:773; Templeton and Eckhart (1984) Mol. Cell Biol. 4:817.
  • the A2b adenosine receptor subtype encoded by the clone hb32C was determined to be the A2b adenosine receptor subtype on the basis of the binding profile of the adenosine receptor agonist NECA and affinities for adenosine receptor antagonists measured on membranes prepared from pSVLhb32C transfected COS7, CHO or HEK 293 cells.
  • ACCATGT (SEQ ID NO. 6) name sequence position clone direction
  • TGCTGCCCCCGCT- (SEQ ID NO. 14) name sequence position clone direction
  • the 1 18bp Sall-Smal fragment of the human ventricle Al PCR product (5 ⁇ VA 1-9) was ligated together with the 1.8 Smal-EcoRI fragment of the human kidney Al adenosine receptor cDNA (hkA l -14) and the 3.0 kb Sail -EcoRI fragment of pBLUESCRIPT ⁇ KS+, resulting in a plasmid containing the contiguous full length coding sequence for the human Al adenosine receptor cDNA and some 5' and 3' untranslated sequence.
  • This plasmid was digested first with EcoRI, the resulting ends were filled in by Klenow enzyme extension and then the plasmid was digested with Xhol to release a fragment of 1.9 kb containing the full length human Al adenosine receptor cDNA. The fragment was subcloned into the expression vector pSVL (PHARMACIA) which had been digested with Xhol-Smal.
  • a contiguous A2a cDNA sequence was constructed before subcloning into the expression vector, pSVL.
  • Primer 131 containing an Xbal recognition site, 14 bp of 5' untranslated sequence of human Al adenosine receptor cDNA, and the first 18 bp of human A2a adenosine receptor cDN A coding sequence was used with primer 75 in PCR with 1 ng of the plasmid containing the human ventricle A2a 2nd round RACE product (5'hvA2-29) as template.
  • the 172 bp Xbal-EagI digestion product of this DNA fragment was ligated together with 1125 bp Eagl-Bglll digestion product of the human striata A2a adenosine receptor cDNA (hbA2-22A) and Xbal-Smal digested pSVL (PHARMACIA), generating the full length human A2a adenosine receptor cDNA coding sequence in a plasmid suitable for its expression in COS, CHO or HEK 293 cells.
  • COS7 cells (ATCC #1651-CRL) were grown in complete medium, Dulbecco's modified Eagles's medium, DMEM (GIBCO, Grand Island, NY) containing 10% fetal bovine serum, lOOU/mL penicillin-streptomycin and 2 mM glutamine, in 5% C0 2 at 37°C Transient transfection of COS7 cells was performed by the CaP ⁇ 4 method (Graham,F.L. and Van Der Erb, A.J. (1973) Virology 52:456- 567) using the Mammalian Transfection Kit (STRATAGENE). See Chen and Okayama Mol. Cell Biol. 7:2745-2752. Plasmid DNA (15 ⁇ g) was precipitated with 125 mM CaCl 2 in BBS (N,N-bis(2-hydroxyethyl)-
  • CHO or HEK 293 cells were co-transfected with 20 ⁇ g of pS VL containing the adenosine receptor cDNA and l ⁇ g of pWLneo (STRATAGENE) containing the neomycin gene. See Southern and Berg (1982) J. Mol. App. Gen. 1 :327-341. Transfection was performed by the CaP ⁇ 4 method. DNA was precipitated at room temperature for 30 minutes, added to the CHO cells and incubated 18h in 5% C0 2 at 37°C The precipitate was removed and the cells were washed twice with serum free DMEM.
  • Membranes were prepared from transiently transfected COS7 cells 48 h after transfection or from G418-selected stably transfected CHO or HEK 293 cells. Cells were harvested in 1 mM
  • EDTA in phosphate buffered saline and centrifuged at 2000 x g for 10 minutes. The cell pellet was washed once with phosphate buffered saline. The cell pellet was resuspended in 2 mL of 5 mM Tris, pH 7.6/ 5mM MgCl 2 . Membranes were prepared from the cells by freeze-thaw lysis in which the suspension was frozen in a dry ice/ethanol bath and thawed at 25°C twice. The suspension was homogenized after adding an additional 2 mL of 5 mM Tris, pH 7.6/5 mM MgCl 2 , in a glass dounce homogenizer with 20 strokes.
  • the membranes were pelleted at 40,000 x g at 4°C for 20 minutes.
  • the membrane pellet was resuspended at a protein concentration of 1 -2 mg/mL in binding assay buffer, 50 mM Tris, pH 7.6/10 mM MgCl 2 . Protein concentration was determined by the method of Bradford ((1976) Anal. Biochem. 72: 248-250).
  • the membranes were incubated with adenosine deaminase (BOEHRINGER MANNHEIM), 2 U/mL for 30 minutes at 37 °C Saturation binding of Hj-cyclohexyladenosine (CHA) was performed on membranes prepared from pSVLAl transfected
  • NECA [3H]5'-N-ethylcarboxamidoadenosine
  • the human A3 adenosine receptor was cloned from a human striata cDNA library. Oligonucleotide probes were designed based on the rat A3 sequence of Zhou et al., Proc. Natl. Acad. Sci. 89, 7432 (1992). The complete sequence of the human A3 adenosine receptor was determined and the protein sequence deduced. The cloned human A3 adenosine receptor is expressed in a heterologous expression system in COS, CHO and HEK 293 cells. Radiolabeled adenosine receptor agonists and antagonists are used to measure the binding properties of the expressed receptor. Stable cell lines can be used to evaluate and identify adenosine receptor agonists, antagonists and enhancers.
  • a synthetic probe homologous to the rat A3 adenosine receptor was generated using the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Three ⁇ l of rat brain cDNA was used as template in a PCR amplification reaction according to the GENEAMP protocol (PERKIN ELMER CETUS, Norwalk, CT) containing 50 pmol of primers 207 (5'- cccaagcttatgaaagccaacaatacc) (SEQ. ID NO: 27) and 208 (5'- tgctctagactctggtatcttcacatt) (SEQ. ID NO: 28) in a total volume of 50 ⁇ l.
  • Primers 207 and 208 are based on the published rat A3 adenosine receptor sequence (Zhou, et al, (1992), Proc. Natl. Acad. Sci. USA, 89:7432-7406). Forty cycles of 40 sec at 94°C, 1 min at 55°C, 3 min at 72°C were performed and the resulting 788 bp fragment was subcloned into Hindffl-Xbal digested pBLUESCRIPT II KS+ (STRATAGENE, La Jolla, CA). The sequence was verified by the SEQUENASE protocol (USBC, Cleveland, OH).
  • the 788 bp PCR fragment was labeled with o.32p_dCTP using the MULTIPRIME DNA LABELLING SYSTEM (AMERSHAM, Arlington Heights, IL) and used to screen a human striata cDNA library (STRATAGENE, La Jolla, CA).
  • E. coli strain XL-1 Blue (STRATAGENE, La Jolla, CA) cells were infected with library phage and grown overnight at 37°C Phage DNA was transferred to HYBOND-N nylon filters according to the manufacturer's protocol (AMERSHAM, Arlington Heights, IL).
  • the probe was incubated with the filters in 5 X SSC, 30% formamide, 5 X Denhardt's solution, 0.5% sodium dodecyl sulfate, and 50 ⁇ g/ml sonicated salmon testis DNA.
  • the filters were washed in 2 X SSC at 55°C
  • a positively hybridizing phage (HS-21a) was identified and plaque purified by two additional rounds of plating and hybridization.
  • the insert was subcloned to the plasmid pBLUESCRIPT II SK- according to the manufacturer's protocol (STRATAGENE, La Jolla, CA).
  • clone HS-21a contained the complete open reading frame conesponding to the human homolog of the rat A3 adenosine receptor.
  • the coding region of the human A3 adenosine receptor cDNA is 78% identical to the rat sequence at the nucleotide level and contains 265 bp and 517 bp of 5' and 3' untranslated sequence, respectively.
  • the 1.7 kb fragment was excised using sites present in the multiple cloning site of pBLUESCRIPT II SK- (STRATAGENE, La Jolla, CA) and subcloned into Xhol/SacI digested pSVL (PHARMACIA, Piscataway, NJ) for its expression in COS and CHO cells.
  • COS7 cells (ATCC #1651-CRL) were grown in complete medium, Dulbecco's modified Eagles's medium, DMEM (GIBCO, Grand Island, NY) containing 10% fetal bovine serum, lOOU/mL penicillin-streptomycin and 2mM glutamine, in 5% C ⁇ 2 at 37°C Transient transfection of COS7 cells was performed by the CaP04 method (Graham, F.L. and Van Der Erb, A.J. (1973) Virology 52:456- 567) using the Mammalian Transfection Kit (STRATAGENE).
  • Plasmid DNA (15 ⁇ g) was precipitated with 125 mM CaCl2 in BBS (N,N-bis(2- hydroxyethyl)-2-aminoethanesulfonic acid buffered saline) at room temperature for 30 minutes. The DNA precipitate was added to the COS7 cells and incubated for 18 h in 5% C02 at 37°C The precipitate was removed and the cells were washed twice with serum free DMEM. Cells were incubated in complete medium in 5% C02 at 37°C for 48 h prior to the binding assay.
  • BBS N,N-bis(2- hydroxyethyl)-2-aminoethanesulfonic acid buffered saline
  • CHO cells were cotransfected with 20 ⁇ g of pSVL containing the adenosine receptor cDNA and 1 ⁇ g of pWLneo (STRATAGENE) containing the neomycin gene. Transfection was performed by the CaP ⁇ 4 method. DNA was precipitated at room temperature for 30 minutes, added to the COS7 cells and incubated 18 h in 5% C02 at 37°C The precipitate was removed and the cells were washed twice with serum free DMEM.
  • pSVL containing the adenosine receptor cDNA
  • pWLneo pWLneo
  • Cells were incubated for 24 h in 5% C ⁇ 2 at 37°C, replated in 24-well dishes at a dilution of 1 :10, and incubated an additional 24 h before adding selection medium, DMEM containing 10% fetal bovine serum, lOOU/mL penicillin-streptomycin, 2 mM glutamine and 1.0 mg/mL G418 (GIBCO). Transfected cells were incubated at 5% C ⁇ 2, 37°C until viable colonies were visible, approximately 14-21 days. Colonies were selected and propagated. The cell clone with the highest number of human adenosine receptors was selected for subsequent application in the binding assay.
  • selection medium DMEM containing 10% fetal bovine serum, lOOU/mL penicillin-streptomycin, 2 mM glutamine and 1.0 mg/mL G418
  • Membranes were prepared from transiently transfected COS7 cells 48 h after transfection or from G418-selected stably transfected CHO or HEK 293 cells. Cells were harvested in 1 mM EDTA in phosphate buffered saline and centrifuged at 2000 x g for 10 minutes. The cell pellet was washed once with phosphate buffered saline. The cell pellet was resuspended in 2 mL of 5 mM Tris, pH 7.6/ 5mM MgCl2- Membranes were prepared from the cells by freeze-thaw lysis in which the suspension was frozen in a dry ice/ethanol bath and thawed at 25°C twice.
  • the suspension was homogenized after adding an additional 2 mL of 5 mM Tris, pH 7.6/ 5mM MgCl2, in a glass dounce homogenizer with 20 strokes.
  • the membranes were pelleted at 40,000 x g at 4°C for 20 minutes.
  • the membrane pellet was resuspended at a protein concentration of 1 -2 mg/mL in binding assay buffer, 50 mM Tris, pH 7.6/10 mM MgCl2- Protein concentration was determined by the method of Bradford ((1976) Anal. Biochem. 72: 248-250).
  • adenosine deaminase BOEHRINGER MANNHEIM
  • Saturation binding of [12 i]-N6-aminobenzyl- adenosine (125I-ABA) or [- ⁇ 5l]-N6-2-(4-amino-3-iodophenyl)ethyl- adenosine (APNEA) was performed on membranes prepared from pSVLA3 transfected COS7 cells.
  • Membranes (100 ⁇ g) were incubated in the presence of 0.2U/mL adenosine deaminase with increasing concentrations of 1 5 ⁇ _ABA in the range of 0.1-30 nM for 120 minutes at 25°C in a total volume of 500 ⁇ L.
  • the binding assay was terminated by rapid filtration and three washes with ice-cold 50 mM Tris, pH 7.6/10 mM MgCl2 on a Skatron cell harvester equipped with a receptor binding filtermat (SKATRON INSTRUMENTS, INC). Non-specific binding was determined on non-transfected cells. Bound radioactivity was measured by scintillation counting in Ready Safe Scintillation Cocktail (BECKMAN).
  • the 1.3 kb XhoI-BamHI fragment of the pSVL expression construct (described in Example 2) containing the full length human A2a adenosine receptor coding sequence was ligated into Sa ⁇ -Spel digested pGEMA (Swanson, et al, ( 1990) Neuron 4:929-939).
  • the resulting plasmid, pGEMA2 was linearized with NotI, forming a template for in vitro transcription with T7 RNA polymerase.
  • the homologous adenosine receptor subtype cDNA in pBluescript SK- was used as a template for in vitro transcription by T3 polymerase after removal of most of the 5' untranslated region, with the exception of 20 bp, as a 0.3 kb Smal fragment.
  • the K + channel cDNA, Kv3.2b was employed as a negative control in the cAMP accumulation assay.
  • the generation of Kv3.2b RNA was described by Luneau, et al, ((1991) FEBS Letters 1 :163-167).
  • Linearized plasmid templates were used with the STRATAGENE mCAP kit according to the manufacturer's protocol, except that the SP6 RNA polymerase reaction was performed at 40°C Oocytes were harvested from mature female Xenopus laevis, treated with collagenase, and maintained at 18°C in ND96 medium (GIBCO) supplemented with 1 mM sodium pyruvate and 100 ⁇ g/mL gentamycin. Fifty nano liters (10 ng) of RNA diluted in H 2 0 was injected and oocytes were incubated at 18°C for 48 hours.
  • Oocytes injected with either human adenosine receptor transcript or the Kv3.2b transcript were transferred to fresh medium supplemented with 1 mM of the phosphodiesterase inhibitor, Ro 20- 1724 (RBI, Natick, MA) and 1 mg/mL bovine serum albumin incubated for 30 minutes and transfened to an identical medium with or without the agonist adenosine (10 mM) for an additional 30 minutes at room temperature.
  • Groups of 5-10 oocytes were lysed by transfer to ND96/100 mM HCl/1 mM Ro 20-1724 in microfuge tubes, shaken, incubated at 95°C for 3 min, and centrifuged at 12000 g for 5 min.
  • the changes in cAMP accumulation can alternatively be measured in stably transfected CHO cells expressing the human adenosine receptor subtypes.
  • CHO cells are washed twice in phosphate buffered saline (PBS) and detached in 0.2% EDTA in PBS.
  • the cells are pelleted at 800 rpm for 10 min and resuspended in KRH buffer (140 mM NaCl/5 mM KC1/2 mM CaCl2/1.2 mM MgS04/1.2 mM KH2PO4/6 mM glucose/25 mM Hepe buffer, pH 7.4).
  • KRH buffer 140 mM NaCl/5 mM KC1/2 mM CaCl2/1.2 mM MgS04/1.2 mM KH2PO4/6 mM glucose/25 mM Hepe buffer, pH 7.4
  • the cells are washed once in KRH buffer and resuspended at 10 ⁇ cells/mL.
  • the cell suspension (100 ⁇ L) is mixed with 100 ⁇ L of KRH buffer containing 200 ⁇ M Ro 20-1724 and incubated at 37°C for 10 minutes.
  • Adenosine (10 ⁇ M) was added in 200 ⁇ L KRH buffer containing 200 ⁇ M Ro 20- 1724 and incubated at 37 °C for 20 minutes.
  • 400 ⁇ L of 0.5 mM NaOAc (pH 6.2) was added and the sample was boiled for 20 minutes.
  • the supernatant was recovered by centrifugation for 15 minutes and stored at -70°C cAMP levels were determined by radioimmunoassay (RIANEN kit, DuPont/NEN) using the acetylation protocol.
  • the effect of antagonists on cAMP accumulation are measured by preincubation for 20 minutes before adding adenosine.
  • the 1.7 kb HS-21a cDNA (A3) was subcloned as a Sall- BamHI fragment into the expression vector pCMV5 (Mumby, S.M., Heukeroth, R.O., Gordon, J.I.and Gilman, A.G. (1990) Proc. Natl. Acad. Sci. USA 87, 728-732.) creating the vector pCMV5-A3.
  • CHO or HEK 293 cells stably expressing the human HS-21a cDNA were prepared by co-transfection of 15 ⁇ g pCMV5-A3 and 1 ⁇ g pWLneo (Stratagene) using the calcium phosphate method.
  • Stable cell lines were also generated using EBV based mammalian expression vectors, pREP (INVITROGEN). Neomycin resistant colonies were selected in 1 mg/mL G418 (GIBCO). Stable colonies were screened for expression of HS-21a by 12 5 I-ABA binding.
  • Membranes were prepared from stable CHO cell lines in 10 mM Hepes, pH 7.4 containing 0.1 mM benzamidine and 0.1 mM PMSF as described (Mahan, L.C, et al., (1991) Mol. Pharmacol. 40, 1- 7). Pellets were resuspended in 5 mM Hepes, pH 7.4/5 mM MgCl 2 /0.1 mM benzamidine/0.1 mM PMSF at a protein concentration of 1-2 mg/mL and were incubated with adenosine deaminase (Boehringer Mannheim), 2U/mL at 37 °C for 20 minutes.
  • adenosine deaminase Boehringer Mannheim
  • the binding properties of the receptor encoded by HS-21a were evaluated on membranes prepared from CHO cells stably expressing the HS-21a cDNA.
  • the radioligand, 125 ⁇ _APNEA was previously used to characterize rat A3 adenosine receptors.
  • high non-specific 125l-APNEA binding to CHO cell membranes was observed which interfered with the measurement of specific binding to expressed receptors.
  • Specific and saturable binding of the adenosine receptor agonist, 1 5j_ABA was measured on membranes prepared from the stably transfected cells ( Figure 11 A). The specific binding of 1 5 ⁇ _ABA could be prevented by either 1 mM nonradioactive I-ABA or 400 ⁇ M NECA.
  • the competition of adenosine receptor agonists and antagonists for binding to HS-21a receptors was determined ( Figures 12A and 12B).
  • the Ki values for agonists (top panel) were calculated to be 26 nM for NECA, 34 nM for R-PIA, 89 nM for CPA and 320 nM for S-PIA, resulting in a potency order profile of NECA >R-PIA > CPA > S-PIA.
  • a number of xanthine antagonists exhibited competition with 12 ⁇ _ABA for binding to the HS-21a receptor (lower panel).
  • I- ABOPX is the highest affinity antagonist yet reported for A3 adenosine receptors.
  • Ki values for antagonists were calculated to be 18 nM for I-ABOPX, 55 nM for BW-A1433, 70 nM for XAC and 750 nM for DPCPX, resulting in a potency order profile of I-ABOPX >BW-A1433 > XAC >DPCPX.
  • adenosine as an agonist.
  • Adenosine (10 ⁇ M) produced a 30 % inhibition of the forskolin-stimulated increase in cAMP.
  • adenosine had no effect on the cAMP levels.
  • adenosine had no effect on cAMP levels when measured with or without forskolin treatment.
  • I-ABA produced only about half as much inhibition of forskolin-stimulated cyclic AMP accumulation in CHO cells as did NECA and other agonists (PIA and CPA). Furthermore, in the presence of I-ABA, the dose response curve of NECA to lower cyclic AMP was right-shifted. These data indicate that I-ABA is a partial agonist in this system. Dose-response curves of NECA-induced inhibition of forskolin- stimulated cAMP accumulation were also right shifted in the presence of competing xanthine antagomsts ( Figures 13A through 13F). Schild analyses were used to estimate the Ki from pA 2 values.
  • Ki values determined by competitive binding for various agonists and antagonists are compared with the KA values in the functional cAMP assay in Table 1.
  • the potency order profiles were nearly identical for the binding and functional assays, however, the K a of agonists to lower cAMP were consistently higher (i.e. lower potency) than Kj values determined from competitive binding assays. Although the conditions of these assays differ, these data suggest that recombinant A3 receptors are not well coupled to inhibition of cyclic AMP accumulation in CHO cells.
  • DNA probes conesponding to nucleotides 512-1614, 936-2168, and 321-1540 of accession numbers X68485(A1), X68486(A2a), and X68487(A2b) respectively, and a 1.7 kb Sall-BamHI fragment of HS-21a were labeled with a32p_dCTP by the random priming method. Filters were washed under high stringency conditions in 0.1XSSC at 65°C.
  • RNA from a number of human tissues was evaluated by Northern blot analyses to establish the distribution of tissue expression for the HS-21a transcript (Figure 14A).
  • a 2 kb transcript was most abundantly expressed in lung and liver, with moderate amounts observed in brain and aorta. Low levels of expression were also observed in testis and heart. No expression was detected in spleen or kidney.
  • a human Al transcript (2.9 kb) is expressed in brain, heart, kidney and lung with the most abundant expression observed in the brain.
  • a second hybridizing band of 4.3 kb is also observed in lower amounts in the brain.
  • the A2a adenosine receptor transcript (2.8 kb) is equally expressed in brain, heart and kidney with slightly higher levels of expression detected in the lung.
  • Tewo hybridizing bands were observed when the full length A2a coding sequence was used as a probe and may be the result of cross-hybridization with the A 1 transcript (upper band)).
  • the expression of the A 1 adenosine receptor subtype is most abundant in the cortex (25) and the expression of the A2a adenosine receptor subtype mRNA has been shown by in situ hybridization to be restricted to the caudate, putamen and nucleus accumbens (26).
  • the human brain mRNA utilized in the Northern analysis was prepared from the brain stem, pons, cerebellum, telencephalon, diencephalon and mesencephalon regions of the brain and does not represent enriched transcripts from those regions of the brain in which the most abundant expression of Al and A2a adenosine receptors has been indicated by radioligand binding or in situ hybridization studies.
  • the human A2b subtype two hybridizing transcripts of 1.7kb and 2.1kb were observed in brain, heart and lung. The smaller 1.7kb transcript was more abundant. In contrast to the expression of the Al and A2a adenosine receptor transcripts, no expression of A2b transcript was observed in the kidney. From the comparison of the distribution of human adenosine receptor transcripts, it can be concluded that the subtype transcripts are widely distributed but differ from each other in the abundance found in particular tissues.
  • mast cell degranulation is a component of: myocardial reperfusion injuryu, hypersensitivity reactions (asthma, allergic rhinitis, and urticaria), ischemic bowel disease, autoimmune inflammationa, and atopic dermatitis.
  • the invention consists of the use of any of a series of highly specific A3 adenosine receptor antagonists to treat or prevent these diseases and pathologic effects that result from mast cell degranulation.
  • Adenosine is a potent vasodilator that has also been shown to cause vasoconstriction.
  • the constrictor response has classically been attributed to Al adenosine receptor stimulation or interactions with the renin-angiotensin system.
  • a previously unreported vasoconstrictor action of adenosine in hamster cheek pouch arterioles is described here, and the specific blockade of this response by A3 adenosine receptor antagonists is demonstrated.
  • Adenosine, inosine, cromolyn, compound 48/80, methylene blue, acetylcholine, and components for saline solutions used to bathe arterioles were obtained from Sigma. 8(p- sulphophenyl)theophylline was obtained from Research Biochemicals, Inc (Natick, MA).
  • Arterioles (luminal diameter approximately 60 ⁇ m) were dissected from male Golden hamster cheek pouches, transferred to a 37°C tissue chamber, and cannulated at both ends (see Duling et al., Am. J. Physiol. 241 (Heart Circ. Physiol. 10): H108-H116, 1981 ; Duling et al., Microcirculatory Technology, edited by CH. Baker and W.G. Nastuk, Orlando Academic Press, 1986, p.265-280).
  • mast cells on the abluminal surface of the arteriole serve as foci of the constrictor response.
  • Pretreatment of vessels with 10 ⁇ M cromolyn (sodium cromoglygate, a mast cell stabilizer) blocked the constrictor response induced by compound 48/80, see figures 18 and 19. Inhibition of degranulation was confirmed by observation of intact methylene blue- stained mast cells. Adenosine A3 receptors have recently been found on mast cells.
  • A3 receptors We tested the involvement of A3 receptors in the following manner: Treatment of the arterioles with the agonist IABA (30 ⁇ M, applied via pipette) resulted in vasoconstriction (see figure 17). Pretreatment with 10 ⁇ M BWA 1433, characterized herein as an A3 specific adenosine receptor antagonist, resulted in complete abolition of the constrictor response and inhibition of mast cell degranulation (see figure 20). The Al specific antagonist, 8PST (10 ⁇ M) had no effect on the IABA induced vasoconstriction, proving that the constrictor response is A3 receptor mediated and that this response can be blocked by contacting the A3 receptors exhibited on the mast cells with an A3 specific antagonist.

Abstract

La présente invention concerne l'utilisation de composés identifiés grâce à l'utilisation des récepteurs A1, A2a, A2b et A3 recombinant l'adénosine humaine. L'invention concerne également les essais fonctionnels permettant de moduler de façon spécifique le rôle physiologique de l'activation de l'adénosine et de ses différents récepteurs.
PCT/US1994/012272 1993-10-29 1994-10-26 Antagonistes du recepteur de l'adenosine humaine WO1995011681A1 (fr)

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Cited By (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2288733A (en) * 1994-04-25 1995-11-01 Merck & Co Inc A3 adenosine receptor subtype specific antagonists for alteration of eosinophil properties
US5780481A (en) * 1996-08-08 1998-07-14 Merck & Co., Inc. Method for inhibiting activation of the human A3 adenosine receptor to treat asthma
EP0884318A1 (fr) * 1996-10-07 1998-12-16 Kyowa Hakko Kogyo Co., Ltd. Derives de purine fondue
WO1999021555A2 (fr) * 1997-10-27 1999-05-06 Takeda Chemical Industries, Ltd. Antagonistes des recepteurs de l'adenosine a¿3?
WO1999042093A2 (fr) * 1998-02-24 1999-08-26 University Of Virginia Antagonistes de recepteurs a2b d'adenosine humaine
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US6232297B1 (en) 1999-02-01 2001-05-15 University Of Virginia Patent Foundation Methods and compositions for treating inflammatory response
US6303619B1 (en) * 1998-03-12 2001-10-16 University Of Virginia Meta-substituted acidic 8-phenylxanthine antagonists of A3 human adenosine receptors
US6322771B1 (en) 1999-06-18 2001-11-27 University Of Virginia Patent Foundation Induction of pharmacological stress with adenosine receptor agonists
US6448235B1 (en) 1994-07-11 2002-09-10 University Of Virginia Patent Foundation Method for treating restenosis with A2A adenosine receptor agonists
US6514949B1 (en) 1994-07-11 2003-02-04 University Of Virginia Patent Foundation Method compositions for treating the inflammatory response
WO2003048120A2 (fr) 2001-11-30 2003-06-12 Osi Pharmaceuticals, Inc. Composes specifiques des recepteurs a1 et a3 de l'adenosine et leurs utilisations
US6664252B2 (en) 1999-12-02 2003-12-16 Osi Pharmaceuticals, Inc. 4-aminopyrrolo[2,3-d]pyrimidine compounds specific to adenosine A2a receptor and uses thereof
US6670334B2 (en) 2001-01-05 2003-12-30 University Of Virginia Patent Foundation Method and compositions for treating the inflammatory response
US6673802B2 (en) 2000-12-01 2004-01-06 Osi Pharmaceuticals, Inc. Compounds specific to adenosine A3 receptor and uses thereof
US6680322B2 (en) 1999-12-02 2004-01-20 Osi Pharmaceuticals, Inc. Compounds specific to adenosine A1 receptors and uses thereof
US6680324B2 (en) 2000-12-01 2004-01-20 Osi Pharmaceuticals, Inc. Compounds specific to adenosine A1 receptors and uses thereof
US6686366B1 (en) 1998-06-02 2004-02-03 Osi Pharmaceuticals, Inc. Compounds specific to adenosine A3 receptor and uses thereof
WO2003042214A3 (fr) * 2001-11-09 2004-03-04 Cv Therapeutics Inc Antagonistes de recepteur d'adenosine a¿2b?
US6800633B2 (en) 1998-06-02 2004-10-05 Osi Pharmaceuticals, Inc. Pyrrolo[2,3d]pyrimidine compositions and their use
WO2004086052A2 (fr) * 2003-03-28 2004-10-07 Bayer Healthcare Ag Produits de diagnostic et de therapeutique pour des maladies liees au recepteur de l'adenosine a2b couple a la proteine g (adora2b)
US6806270B2 (en) 1999-08-31 2004-10-19 Vanderbilt University Selective antagonists of A2B adenosine receptors
WO2004106337A1 (fr) * 2003-05-06 2004-12-09 Cv Therapeutics, Inc. Derives de xanthine servant d'antagonistes du recepteur de l'adenosine a2b
US6878716B1 (en) 1998-06-02 2005-04-12 Osi Pharmaceuticals, Inc. Compounds specific to adenosine A1 receptor and uses thereof
WO2005063246A1 (fr) * 2003-12-29 2005-07-14 Can-Fite Biopharma Ltd. Methode de traitement de la sclerose en plaques
JP2005533054A (ja) * 2002-06-12 2005-11-04 バイオジェン・アイデック・エムエイ・インコーポレイテッド アデノシンレセプターアンタゴニストを使用して虚血再灌流障害を処置する方法
US6977300B2 (en) 2001-11-09 2005-12-20 Cv Therapeutics, Inc. A2B adenosine receptor antagonists
EP1666041A2 (fr) * 1997-09-05 2006-06-07 Kyowa Hakko Kogyo Co., Ltd. Derivés de la xanthine contre désordres neurodégénératifs
US7125993B2 (en) 2001-11-09 2006-10-24 Cv Therapeutics, Inc. A2B adenosine receptor antagonists
EP1731520A1 (fr) 1999-12-02 2006-12-13 OSI Pharmaceuticals, Inc. Dérivés de pyrrolo[2,3-d]pyrimidine comme antagonistes d'adénosine A1, A2A et A3
US7160890B2 (en) 1999-12-02 2007-01-09 Osi Pharmaceuticals, Inc. Compounds specific to adenosine A3 receptor and uses thereof
US7214665B2 (en) 2001-10-01 2007-05-08 University Of Virginia Patent Foundation 2-propynyl adenosine analogs having A2A agonist activity and compositions thereof
US7253176B1 (en) * 2000-05-04 2007-08-07 K.U. Leuven Research & Development Immunosuppressive effects of 8-substituted xanthine derivatives
US7378400B2 (en) 1999-02-01 2008-05-27 University Of Virginia Patent Foundation Method to reduce an inflammatory response from arthritis
US7423144B2 (en) 2004-05-26 2008-09-09 Inotek Pharmaceuticals Corporation Purine Derivatives as adenosine A1 receptor agonists and methods of use thereof
US7427606B2 (en) * 1999-02-01 2008-09-23 University Of Virginia Patent Foundation Method to reduce inflammatory response in transplanted tissue
US7442687B2 (en) 2004-08-02 2008-10-28 The University Of Virginia Patent Foundation 2-polycyclic propynyl adenosine analogs having A2A agonist activity
US7501407B2 (en) 2001-12-20 2009-03-10 Osi Pharmaceuticals, Inc. Pyrimidine A2B selective antagonist compounds, their synthesis and use
US7576069B2 (en) 2004-08-02 2009-08-18 University Of Virginia Patent Foundation 2-polycyclic propynyl adenosine analogs having A2A agonist activity
US7605143B2 (en) 2004-08-02 2009-10-20 University Of Virginia Patent Foundation 2-propynyl adenosine analogs with modified 5′-ribose groups having A2A agonist activity
US7645754B2 (en) 2001-12-20 2010-01-12 Osi Pharmaceuticals, Inc. Pyrrolopyrimidine A2B selective antagonist compounds, their synthesis and use
US7732424B2 (en) 2005-11-30 2010-06-08 Inotek Pharmaceuticals Corporation Purine derivatives and methods of use thereof
US7863253B2 (en) 2004-09-20 2011-01-04 Inotek Pharmaceuticals Corporation Purine Derivatives and methods of use thereof
US7956179B2 (en) 2006-02-03 2011-06-07 Gilead Sciences, Inc. Process for preparing an A2A-adenosine receptor agonist and its polymorphs
US8071566B2 (en) 2000-02-23 2011-12-06 Gilead Sciences, Inc. Methods of coronary imaging
US8106029B2 (en) 2004-10-20 2012-01-31 Gilead Sciences, Inc. Use of A2A adenosine receptor agonists
US8133879B2 (en) 2002-07-29 2012-03-13 Gilead Sciences, Inc. Myocardial perfusion imaging methods and compositions
US8183226B2 (en) 2002-07-29 2012-05-22 Gilead Sciences, Inc. Myocardial perfusion imaging method
US8193200B2 (en) 2007-01-04 2012-06-05 University Of Virginia Patent Foundation Antagonists of A2B adenosine receptors for treatment of inflammatory bowel disease
AU2011224104B2 (en) * 2003-05-06 2012-06-28 Gilead Sciences, Inc. A2B adenosine receptor antagonists
US8324224B2 (en) 2001-11-09 2012-12-04 Gilead Sciences, Inc. A2B adenosine receptor antagonists
US8470801B2 (en) 2002-07-29 2013-06-25 Gilead Sciences, Inc. Myocardial perfusion imaging methods and compositions
US9045519B2 (en) 1999-06-22 2015-06-02 Gilead Sciences, Inc. N-pyrazole A2A receptor agonists
US9278991B2 (en) 2012-01-26 2016-03-08 Inotek Pharmaceuticals Corporation Anhydrous polymorphs of [(2R,3S,4R,5R)-5-(6-(cyclopentylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)} methyl nitrate and processes of preparation thereof
US9289383B2 (en) 2010-03-26 2016-03-22 Inotek Pharmaceuticals Corporation Method of reducing intraocular pressure in humans
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USRE47351E1 (en) 1999-06-22 2019-04-16 Gilead Sciences, Inc. 2-(N-pyrazolo)adenosines with application as adenosine A2A receptor agonists

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4879296A (en) * 1985-04-27 1989-11-07 Burroughs Wellcome Co. Xanthine derivatives
US5298508A (en) * 1988-07-19 1994-03-29 The United States Of America As Represented By The Department Of Health And Human Services Irreversible inhibitors of adenosine receptors

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4879296A (en) * 1985-04-27 1989-11-07 Burroughs Wellcome Co. Xanthine derivatives
US5298508A (en) * 1988-07-19 1994-03-29 The United States Of America As Represented By The Department Of Health And Human Services Irreversible inhibitors of adenosine receptors

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BRITISH JOURNAL OF PHARMACOLOGY, Volume 109, No. 1, issued May 1993, FOZARD et al., "Adenosine A3 Receptors Mediate Hypotension in the Angiotensin II-Supported Circulation of the Pithed Rat", pages 3-5. *
DRUG DEVELOPMENT RESEARCH, Volume 30, issued 1993, CARRUTHERS et al., "Hypotensive Responses to the Putative Adenosine A3 Receptor Agonist N6-2-(4-Aminophenyl)-Ethyladenosine in the Rat", pages 147-152. *
INTERNATIONAL ARCHIVE OF ALLERGY AND APPLIED IMMUNOLOGY, Volume 98, issued 1982, OTT et al., "Effects of Adenosine on Histamine Release From Human Lung Fragments", pages 50-56. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 268, No. 23, issued 15 August 1993, RAMKUMAR et al., "The A3 Adenosine Receptor is the Unique Adenosine Receptor Which Facilitates Release of Allergic Mediators in Mast Cells", pages 16887-16890. *
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, Volume 258, No. 3, issued 1991, ALI et al., "Methylxanthines Block Antigen-Induced Responses In RBL-2H3 Cells Independently of the Adenosine Receptors or Cyclic AMP: Evidence for the Inhibition of Antigen Binding to IgE", pages 954-962. *
MOLECULAR PHARMACOLOGY, Volume 44, issued 20 September 1993, LINDEN et al., "Molecular Cloning and Functional Expression of a Sheep A3 Adenosine Receptor With Widespread Tissue Distribution", pages 524-532. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 80, issued April 1983, BRUNS et al., "Adenosine Receptor Binding: Structure-Activity Analysis Generates Extremely Potent Xanthine Antagonists", pages 2077-2080. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 89, issued August 1992, ZHOU et al., "Molecular Cloning and Characterization of an Adenosine Receptor: the A3 Adenosine Receptor", pages 7432-7436. *

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EP2050751A1 (fr) 2001-11-30 2009-04-22 OSI Pharmaceuticals, Inc. Composés spécifiques aux récepteurs A1 et A3 de l'adénosine et leur utilisation
US7645754B2 (en) 2001-12-20 2010-01-12 Osi Pharmaceuticals, Inc. Pyrrolopyrimidine A2B selective antagonist compounds, their synthesis and use
US7501407B2 (en) 2001-12-20 2009-03-10 Osi Pharmaceuticals, Inc. Pyrimidine A2B selective antagonist compounds, their synthesis and use
JP2005533054A (ja) * 2002-06-12 2005-11-04 バイオジェン・アイデック・エムエイ・インコーポレイテッド アデノシンレセプターアンタゴニストを使用して虚血再灌流障害を処置する方法
US9289446B2 (en) 2002-07-29 2016-03-22 Gilead Sciences, Inc. Myocardial perfusion imaging methods and compositions
US8906878B2 (en) 2002-07-29 2014-12-09 Gilead Sciences, Inc. Myocardial perfusion imaging methods and compositions
US8133879B2 (en) 2002-07-29 2012-03-13 Gilead Sciences, Inc. Myocardial perfusion imaging methods and compositions
US8470801B2 (en) 2002-07-29 2013-06-25 Gilead Sciences, Inc. Myocardial perfusion imaging methods and compositions
US8183226B2 (en) 2002-07-29 2012-05-22 Gilead Sciences, Inc. Myocardial perfusion imaging method
WO2004086052A2 (fr) * 2003-03-28 2004-10-07 Bayer Healthcare Ag Produits de diagnostic et de therapeutique pour des maladies liees au recepteur de l'adenosine a2b couple a la proteine g (adora2b)
WO2004086052A3 (fr) * 2003-03-28 2005-03-17 Bayer Healthcare Ag Produits de diagnostic et de therapeutique pour des maladies liees au recepteur de l'adenosine a2b couple a la proteine g (adora2b)
WO2004106337A1 (fr) * 2003-05-06 2004-12-09 Cv Therapeutics, Inc. Derives de xanthine servant d'antagonistes du recepteur de l'adenosine a2b
AU2011224104B2 (en) * 2003-05-06 2012-06-28 Gilead Sciences, Inc. A2B adenosine receptor antagonists
US7465715B2 (en) 2003-12-29 2008-12-16 Can-Fite Biopharma, Ltd. Method for treatment of multiple sclerosis
WO2005063246A1 (fr) * 2003-12-29 2005-07-14 Can-Fite Biopharma Ltd. Methode de traitement de la sclerose en plaques
US8183224B2 (en) 2004-05-26 2012-05-22 Inotek Pharmaceuticals Corporation Purine derivatives as adenosine A1 receptor agonists and methods of use thereof
US7423144B2 (en) 2004-05-26 2008-09-09 Inotek Pharmaceuticals Corporation Purine Derivatives as adenosine A1 receptor agonists and methods of use thereof
US8609833B2 (en) 2004-05-26 2013-12-17 Inotek Pharmaceuticals Corporation Purine derivatives as adenosine A1 receptor agonists and methods of use thereof
US7605143B2 (en) 2004-08-02 2009-10-20 University Of Virginia Patent Foundation 2-propynyl adenosine analogs with modified 5′-ribose groups having A2A agonist activity
US7442687B2 (en) 2004-08-02 2008-10-28 The University Of Virginia Patent Foundation 2-polycyclic propynyl adenosine analogs having A2A agonist activity
US7576069B2 (en) 2004-08-02 2009-08-18 University Of Virginia Patent Foundation 2-polycyclic propynyl adenosine analogs having A2A agonist activity
US7989431B2 (en) 2004-08-02 2011-08-02 University Of Virginia Patent Foundation 2-propynyl adenosine analogs with modified 5′-ribose groups having A2A agonist activity
US7863253B2 (en) 2004-09-20 2011-01-04 Inotek Pharmaceuticals Corporation Purine Derivatives and methods of use thereof
US8133880B2 (en) 2004-09-20 2012-03-13 Inotek Pharmaceuticals Corporation Purine derivatives and methods of use thereof
US8106029B2 (en) 2004-10-20 2012-01-31 Gilead Sciences, Inc. Use of A2A adenosine receptor agonists
US7732424B2 (en) 2005-11-30 2010-06-08 Inotek Pharmaceuticals Corporation Purine derivatives and methods of use thereof
US7956179B2 (en) 2006-02-03 2011-06-07 Gilead Sciences, Inc. Process for preparing an A2A-adenosine receptor agonist and its polymorphs
US9085601B2 (en) 2006-02-03 2015-07-21 Gilead Sciences, Inc. Process for preparing an A2A-adenosine receptor agonist and its polymorphs
US8106183B2 (en) 2006-02-03 2012-01-31 Gilead Sciences, Inc. Process for preparing an A2A-adenosine receptor agonist and its polymorphs
USRE47301E1 (en) 2006-02-03 2019-03-19 Gilead Sciences, Inc. Process for preparing an A2A-adenosine receptor agonist and its polymorphs
US8193200B2 (en) 2007-01-04 2012-06-05 University Of Virginia Patent Foundation Antagonists of A2B adenosine receptors for treatment of inflammatory bowel disease
US9370530B2 (en) 2010-01-11 2016-06-21 Inotek Pharmaceuticals Corporation Combination, kit and method of reducing intraocular pressure
US9289383B2 (en) 2010-03-26 2016-03-22 Inotek Pharmaceuticals Corporation Method of reducing intraocular pressure in humans
US9718853B2 (en) 2012-01-26 2017-08-01 Inotek Pharmaceuticals Corporation Anhydrous polymorphs of [(2R,3S,4R,5R)-5-(6-(cyclopentylamino)-9H-purin-9-YL)-3,4-dihydroxytetrahydrofuran-2-YL)] methyl nitrate and processes of preparation thereof
US9278991B2 (en) 2012-01-26 2016-03-08 Inotek Pharmaceuticals Corporation Anhydrous polymorphs of [(2R,3S,4R,5R)-5-(6-(cyclopentylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)} methyl nitrate and processes of preparation thereof
US9522160B2 (en) 2013-03-15 2016-12-20 Inotek Pharmaceuticals Corporation Ophthalmic formulations

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