WO2012125400A1 - Compositions and methods using adenosine a3 receptor antagonists for the treatment of inflammatory eye diseases - Google Patents

Compositions and methods using adenosine a3 receptor antagonists for the treatment of inflammatory eye diseases Download PDF

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WO2012125400A1
WO2012125400A1 PCT/US2012/028247 US2012028247W WO2012125400A1 WO 2012125400 A1 WO2012125400 A1 WO 2012125400A1 US 2012028247 W US2012028247 W US 2012028247W WO 2012125400 A1 WO2012125400 A1 WO 2012125400A1
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group
adenosine
eye
alkyl
receptor antagonist
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PCT/US2012/028247
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French (fr)
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Alan Jeffrey Jacobs
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Acorn Biomedical, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4422—1,4-Dihydropyridines, e.g. nifedipine, nicardipine
    • 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
    • A61K31/70—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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the invention relates to compositions of adenosine A3 receptor antagonists and methods of their use for the treatment of inflammatory eye diseases.
  • Inflammatory eye diseases such as dry eye and uveitis, are serious conditions that can cause eye pain, redness, discomfort, or sensitivity to light.
  • the inflammatory eye disease can be unresponsive to treatment and may lead to blindness with glaucoma, cataract or retinopathy.
  • Treatment options for inflammatory eye disease are limited to steroids, non-steroid anti-inflammatory drugs (NSAIDs), immunosuppressive chemotherapy medications or immunomodulatory therapy (IMT).
  • NSAIDs non-steroid anti-inflammatory drugs
  • IMT immunomodulatory therapy
  • Conventional treatments may not reverse or stop progression of the eye disease.
  • inflammatory eye diseases can be difficult to treat and may recur after treatment.
  • Adenosine receptors belong to the G protein-coupled family of proteins and are characterized by seven transmembrane helical domains. Receptor activation leads to its internalization and the subsequent inhibition of: (i) adenylyl cyclase activity, (ii) cAMP formation and (iii) protein kinase A (PKA) expression, resulting in the initiation of various signaling pathways.
  • PKA protein kinase A
  • Adenosine A3 receptors are involved in a variety of important patho-physiological processes, including modulation of cerebral and cardiac ischemic damage, inflammation, modulation of intraocular pressure, regulation of normal and tumor cell growth, and immune suppression.
  • adenosine A3 receptor agonists and antagonists have been identified, such as adenosine derivatives, fiavonoids, and triazoloquinazolines, which specifically bind to the adenosine A3 receptor. See e.g., PCT/KR2007/001131; U.S. Patent No. 6,376,521; WO 2010/014921; U.S. Patent No. 6,199,127.
  • Agonists of the adenosine A3 receptor have been shown to desensitize the adenosine A3 receptor. Palmer and Stiles. (1999) Molecular Pharmacology; Palmer, T.M. et al. (1995) J. Biol. Chem.
  • CF101 adenosine A3 receptor agonist
  • the invention addresses these and other shortcomings of the prior art by providing methods for treating inflammatory eye disease using adenosine A3 receptor antagonist compositions.
  • Methods of the invention include treating an inflammatory eye disease by
  • the inflammatory eye disease is selected from the group consisting of: dry eye, uveitis, scleritis, blepharitis, keratitis, conjunctivitis, chorioretinital inflammation, choroiditis, retinitis, iridocyclitis, ulceris, posterior cyclitis, diabetic retinopathy, a post-surgery condition, allergies, eye trauma, eye bruises, foreign body, toxin exposure, chemical exposure, food allergies, hives, rheumatoid arthritis, sarcoidosis, juvenile idiopathic arthritis, lupus, Behcet's disease, viral infection, fungal infection, mycobacterial infection, parasite infection, bacterial infection, acanthamoeba infection, spirochete infection, Crohn's disease, arthritis, ulcerative colitis, lympho
  • the adenosine A3 receptor antagonist comprises any of the compositions described herein.
  • the adenosine A3 receptor antagonist is selected from the group consisting of: ACN-1052, LJ1251, MRS3820, MRS3771, LJ 979, MRS 3826, MRS 3827, MRS1191 or MRS 1292.
  • the inflammatory eye disease is uveitis
  • the adenosine A3 receptor antagonist is selected from the group consisting of: ACN-1052, LJ1251, or MRS3820.
  • the inflammatory eye disease is dry eye and the adenosine A3 receptor antagonist is selected from the group consisting of: ACN-1052, LJ1251, MRS3820, MRS 3771, LJ 979, MRS 3826, or MRS3827.
  • the adenosine A3 receptor antagonist is ACN-1052.
  • administering an effective amount of an adenosine A3 receptor antagonist reduces corneal inflammation. In one embodiment, administering an effective amount of an adenosine A3 receptor antagonist reduces inflammatory cytokine levels.
  • the mammalian subject is a human subject.
  • the adenosine A3 receptor antagonist is formulated for topical delivery to an eye of the mammalian subject. In other embodiments, the adenosine A3 receptor antagonist is formulated for topical delivery, intraocular injection, intravitreal injection, oral delivery, sublingual delivery, intranasal delivery, intravenous injection, intramuscular injection, intraperitoneal injection, or rectal delivery.
  • the method includes co-administering to the mammalian subject an effective amount of a steroid, a non-steroidal anti-inflammatory, or an
  • the immunosuppressant is cyclosporine A.
  • FIG. la shows a graph of tear reduction in mice after scopolamine patch application under dry environmental conditions.
  • FIG. lb shows histopathology scores 12 days after scopolamine administration in mice.
  • “a” indicates that the p-value is less than 0.05 relative to the vehicle control group
  • “b” indicates that the p-value was less than 0.05 reltaive to the no- treatment group.
  • N 10 female C57BL/6 mice per group.
  • FIG. 2 shows the effects of Dexamethasone (DEX) and cyclosporine (CSA, Restasis) in a Tear Break Up Test (TBUT) in concanavalin A (ConA)-induced dry eye in rabbits.
  • DEX Dexamethasone
  • CSA cyclosporine
  • ConA concanavalin A
  • FIG. 3a shows the effects of ConA injection on the Schirmer Tear Test (STT) in rabbits.
  • FIG. 3b shows the use of DEX and CSA in the Schirmer Tear Test (STT) in rabbits.
  • FIGs. 4a-4c shows representative photographs of the anterior area of the eye of a rat with a melanin-associated antigen (MAA) induced anterior uveitis.
  • MAA melanin-associated antigen
  • FIG. 5 shows histopathology scores 18 days after MAA injection in untreated rats and rats treated with vehicle, DEX, CSA, or Restasis.
  • FIGs. 6a-6c illustrates the photomicroscopy of histopathology findings in MAA- treated rats.
  • FIG. 7 shows inhibition of corneal injury by ACN-1052 in a mouse model of dry eye syndrome measured by corneal staining.
  • FIG. 8 shows the effect of ACN-1052 on dry eye syndrome in a mouse model measured by corneal staining.
  • Adenosine refers to a purine nucleoside and a metabolite of adenosine triphosphate (ATP).
  • Adenosine is a nucleoside composed of a molecule of adenine attached to a ribose sugar molecule (ribofuranose) moiety via a P-N9-glycosidic bond.
  • Adenosine plays an important role in biochemical processes, such as energy transfer, as adenosine triphosphate (ATP) and adenosine diphosphate (ADP), as well as in signal transduction as cyclic adenosine monophosphate, cAMP.
  • the IUPAC name is (2R,3R,4S,5R)-2-(6-amino-9H- purin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol.
  • Adenosine mediates a wide variety of physiological functions by interacting with four cell surface receptors namely Al, A2a, A2b and A3.
  • Adenosine receptor refers to a class of purinergic receptors or G protein-coupled receptors that bind adenosine as an endogenous ligand.
  • Adenosine receptors include four types: Al, A2a, A2b, and A3.
  • Adenosine A3 receptor or "A3AR” or “A3 Adenosine Receptor” refers to a G protein-coupled receptor that is involved in a variety of intracellular signaling pathways and physiological functions.
  • Adenosine A3 receptors are ubiquitously expressed in a wide variety of tissues in human body, with high levels in peripheral organs and low levels in the brain.
  • Adenosine A3 receptors are involved in a variety of important patho-physiological processes, including modulation of cerebral and cardiac ischemic damage, inflammation, modulation of intraocular pressure, regulation of normal and tumor cell growth, and immunosuppression.
  • Adenosine A3 receptors are also involved in the inhibition of neutrophil degranulation in neutrophil-mediated tissue injury, have been implicated in both neuroprotective and neurodegenerative effects, and may also mediate both cell proliferation and cell death.
  • the adenosine A3 receptor is encoded by the mR A with
  • GenBank Accession No. NM 000677 and has the protein sequence of GenBank Accession No. NP 000668.
  • the adenosine A3 receptor is encoded by the mRNA with
  • GenBank Accession No. NM 009631 Citations in this specification to GenBank Accession Numbers refer to those sequence versions current as of the filing date of this specification.
  • Adenosine A3 receptor antagonist or “A3AR Ag” refers to a ligand, molecule, composition, or drug that binds to an adenosine A3 receptor and inhibits or dampens agonist- mediated responses.
  • the A3AR Ag exerts its effect through binding and inactivation of the A3AR.
  • Adenosine A3 receptor antagonists can have affinity but no efficacy for their cognate receptors, and binding can disrupt the interaction and inhibit the function of an agonist or inverse agonist at the receptors.
  • Antagonists refer to receptor ligands, molecules, compositions or drugs that activate a cellular response by binding to a receptor.
  • Antagonists refer to receptor ligands, molecules, compositions or drugs that inhibit the receptor's agonist response, in some cases by blocking the receptor from the agonist.
  • Competitive antagonists or surmountable antagonists
  • the level of receptor inhibition is determined by the relative affinity of each molecule for the site and their relative concentrations.
  • Noncompetitive antagonists or non-surmountable antagonists are allosteric antagonists. These antagonists bind to a distinctly separate binding site from the agonist, exerting their action to that receptor via the other binding site.
  • Inflammatory eye disease refers to disorders, diseases, or conditions that affect the eye, including, but not limited to, dry eye, uveitis, scleritis, blepharitis, keratitis,
  • conjunctivitis chorioretinital inflammation, choroiditis, retinitis, iridocyclitis, ulceris, posterior cyclitis, diabetic retinopathy, post-surgery, allergy, trauma, bruises, foreign body, exposure to toxins or chemicals, food allergies, hives, autoimmune disorders (such as rheumatoid arthritis, sarcoidosis, juvenile idiopathic arthritis, lupus, Behcet's disease, etc.), infection (such as viral, fungal, mycobacteria, parasite, bacteria, acanthamoeba, spirochete, etc.), inflammatory conditions (such as Crohn's disease, arthritis and ulcerative colitis) and cancers (such as lymphoma, eye tumors, cancer of blood vessels, etc.), or an inflammatory retinal disease (such as neuroretinitis, Sarcoidosis-related retinitis, Behcet' s-related retinitis, or acute retinal
  • In vivo refers to processes that occur in a living organism.
  • Effective amount refers to a sufficient amount of an agent to provide the desired biological result.
  • the result can be a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • an "effective amount” for therapeutic uses is the amount of the composition comprising an active composition herein required to provide a clinically significant decrease in inflammation in the eye, and thereby reducing symptoms of an eye disease, e.g. , uveitis, such as blurred vision or eye pain.
  • Treatment or “treatment” are used interchangeably and are meant to indicate
  • compositions in accordance with the methods of the invention further include ameliorating existing eye symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, and/or promoting resolution of the disease or condition.
  • “Ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., an eye disease, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.
  • pharmaceutically acceptable or “pharmacologically acceptable” is meant a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual without causing any substantial undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • a “subject” encompasses mammals.
  • a “mammal” includes any member of the Mammalia class: humans, non-human primates (chimpanzees, and other apes and monkey species), canines, felines, murines, bovines, equines, porcines, etc.
  • Other examples of mammals include cattle, horses, sheep, goats, swine, rabbits, dogs, cats, and rodents, such as rats, mice and guinea pigs, and the like.
  • the terms "subject” or “mammal” do not denote a particular age or gender.
  • Aryl refers to aromatic moieties such as phenyl, naphthyl, anthracenyl, and biphenyl.
  • Heterocyclyl refers to 3-7 membered rings which can be saturated or unsaturated or heteroaromatic, comprising carbon and one or more heteroatoms such as O, N, and S, and optionally hydrogen; optionally in combination with one or more aromatic rings.
  • heterocyclyl groups include pyridyl, piperidinyl, piperazinyl, pyrazinyl, pyrolyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, pyrrolidinyl, furanyl, tetrahydrofuranyl, thienyl, furyl, thiophenyl, tetrahydrothiophenyl, purinyl, pyrimidinyl, thiazolyl, thiazolidinyl, thiazolinyl, oxazolyl, tetrazolyl, tetrazinyl, benzoxazolyl, morpholinyl, thiomorpholinyl, quinolinyl, and isoquinolinyl.
  • heteroaryl alkyl include heteroaryl methyl such as 2- or 3- methyl substituted groups, e.g., thienylmethyl, pyridylmethyl,
  • Alkyl, alkoxy, and alkylamino groups can be linear or branched.
  • a substituent e.g., halo, amino, alkyl, hydroxyl, alkoxy, and others
  • the aromatic ring hydrogen is replaced with the substituent and this can take place in any of the available hydrogens, e.g., 2, 3, 4, 5, and/or 6-position wherein the 1 -position is the point of attachment of the aryl group in the composition of the present invention.
  • Halo refers to fluorine, chlorine, bromine, and iodine.
  • compositions of the invention are provided.
  • compositions of the invention comprise adenosine A3 receptor antagonists.
  • compositions of the invention comprise adenosine derivatives, dihydropyridine derivatives, pyridine derivatives, or purine nucleosides.
  • composition of the invention comprises:
  • R is 3-iodobenzyl.
  • ACN-1052 also called LJ1251 or MRS3820
  • 2R,3R,4S is the chemical name (2R,3R,4S)-2-(6-(3-iodobenzylamino)-2- chloro-9H-purin-9-yl)-tetrahydrothiophene-3,4-diol. It is described in Jeong et al. (2007) Journal of Medicinal Chemistr .
  • ACN-1052 is also shown below:
  • ACN-1052 is freely soluble in DMSO and various mixtures of organic solvents such as DCM and DMSO. It is not appreciably soluble in water-based vehicles with low concentrations of organics such as DMSO or ethanol.
  • ACN-1052 can be synthesized by the following steps:
  • composition of the invention is selected from the group comprising:
  • a 3 ⁇ 4 antagonist prodrugs A antagonist ⁇ truncated ⁇
  • composition of the invention comprises:
  • MRS 1292 is derived from the agonist IB-MECA (N 6 -(3-iodobenzyl)-adenosine-5'-N- methyluronamide). MRS 1292 is described in Yang et al. (2005) Current Eye Research. 30:747-754. MRS 1292 can be synthesized by the following steps, described in Besada et al. (2006) Collect. Czech. Chem. Commun. 71(6):912-928.
  • composition of the invention comprises:
  • MRS 1191 has the IUPAC name 03-ethyl 05-(phenylmethyl) 2-methyl-6-phenyl-4- (2-phenylethynyl)-l ,4-dihydropyridine-3,5-dicarboxylate. MRS 1191 is described in Jacobson et al. (1997) Neuropharmacology. 36(9):1 157-1165. MRS 1191 can be synthesized by the following steps, wherein R 4 is phenylethynyl.
  • composition of the invention comprises:
  • R 1 is selected from the group consisting of hydrogen, Ci-C 6 alkyl, Ci-C 6 alkoxy, hydroxyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkyl Ci-C 6 alkyl, C 3 -C 8 dicycloalkyl Ci-C 6 alkyl, C 7 -C 12 bicycloalkyl Ci-C 6 alkyl, C 7 -C 14 tricycloalkyl Ci-C 6 alkyl, C 6 -Ci 4 aryl, C 6 -Ci 4 aryl Ci- C 6 alkyl, C 6 -Ci 4 diaryl Ci-C 6 alkyl, C 6 -Ci 4 aryl Ci-C 6 alkoxy, Ci-C 6 alkyl carbonyl, sulfonyl, Ci-C 6 alkyl sulfonyl, C 6 -Cj 4 aryl sulfonyl, heterocyclyl Ci-C 6 alkyl carbony
  • R 2 is selected from the group consisting of hydrogen, halo, amino, hydrazido, mercapto, C1-C20 alkylamino, C 6 -Ci4 aryl amino, C 6 -Ci4 aryloxy, C1-C20 alkyl, C1-C20 alkoxy, C1-C20 thioalkoxy, pyridylthio, C7-C12 cycloalkyl C1-C20 alkyl, C7-C12 bicycloalkyl C1-C20 alkyl, C7-C2 bicycloalkenyl C1-C20 alkyl, C 6 -Ci4 aryl C1-C20 alkyl, C2-C20 alkenyl, C7- C12 cycloalkyl C 2 -C 2 o alkenyl, C7-C12 bicycloalkyl C 2 -C 2 o alkenyl, C7-C12 bicycloalkyl C 2
  • R 3 and R 4 are independently selected from the group consisting of hydroxyl, amino, thiol, ureido, Ci-C 6 alkyl carbonylamino, hydroxy Ci-C 6 alkyl, and hydrazinyl; and R 5 is selected from the group consisting of hydrogen, Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, heteroaryl, and Ci-C 6 aminoalkyl; or a pharmaceutically acceptable salt.
  • R 1 is selected from the group consisting of C 6 -Ci4 aryl Ci-C 6 alkyl and C 6 -Ci4 aryl C3-C8 cycloalkyl, wherein the aryl portion of R 1 is optionally substituted with one or more substituents selected from the group consisting of halo, amino, Ci-C 6 alkyl, Ci-C 6 alkoxy, C 6 -Ci4 aryloxy, hydroxy Ci-C 6 alkyl, hydroxy C 2 -C 6 alkenyl, hydroxy C 2 -C 6 alkynyl, aminocarbonyl Ci-C 6 alkoxy, and C 6 -Ci4 aryl Ci-C 6 alkoxy; and in one embodiment, R 1 is selected from the group consisting of benzyl, phenyl cyclopropyl, or 1-naphthyl methyl, wherein the phenyl or naphthyl portion of R 1 is optionally substituted with
  • R 1 is benzyl, phenyl cyclopropyl, or 1- naphthyl methyl, wherein the phenyl or naphthyl portion of R 1 is optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, and alkoxy.
  • R 1 are benzyl and benzyl substituted with one or more substituents selected from the group consisting of halo and Ci-C 6 alkoxy.
  • R 1 is selected from the group consisting of 3-chlorobenzyl, 3-bromobenzyl, 3-iodobenzyl, 2-hydroxy-5-methoxy- benzyl, and 2,5-dimethoxybenzyl.
  • the phenyl cyclopropyl is traro-2- phenyl- 1 -cyclopropyl.
  • R 3 and R 4 are particularly hydroxyl.
  • R 5 is hydrogen.
  • adenosine A3 receptor antagonists include those described in WO 2010/014921 A2 (PCT/US2009/052439), which is incorporated by reference in its entirety.
  • the composition comprises:
  • R is a linear or branched Ci-C 5 alkyl which is non-substituted or is independently or selectively substituted with one or more C 6 -Cio aryl groups, a benzyl which is non-substituted or is independently or selectively substituted with halogen or one or more linear or branched C 1 -C4 alkoxy groups, or a hydroxycarbonyl-substituted benzyl; and
  • Y is H or a halogen atom.
  • A is O or S
  • R is methyl, ethyl, propyl, naphthylmethyl, benzyl, benzyl independently or selectively substituted with a substituent selected from a group consisting of F, CI, Br, I, C 1 -C3 alkoxy and combinations thereof, or toluic acid
  • Y is H or CI.
  • A is O or S
  • R is methyl, ethyl, 1- naphthylmethyl, benzyl, 2-chlorobenzyl, 3-fluorobenzyl, 3-chlorobenzyl, 3-bromobenzyl, 3- iodobenzyl, 2-methoxy-5-chlorobenzyl, 2-methoxybenzyl, or 3-toluic acid
  • Y is H or CI.
  • compositions are described in U.S. Publication No. 2010/0137577.
  • the adenosine derivatives comprise: (2R,3R,4S)-2-(2-chloro-6-
  • adenosine derivatives for use as adenosine A3 receptor antagonists and synthesis are described in U.S. Publication No. 2010/0137577, which is incorporated by reference in its entirety.
  • the composition comprises:
  • Ri and R 3 are selected from the group consisting of hydrogen, hydroxy, Ci- C 6 alkyloxy, and Ci-C 6 alkylcarbonyloxy; R 2 is selected from the group consisting of:
  • the composition comprises:
  • Ri is selected from the group consisting of hydroxyl and Ci-C 6 alkoxy
  • M is a divalent radical selected from the group consisting of— CH(OH)— CH(R 2 )— and—
  • composition comprises:
  • R 2 is a Ci -C 6 alkyl
  • R ⁇ 5 is selected from the group consisting Ci-C 6 alkyl, Ci-C 6 haloalkyl, and phenyl which may be further substituted with Ci-C 6 alkyl, halo, nitro, furyl, or thienyl
  • R 3 is selected from the group consisting of Ci-C 6 alkyl, Ci-C 6 alkyloxycarbonyl, aryl Ci-C 6 alkyloxycarbonyl, Ci-C 6 alkylthiocarbonyl, Ci-C 6 alkylaminocarbonyl, and Ci-C 6 alkyloxy Ci-C 6 alkylcarbonyl, or R 3 together with R 2 forms a ring having 2-4 methylene groups, and Ci-C 6 alkenyloxycarbonyl;
  • R4 is selected from the group consisting of Ci-C 6 alkyl, aryl Ci-C 6 alkenyl, Ci-C 6 alkyl
  • the composition comprises:
  • R 2 is selected from the group consisting of hydrogen and Ci-C 6 alkyl
  • R 3 is selected from the group consisting of hydrogen and Ci-C 6 alkyloxycarbonyl
  • R4 is selected from the group consisting of Ci-C 6 alkyl, phenyl C 2 -C 6 alkenyl, phenyl C 2 -C 6 alkynyl, aryl, and aryl substituted with one or more substituents selected from the group consisting of nitro and Ci-C 6 alkyloxy
  • R 5 is selected from the group consisting of hydrogen, Ci-C 6 alkyloxycarbonyl, and aryl Ci-C 6 alkyloxy carbonyl
  • 5 is selected from the group consisting of hydrogen, aryl, and Ci-C 6 alkyl; with the proviso that when R4 is not alkyl.
  • the composition comprises:
  • Ri is selected from the group consisting of Ci-C 6 alkylcarbonyl, Ci-C 6 alkyloxycarbonyl, amino Ci-C 6 alkylcarbonyl, and arylcarbonyl wherein the aryl may be further substituted with halo, nitro, hydroxy, amino or cyano; and R 2 is hydrogen or halogen.
  • This composition is described in U.S. Patent No. 6,066,642.
  • Other embodiments of compositions including dihydronepyridine-, pyridine-, benzopyran-4-one- and
  • composition comprises:
  • R 2 is selected from the group consisting of Ci-C 6 alkyl, C3-C7 cycloalkyl, and Ci-C 6 alkoxy Ci-C 6 alkyl
  • R 3 is selected from the group consisting of Ci-C 6 alkoxy, Ci-C 6 alkylsulfanyl, hydroxy, Ci-C 6 alkoxy Ci-C 6 alkylsulfanyl, hydroxy Ci-C 6 alkylsulfanyl, and halo Ci-C 6 alkylsulfanyl, or R 3 together with R4 forms a 3-7 membered heterocyclic ring containing O, N, or S
  • R 4 is selected from the group consisting of Ci-C 6 alkyl, halo Ci-C 6 alkyl, hydroxy Ci-C 6 alkyl, Ci-C 6 alkoxy, Ci-C 6 alkylsulfanyl, Ci-C 6 alkylamino, Ci-C 6 alkylcarbonyl sulfanyl Ci-
  • the composition comprises:
  • R 2 is a Ci-C 6 alkyl
  • R 3 is selected from the group consisting of Ci -C 6 alkoxy, Ci-C 6 alkoxy Ci-C 6 alkylsulfanyl, and Ci -C 6 alkylsulfanyl
  • R 4 is selected from the group consisting of Ci -C 6 alkyl, acetal, formyl, aryl C 2 -C 6 alkenyl, and aryl C 2 -C 6 alkynyl
  • R 5 is selected from the group consisting of Ci-C 6 alkyl and aryl Ci-C 6 alkyl
  • 3 ⁇ 4 is selected from the group consisting of aryl and C 3 -C 6 cycloalkyl; wherein said aryl is a phenyl or naphthyl; or a pharmaceutically acceptable salt thereof.
  • This composition is described in U.S. Patent No. 6,376,521. Other embodiments are described in U.S. Patent No. 6,376,521, which is described
  • composition comprises:
  • X is sulfur or oxygen
  • Ri is hydrogen, C 1 -C 5 alkyl, benzyl, halobenzyl, or phenyl C 1 -C 5 alkyl
  • R 2 is hydrogen, halogen, C 1 -C 5 alkoxy group, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 5 alkylthio, or thio
  • R 3 and R 3 ' may be the same or different and are hydrogen, hydroxy C 1 -C 5 alkyl, C 1 -C 5 alkoxycarbonyl, or C 1 -C 5 alkylaminocarbonyl
  • R 4 is hydrogen or C 1 -C 5 alkyl; or a pharmaceutically acceptable salt, or isomer.
  • R3 and R 3' are not the same.
  • X is sulfur.
  • Ri is 3-iodobenzyl
  • R 2 is chloride
  • R 3 is methylaminocarbonyl
  • R 3 > and R4 are hydrogen
  • X is sulfur.
  • composition comprises:
  • R ls R 2 and R 3 are as defined by the preceding formula.
  • the adenosine A3 receptor antagonist is (2R,3S,4R)-2-[2-chloro-6-(3-iodobenzylamino)purin- 9-yl]-3,4-dihydroxy-tet- rahydrothiophene-2-carboxylic acid methyl amide.
  • composition comprises:
  • R ls R 2 and R 3 are as defined above for the preceding formula, and specifically, Ri is hydrogen, methyl group, or 3-iodobenzyl group, R 2 is chloride, R 3 and R 4 are hydrogen, R 3 ' is methylaminocarbonyl group or hydroxymethyl group, and X is sulfur.
  • compositions include (2R,3R,4S,5R)-2-[2-chloro-6- (3iodobenzylamino)purin-9-yl]-5-hydroxymethyl- tetrahydrothiophene-3,4-diol,
  • the composition comprises:
  • R ls R 2 and R 3 are as defined by the preceding formula.
  • An example of the composition is (2S,3R,4S,5R)-2-[2-chloro-6-(3-iodobenzylamino)purin-9-yl]-5- hydroxymethy- ltetrahydrothiophene-3,4-diol.
  • This composition is described in U.S. Patent No. 7,199,127.
  • Other embodiments of compositions and their synthesis are described in U.S. Patent No. 7,199,127, which is incorporated by reference in its entirety.
  • composition of the invention comprises:
  • A is O or S
  • R is a linear or branched Ci-C 5 alkyl which is non-substituted or is independently or selectively substituted with one or more C 6 -Cio aryl groups, a benzyl which is non-substituted or is independently or selectively substituted with halogen or one or more linear or branched C1-C4 alkoxy groups, or a hydroxycarbonyl-substituted benzyl; and Y is H or a halogen atom.
  • A is O or S
  • R is methyl, ethyl, propyl, naphthylmethyl, benzyl, benzyl independently or selectively substituted with a substituent selected from a group consisting of F, CI, Br, I, C1-C3 alkoxy and combinations thereof, or toluic acid
  • Y is H or CI.
  • A is O or S
  • R is methyl, ethyl, 1- naphthylmethyl, benzyl, 2-chlorobenzyl, 3-fluorobenzyl, 3-chlorobenzyl, 3-bromobenzyl, 3- iodobenzyl, 2-methoxy-5-chlorobenzyl, 2-methoxybenzyl, or 3-toluic acid
  • Y is H or CI.
  • adenosine derivatives include: (2R, 3R, 4S)-2-(2-chloro-6-(3- fluorobenzylamino)-9H-purin-9-yl) tetrahydrothiophen-3, 4-diol; (2R, 3R,4S)-2-(2-chloro-6- (3-chlorobenzylamino)-9H-purin-9-yl) tetrahydrothiophen-3, 4-diol; (2R, 3R,4S)-2-(6- (3- bromobenzylamino)-2-chloro-9H-purin-9-yl) tetrahydrothiophen-3, 4-diol; (2R, 3R, 4S)-2- (2-chloro-P-(3-iodobenzylamino)-9H-purin-9-yl) tetrahydrothiophen-3, 4-diol; (2R, 3R,4S)-2- (2-chlor
  • compositions of the invention comprise 2-(6-Cyano-l- yl)adenosine, 2-(6-cyano-l-hexyn-l-yl)adenosine 5 '-monophosphate, or salts thereof, as described in U.S. Patent No. 7,132,409 and U.S. Patent No. 6,936,596, which are incorporated by reference in their entireties.
  • composition of the invention comprises 2-(6-cyano-l-hexyn-
  • 1- yl)adenosine which can be produced by the following steps. Under argon, into a solution of 2-iodoadenosine (420 mg, 1.07 mmol) and bis(triphenylphosphine)palladium dichloride (75 mg, 10 mol %) dissolved in N,N-dimethylformamide (10 mL), diisopropylamine (0.18 mL, 1.28 mmol, 1.2 eq.) and 6-cyano-l-hexyne (137 mg, 1.28 mmol, 1.2 eq.) are added. Under ice-cooling conditions, cuprous iodide (10 mg, 5 mol %) is added, followed by stirring at 50°C for 24 hours.
  • reaction mixture is cooled to room temperature, and the solvent is removed under reduced pressure.
  • composition of the invention comprises triethylammonium
  • 2- (6-cyano-l-hexyn-l-yl)adenosine 5 '-monophosphate which can be produced by the following steps. Under argon, a 2-(6-cyano-l-hexyn-l-yl)adenosine (2.41 g) can be dissolved in triethyl phosphate (15 mL), and under ice-cooling conditions, phosphorous oxychloride (25.2 mL, 3.9 eq.) is added, followed by stirring at the same temperature for 5 hours. The reaction mixture is added drop-wise to a 10% sodium hydroxide solution (300 mL), and the resultant mixture is washed with ether.
  • IR(KBr)o max 3337, 3179, 2938, 2678, 2243, 1654, 1637, 1594, 1457, 1380, 1068, 919 cm "1 .
  • the composition of the invention comprises 2-(6-cyano-l- hexyn-l-yl)adenosine 5 '-monophosphate , which can be synthesized by the following steps.
  • a triethylammonium 2-(6-cyano-l-hexyn-l-yl)adenosine 5 '-monophosphate (1.1 g) is dissolved in water (20 mL), and, under ice-cooling conditions, IN HC1 solution (2 mL) was added thereto, followed by standing for 10 minutes. After having been diluted with water (20 mL), the resultant mixture is filtered.
  • the composition of the invention comprises monosodium 2-(6-cyano-l-hexyn-l-yl)adenosine 5 '-monophosphate or disodium 2-(6-cyano-l -hexyn- 1- yl)adenosine 5 ' -monophosphate.
  • a 2-(6-Cyano- 1 -hexyn- 1 -yl)adenosine 5 ' -monophosphate (800 mg) is dispersed in water (8 mL), and IN sodium hydroxide solution (1.8 mL) is added thereto, followed by stirring for 0.5 hours. After having been diluted with water (10 mL), the resultant mixture is purified by C 18 silica gel column chromatography
  • compositions of the invention include purine derivatives described in U.S. Patent No. 7,465,715, which is incorporated by reference in its entirety.
  • composition of the invention comprises the purine derivative of the formula:
  • Rl represents an alkyl, hydroxyalkyl, carboxyalkyl or cyanoalkyl or a group of the following general formula:
  • Y represents oxygen, sulfur or CH 2 ;
  • R a and R b may be the same or different and are selected from the group consisting of hydrogen, alkyl, amino, haloalkyl, aminoalkyl, BOC-aminoalkyl, and cycloalkyl or are joined together to form a heterocyclic ring containing two to five carbon atoms; and R c is selected from the group consisting of alkyl, amino, haloalkyl, aminoalkyl,
  • X 2 is H, hydroxyl, alkylamino, alkylamido or
  • X 3 and X 4 represent independently hydrogen, hydroxyl, amino, amido, azido, halo, alkyl, alkoxy, carboxy, nitrilo, nitro, trifluoro, aryl, alkaryl, thio, thioester, thioether,—
  • R and R" represent independently an alkyl group
  • R 2 is selected from the group consisting of hydrogen, halo, alkylether, amino, hydrazido, alkylamino, alkoxy, thioalkoxy, pyridylthio, alkenyl; alkynyl, thio, and alkylthio
  • R 3 is a group of the formula --NR 4 R 5 wherein R4 is a hydrogen atom or a group selected from alkyl, substituted alkyl or aryl-NH— C(Z) ⁇ , with Z being O, S, or NR a with R a having the above meanings; wherein when R4 is hydrogen, then R 5 is selected from the group consisting of R- and S-l-phenylethyl, benzyl, phenylethyl or anilide groups unsubstituted or substituted in one or more positions with a substituent selected from the group consisting of alkyl, amino, halo
  • R 5 is selected from the group consisting of heteroaryl-NR a -C(Z)-, heteroaryl-C(Z)-, alkaryl-NR a ⁇ C(Z)-, alkaryl-C(Z)--, aryl-NR--C(Z)-- and aryl-C(Z)— ; Z representing an oxygen, sulfur or imine; or a
  • composition of the invention comprises the nucleoside derivative, described in U.S. Patent No. 7,465,715:
  • the composition of the invention comprises N 6 -2- (4-aminophenyl)ethyladenosine (APNEA), N 6 -(4-amino-3-iodobenzyl) adenosine-5'-(N- methyluronanide) (AB-MECA), N 6 -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB- MECA) and 2-chloro-N 6 -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), which are described in U.S. Patent No. 7,465,715.
  • APIA N 6 -2- (4-aminophenyl)ethyladenosine
  • AB-MECA N 6 -(4-amino-3-iodobenzyl) adenosine-5'-(N- methyluronanide)
  • IB- MECA
  • compositions of the invention include adenosine A3 receptor antagonists described in U.S. Patent No. 7,064,112, which is incorporated by reference in its entirety.
  • composition of the invention comprises:
  • compositions of the invention include adenosine A3 receptor inhibitors described in U.S. Patent No. 6,673,802, which is incorporated by reference in its entirety.
  • composition of the invention comprises:
  • Ri and R 2 are each independently a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety or together form a substituted or unsubstituted heterocyclic ring.
  • R 3 is a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety.
  • R 4 is a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety.
  • R5 and R 6 are each independently a halogen atom, e.g., chlorine, fluorine, or bromine, a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety or R 4 and R 5 or R5 and R6 together form a substituted or unsubstituted heterocyclic or carbocyclic ring.
  • Ri and R 2 can each independently be a substituted or unsubstituted cycloalkyl or heteroarylalkyl moieties.
  • R 3 is a hydrogen atom or a substituted or unsubstituted heteroaryl moiety.
  • R 4 , R 5 and R 6 can each be independently a heteroaryl moieties.
  • Ri is a hydrogen atom
  • R 2 is a cyclohexanol, e.g., trans- cyclohexanol
  • R 3 is phenyl
  • R 4 is a hydrogen atom
  • R 5 is a methyl group
  • 5 is a methyl group.
  • Ri is a hydrogen atom
  • R 2 is:
  • R 3 is phenyl
  • R 4 is a hydrogen atom
  • R 5 and R 6 are methyl groups.
  • composition of the invention comprises:
  • Ri is hydrogen
  • R 2 is substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkyl, or Ri and R 2 together form a substituted or unsubstituted heterocyclic ring
  • R 3 is unsubstituted or substituted aryl
  • R 4 is hydrogen
  • R 5 and R 6 are each independently hydrogen or alkyl, and pharmaceutically acceptable salts thereof.
  • composition of the invention comprises:
  • Ri and R 2 are each independently hydrogen, or substituted or unsubstituted alkoxy, aminoalkyl, alkyl, aryl, or alkylaryl, or together form a substituted or unsubstituted heterocyclic ring, provided that both Ri and R 2 are both not hydrogen;
  • R 3 is substituted or unsubstituted alkyl, arylalkyl, or aryl;
  • R4 is hydrogen or substituted or unsubstituted Ci-C 6 alkyl;
  • L is hydrogen, substituted or unsubstituted alkyl, or R 4 and L together form a substituted or unsubstituted heterocyclic or carbocyclic ring;
  • R 6 is hydrogen, substituted or unsubstituted alkyl, or halogen;
  • Q is CH 2 , O, S, or NRg, wherein R 8 is hydrogen or substituted or unsubstituted
  • composition of the invention comprises:
  • R 3 is a substituted or unsubstituted four to six membered ring, pyrrole, thiophene, furan, thiazole, imidazole, pyrazole, 1 ,2,4-triazole, pyridine, 2(lH)-pyridone, 4(lH)-pyridone, pyrazine, pyrimidine, pyridazine, isothiazole, isoxazole, oxazole, tetrazole, naphthalene, tetralin, naphthyridine, benzofuran, benzothiophene, indole, 2,3-dihydroindole, lH-indole, indoline, benzopyrazole, 1 ,3-benzodioxole, benzoxazole, purine, coumarin, chromone, quinoline, tetrahydroquinoline, isoquinoline, benzo
  • compositions of the invention include nucleoside 5 '-pyrophosphate pyranosides and analogues described in U.S. Patent No. 6,897,201 , which is incorporated by reference in its entirety.
  • the composition of the invention comprises:
  • G 3 CH 2 , CHF, CF 2 , CH(OH) or CH(NHJ 7 );
  • G 4 CH 2 , CHF, CF 2 , CH(OH) or CH(NHJi 3 );
  • G 5 CH 2 , CHF, CF 2 , CH(OH) or CH(NHJi 7 );
  • G 6 CH 2 , CH(CH 2 OH); the number of hydrogen atoms bonded to the Gi-G 6 ring atoms is limited to a maximum of 8; also with the provision that the number of nitrogen atoms bonded to the Gi-G 6 ring atoms are limited to a maximum of 2;
  • J 7 , Ji 3 , and Ji 7 are independently H, C(0)H, or C(0)alkyl.
  • compositions of the invention include triazolopurine derivatives described in U.S. Patent Nos. 6,288,070 and 6,686,343, which are incorporated by reference in their entireties.
  • composition of the invention comprises:
  • Ri represents an alkyl group, or a phenyl group which is optionally substituted with a lower alkyl group
  • R 2 represents a pyridyl group, a furyl group, a thienyl group, a lower alkyl group, a phenyl lower alkyl group which optionally has 1 to 3 lower alkoxy groups as a substituent, a styryl group which optionally has 1 to 3 lower alkoxy groups as a substituent, a naphthyl group which optionally has a hydroxy groups as a substituent, or a phenyl group which optionally has 1 to 3 groups selected from lower alkyl group, lower alkoxy group, nitro group, hydroxyl group, amino group, N-lower alkylamino group, N,N-di lower alkylamino group, N-phenyl lower alkylamino group, N,N-bisphenyl lower alkylamino group, phenyl group, phen
  • alkylphosphorylmethyl group lower alkylthio group, lower alkoxy lower alkyl group, phenyl lower alkoxy lower alkyl group and halogen atom as a substituent; and
  • A represents a group:
  • R 3 represents a lower alkyl group or a phenyl lower alkyl group.
  • compositions of the invention include compositions described by Okamura et al. (2004) Bioorg. & Med. Chem. Lett. 14:2443-2446.
  • the composition of the invention is selected from the group consisting of: Monocyclic
  • Pyrazolo[4,3-e]-l,2,4-triazolo[l,5-c]pyrimidine and l ,2,4-triazolo[l,5- c]quinazoline rings can be synthesized bearing various substituents at the 2 and 5 positions of the ring to generate the A3 receptor antagonists of the invention.
  • the fused 1,2,4- triazolo[l,5-c]pyrimidine 9 can be synthesized by condensation of iminoester 8 and acylhydrazine based on the synthetic strategy, as shown in the Scheme below:
  • the iminoester 8 can be prepared by the reaction of primary amine analog 7 with a large excess of substituted orthoester. Iminoesterification of various aromatic or heterocyclic amines 7a-c were accomplished by condensation of trimethyl orthovalerate under the three different conditions, as shown in the table below.
  • 5-Amino-4- cyanoimidazole 7a can be converted to iminoester 8a in good yields (62-86%) at room temperature, at 90°C, or in the presence of a catalytic amount of dl-camphorsulfonic acid (CSA) at room temperature.
  • 3-Amino-4-cyanopyrazole 7b can be converted to 8b quantitatively by heating at 90°C or in the presence of a catalytic amount of CSA at room temperature, but there is no yield at room temperature without CSA.
  • 2-aminobenzonitrile 7c does not afford 8c at room temperature or by heating at 90°C.
  • the reaction can proceed in the presence of CSA at room temperature.
  • the reaction conditions using CSA can be chosen for the one-pot synthesis of fused l,2,4-triazolo[l,5-c]pyrimidine derivatives 9a-c.
  • Amines 7a-c can be transformed into the fused l,2,4-triazolo[l,5- c]pyrimidine derivatives 9a-c via intermediates 8a-c in situ by treatment with l . lmol equiv. of the corresponding orthoesters in the presence of 1% of CSA in DMF at room temperature.
  • compositions of the invention include compositions described by Okamura et al. (2004) Bioorg. & Med. Chem. Lett. 14:3775-3779. Okamura et al.
  • the composition of the invention comprises an adenosine A3 receptor antagonist selected from the group of:
  • R 1 and R 2 are selected from the table below
  • compositions of the invention include isoquinolines or quinazolines described by van Muijlwijk-Koezen, J.E. et al. (2000) J. Med. Chem. 43(11):2227-2238.
  • the composition of the invention comprises an adenosine A3 receptor antagonist selected from the group of:
  • compositions of isoquinolines or quinazolines described by van Muijlwijk- Koezen, J.E. et al. can be synthesized by the following steps.
  • the preparation of compounds 5a-k can be performed following the general synthetic strategy depicted the scheme shown above.
  • the intermediates 4a-k are synthesized initially based on a method described by Linschoten et al. with some modifications (Eur. J. Med. Chem.-Chim. Ther. 1984, 19, 137-142).
  • Treatment of 2-aminobenzonitrile (1) with strong base yielded a relatively stable anion as intermediate. This nucleophile reacts with nitriles 3a-k, and after hydrolysis the quinazoline derivatives 4a-k are obtained.
  • the yield of the derivatives is dependent on the bulkiness of substituents Rl of nitriles 3a-k due to steric hindrance in the nucleophilic attack of the anion.
  • the anion can react with the starting nitrile 1 which, after hydrolysis, yields the dimeric side product 2-(2-aminophenyl)- 4-quinazolineamine (2).
  • the course of the condensation reaction's dependence on the accessibility of the participating nitrile group to undergo nucleophilic attack is demonstrated by a mixture of 1 and trimethylacetonitrile (3c), which yields only dimer 2.
  • the derivatives 5a-k are prepared by reaction of phenyl isocyanate (7i) with 4-aminoquinazolines 4.
  • the substituted phenylurea derivatives 9a- h as well as compounds 8, 10, and 11 are prepared as depicted in the scheme shown below. Reaction of quinazolines 4 and isoquinolines 6 in dry acetonitrile at 30-50 °C with the appropriate isocyanate 7a-i affords the products 8-11 in good overall yields. The low solubility of the products in acetonitrile simplified the isolation and purification.
  • compositions of the invention include derivatives of triazoloquinazoline and pyrazolotriazolopyrimidine described in Canadian Patent Application No. 2437437.
  • the composition of the invention comprises:
  • Rl represents a lower alkyl group, a phenyl group, a lower alkoxy carbonyllower alkyl group, or a carboxy lower alkyl group
  • R2 represents a pyridyl group, a furyl group, a thienyl group, or a phenyl group which optionally has 1 to 3 groups selected from lower alkyl group, halogen atom, phenyl group, halogen-substituted lower alkyl group, hydroxyl group, lower alkoxy group, N,N-di lower alkylamino group, lower alkylthio group, lower alkanoyloxy group and nitro group as a substituent;
  • A represents a pyrazole ring which is optionally substituted with a group selected from lower alkyl group, phenyl lower alkyl group, lower alkoxycarbonyllower alkyl group, carboxy lower alkyl group and
  • composition of the invention comprises:
  • R 1 represents a lower alkyl group, a phenyl group, a lower alkoxycarbonyllower alkyl group, or a carboxy lower alkyl group
  • R 2 represents a pyridyl group, a furyl group, a thienyl group, or a phenyl group which optionally has 1 to 3 groups selected from lower alkyl group, halogen atom, phenyl group, halogen-substituted lower alkyl group, hydroxyl group, lower alkoxy group, N,N-di lower alkylamino group, lower alkylthio group, lower alkanoyloxy group and nitro group as a substituent.
  • compositions described in Canadian Patent Application No. 2437437 can be synthesized by the following steps:
  • R 2 is as defined above;
  • R la represents a lower alkyl group or a phenyl group;
  • Al represents a non-substituted pyrazole ring, or a benzene ring which is optionally substituted with 1 to 2 groups selected from halogen atom, lower alkyl group, nitro group and lower alkoxy group as a substituent; and
  • Z represents a lower alkyl group.
  • R 2 and Al are as defined above;
  • R la represents a lower alkyl group;
  • R 1 represents a lower alkyl group, a phenyl group or a lower alkoxy carbonyllower alkyl group,
  • R 2b represents a pyridyl group, a furyl group, a thienyl group, or a phenyl group which optionally have 1 to 3 groups selected from lower alkyl group, halogen atom, phenyl group, halogen-substituted lower alkyl group, hydroxy group, lower alkoxy group, N ,N-di lower alkylamino group, lower alkylthio group and nitro group as a substituent; and
  • R 2c represents a pyridyl group, a furyl group, a thienyl group, or a phenyl group which optionally have 1 to 3 groups selected from lower alkyl group, halogen atom, phenyl group, halogen
  • the compound (lc) of the present invention can be converted into compounds (ld-1) and (ld-2) wherein a substituent is introduced into the pyrazole ring by treating with a halide (9), as shown in the following reaction scheme.
  • R lb is as defined above;
  • R 2a represents a pyridyl group, a furyl group, a thienyl group, or a phenyl group which optionally has 1 to 3 groups selected from lower alkyl group, halogen atom, phenyl group, halogen-substituted lower alkyl group, lower alkoxy group, N,N-di lower alkylamino group, lower alkylthio group, lower alkanoyloxy group and nitro group as a substituent;
  • R 3 represents a lower alkyl group, a phenyl lower alkyl group, a lower alkoxycarbonyllower alkyl group, or a hydroxy lower alkyl group,
  • R 3a represents a lower alkyl group, a phenyl lower alkyl group, a lower alkoxy carbonyl lower alkyl group, or a trimethylsilyloxy-lower alkyl group;
  • X represents a
  • compositions of the invention include 2,6,9- trisubstituted purines.
  • 2,6,9-trisubstituted purines The synthesis of 2,6,9-trisubstituted purines is described by
  • compositions of the invention are provided.
  • compositions of the invention can be formulated in pharmaceutical compositions.
  • These compositions can comprise pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material can depend on the route of administration, e.g., topical or intraocular injection.
  • Administration is preferably in a "therapeutically effective amount" or
  • prophylaxis can be considered therapy
  • prophylaxis can be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the inflammatory eye disease being treated. Prescription of treatment, e.g. , decisions on dosage, etc. , is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington 's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack
  • compositions comprising the molecules described above, together with one or more pharmaceutically-acceptable excipients or vehicles, and optionally other therapeutic and/or prophylactic ingredients.
  • excipients include liquids such as water, saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol, cyclodextrins, modified cyclodextrins ⁇ i.e., sufobutyl ether cyclodextrins), etc.
  • Suitable excipients for non-liquid formulations are also known to those of skill in the art.
  • compositions of the present invention include, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • auxiliary substances such as wetting or emulsifying agents, biological buffering substances, surfactants, and the like, may be present in such vehicles.
  • a biological buffer can be virtually any solution which is pharmacologically acceptable and which provides the formulation with the desired pH, i.e., a pH in the physiologically- acceptable range.
  • buffer solutions include saline, phosphate buffered saline (PBS), Tris buffered saline, Hank's buffered saline, and the like.
  • compositions of this invention will be administered as pharmaceutical formulations including those suitable for oral (including buccal and sublingual), topical, intravitreal administration, intraocular injection, intravenous injection, intramuscular injection, rectal, intranasal administration, intraperitoneal administration, administration by inhalation or insufflation, or in other forms suitable for administration to an eye.
  • the preferred modes of administration are topical or intraocular administration, preferably using a convenient daily dosage regimen which can be adjusted according to the degree of the condition or disease.
  • the pharmaceutical compositions may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, creams, ointments, lotions or the like, preferably in unit dosage form suitable for single
  • compositions will include an effective amount of the selected compound in combination with a pharmaceutically acceptable carrier and, in addition, may include other pharmaceutical agents, adjuvants, diluents, buffers, etc.
  • Formulations also include compositions of the invention administered as eye-drops, injectable liquids or solutions, or topical eye creams.
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions.
  • Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.
  • Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and cornstarch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid,
  • micro crystalline cellulose acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.
  • a flavor usually sucrose and acacia or tragacanth
  • pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.
  • the invention includes a pharmaceutical composition
  • a pharmaceutical composition comprising a composition of the present invention including isomers, racemic or non-racemic mixtures of isomers, or pharmaceutically acceptable salts or solvates thereof together with one or more
  • compositions can be presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.
  • the composition that is administered is in powder or particulate form.
  • the active compounds may be in powder form for constitution with a suitable vehicle, e.g. , a buffered saline, before use.
  • Effective amounts of the compositions of the invention are administered to a subject in need of such treatment. Effective amounts are those amounts, which will result in a desired improvement in the condition, disease or disorder or symptoms of the condition, disease or disorder.
  • a suitable dose of a formulation of an adenosine A3 receptor antagonist will be in the range of 0.001 to 1000.0 milligrams per kilogram body weight of the recipient per day, in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, or in the range of 1 to 50 mg per kilogram body weight per day.
  • the formulation of an adenosine A3 receptor antagonist can be administered at 0.001 mg/kg, 0.01 mg.kg, 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg per single dose.
  • the formulation of an adenosine A3 receptor antagonist can be administered at 0.001 mg/kg, 0.01 mg.kg, 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg
  • the pharmaceutical composition may be administered once daily, or the adenosine A3 receptor antagonist may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation.
  • the adenosine A3 receptor antagonist contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage.
  • the dosage unit can also be compounded for delivery over several days. The absolute amount will depend upon a variety of factors (including whether the administration is in conjunction with other methods of treatment, the number of doses and individual patient parameters including age, physical condition, size and weight) and can be determined with routine experimentation. In some embodiments, a maximum dose can be used, that is, the highest safe dose according to sound medical judgment.
  • the pharmaceutical composition is formulated for administration of a dosage regimen described herein, e.g., not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week.
  • a dosage regimen described herein e.g., not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week.
  • composition can be maintained for a month or longer, e.g., one, two, three, or six months, or one year or longer.
  • Methods for the use of adenosine A3 receptor antagonists and methods of treatment of various inflammatory eye diseases using adenosine A3 receptor antagonists are encompassed by the present invention.
  • Said methods of the invention include administering an effective amount of a composition comprising an adenosine A3 receptor antagonist to a subject that has an inflammatory eye disease.
  • Any of the adenosine A3 receptor antagonists described above may be used in the methods of use and methods of treatment of the invention.
  • Inflammatory eye diseases include, but are but not limited to, dry eye, uveitis, scleritis, blepharitis, keratitis, conjunctivitis, chorioretinital inflammation, choroiditis, retinitis, iridocyclitis, ulceris, posterior cyclitis, diabetic retinopathy, post-surgery, allergy, trauma, bruises, foreign body, exposure to toxins or chemicals, food allergies, hives, autoimmune disorders (such as rheumatoid arthritis, sarcoidosis, juvenile idiopathic arthritis, lupus, Behcet's disease, etc.), infection (such as viral, fungal, mycobacteria, parasite, bacteria, acanthamoeba, spirochete, etc), inflammatory conditions (such as Crohn's disease, arthritis and ulcerative colitis) and cancers (such as lymphoma, eye tumors, cancer of blood vessels, etc.), or an
  • animal models are used to induce inflammatory eye disease. See Barabino, S. et al. (2004) Investigative Opthalmo logy & Visual Science. 45(6): 1641-1646.
  • Dry eye syndrome is a prevalent inflammatory disorder of the lacrimal functional unit of multifactorial origin leading to chronic ocular surface disease, impaired quality of vision, and a wide range of complications, eventually causing a reduction in quality of life.
  • Some dry eye animal models have been developed in mice, rats, and rabbits, e.g., for lacrimal inflammation or human Sjogren's syndrome. Animal models for dry eye syndrome are discussed herein and in detail in Barabino, S. et al. (2004) Investig. Opthal. & Visual Sci.
  • a mouse model of botulinum (BTX)-induced dry eye was used to induce dry eye.
  • Botulinum toxin was injected into the lacrimal gland of mice. Dry eye developed and persisted over about 3-4 weeks with resultant decreases in tear production and
  • Dexamethasone was used as an appropriate positive control (cyclosporine is not as effective, but is still active).
  • a Phenol Red Thread Test for tear production was performed, and a fluorescein slit lamp examination was used to assess corneal damage.
  • a histopathology exam was performed on the eye and adenexal structures.
  • the Phenol Red Thread Test was used.
  • the Phenol Red Thread Test is a commercially available test (Zone-Quick, Showa Yakuhin Kako Co., Ltd). A cotton thread impregnated with phenol red dye was used. Phenol red is pH sensitive and changes from yellow to red when wetted by tears. The crimped end of a 70mm long thread was placed in the lower conjunctival fornix. Forceps were used to insert the folded portion of the thread into the palpebral conjunctiva of the eye 1/3 of the distance from the lateral canthus of the lower eye lid.
  • the length of the color change on the thread (which indicated the length of the thread wetted by the tears) was measured in millimeters. Wetting lengths should normally be between 9mm and 20mm. Patients with dry eyes have wetting values of less than 9 mm. A reading of less than 10 mm was used to indicate dry eyes, less than 20 mm indicates marginally dry eyes, and more than 20 mm indicated normal tear volume.
  • Botulinum injected and treatment started on Day 0 and for all groups. ANOVA followed by post hoc.
  • Botulinum injected and treatment started on Day 0 for all groups. ANOVA followed by post hoc Dunnett's: Statistically significant (**) p value less than 0.01 ; (*) p value less than 0.05. 50 ⁇ of 1% sodium fluorescein applied to corneal for slit lamp examination with cobalt light.
  • Botulinum injected and treatment started on Day 0 for all groups. ANOVA followed by post hoc Dunnett's: Statistically significant (**) p value less than 0.01 ; (*) p value less than 0.05.
  • stroma increased thickness of inflammatory cells in gland epithelium, inflammatory cell
  • a mouse model of Scopolamine -induced dry eye syndrome can be used.
  • Scopolamine patches were applied to the tails of mice, and the mice were maintained in an environment with blowing air and low humidity. Dry eye developed over about 2 weeks with resultant decreases in tear production and histopathologic damage to the cornea. The dry eye continued as long as the patches were applied.
  • the study design included experimental groups with at least 10 mice per group. A scopolamine patch was applied, and a dry environment was initiated. A topical
  • dexamethasone (DEX) was administered as an appropriate positive control (or cyclosporine (CSA) (Restasis) offers some protection as well).
  • CSA cyclosporine
  • Regular assessments of the Phenol Red Thread Test for tear production was performed.
  • a fluorescein slit lamp assessment was performed to determine corneal changes.
  • a histopathology was performed on eye and adenexal structures.
  • FIG. la shows a graph of typical tear reduction after scopolamine patch application and a dry environment.
  • FIG. lb also shows histopathology scores 12 days after scopolamine administration.
  • Four groups are shown for no-treatment, vehicle, DEX, and Restasis.
  • the p-value is less than 0.05 relative to the vehicle control group.
  • the p-value was less than 0.05 reltaive to the no-treatment group.
  • N 10 female C57BL/6 mice per group.
  • Scopolamine and air draft started on Day 0 for all groups. ANOVA followed by post hoc Dunnett's: Statistically significant (**) p value less than 0.01; (*) p value less than 0.05.
  • a corneal edema refers to the presence of edema in the corneal stroma as indicated by separation and spreading of the fibers.
  • Epithelial edema refers to the presence of intracellular or extracellular edema in the corneal epithelium.
  • Corneal thickness refers to the number of epithelial cell layers in the corneal epithelium, normally about 2-3 cell layers thick. In cases of injury to the corneal epithelium, the number of cell layers may be increased, and there may be other indications of epithelial proliferation, such as increased mitosis on the basal layer or cellular hypertrophy or hyperplasia.
  • Cyclosporine (CSA) produced a similar trend, but was generally not statistically significant.
  • a controlled adverse environment (CAE) model combines multiple
  • the CAE model is based on a clinical model, which has been widely used in human clinical dry eye studies and has been pivotal in determining treatment efficacy in various dry eye development programs.
  • mice were housed in polycarbonate cages with direct contact bedding.
  • the cages conformed to standards set forth in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences). Space recommendations for animals were in accordance with PHS policy and the Animal Welfare Act. Litter or bedding in animal cages was changed as often as necessary to keep animals dry and clean and to minimize offensive odors.
  • n ma s were ran om ze a ter uoresce n sta n ng on ay . os ng egan at least 1 hour after staining. [00181] ** Scopolamine injections began after staining evaluations.
  • mice were evaluated on the morning of day 5. Mice were assigned to treatment groups such that the average total staining between the groups was approximately equivalent ( ⁇ 2).
  • Topical treatment was administered according to the Guidance document: Topical Administration of Ocular Treatments in Preclinical Studies. On days 5 through 19, topical treatment with test article, positive control, or the control article (vehicle control) was administered as outlined in the table below. Group 1 received ocular treatment four times daily at approximately 7am, 1 lam, 2pm, and 6pm. All other groups were dosed twice daily at approximately 7am and 6pm. All times listed were ⁇ 1 hour, and the exact time of dosing was recorded in the datasheets. Mice were dosed topically to the central cornea using a calibrated micropipette, with a 3 drop of treatment (test or vehicle control article) in each eye. For example, the mice were dosed topically with either ACN- 1052 or a vehicle control.
  • test article tubes were masked throughout the experiment (labeled A-D) such that personnel dosing or performing the examinations did know the identity of the treatment groups.
  • Scopolamine Hydrobromide Injection Subcutaneous injection of scopolamine was administered according to the Technical Procedure: Injection of Scopolamine in a Murine Model of Dry Eye. Dursun D, Wang M, Monroy D, et al. "A mouse model of keratoconjunctivitis sicca.” Invest Ophthalmol Vis Sci. 2002/43. -632-638.
  • Corneal Fluorescein Staining Corneal staining was assessed according to the Guidance: Fluorescein Staining and Grading Procedure. 0.6 ⁇ ⁇ ⁇ ⁇ % sodium fluorescein was applied via a micro-pipette to the central cornea. Eight to ten minutes following application, corneal fluorescein staining was evaluated under magnification. The degree of fluorescein staining was assessed using a modified 0-4 grading scale (Ora Scale, modified from NEI), similar to that used in clinical dry eye studies, where five distinct areas of the cornea were graded individually. The sum of scores and the differences in the different regions of the cornea was assessed.
  • Guidance Fluorescein Staining and Grading Procedure. 0.6 ⁇ ⁇ ⁇ ⁇ % sodium fluorescein was applied via a micro-pipette to the central cornea. Eight to ten minutes following application, corneal fluorescein staining was evaluated under magnification. The degree of fluorescein staining was
  • Body weight was measured at baseline, at pre-scopolamine injection, and at sacrifice.
  • Eyes were monitored at baseline as well as during dosing for any abnormalities (e.g., conjunctival hyperemia, chemosis, discharge, lid edema and ulceration).
  • abnormalities e.g., conjunctival hyperemia, chemosis, discharge, lid edema and ulceration.
  • Unscheduled Deaths If an animal appears in poor condition or in extremis, it may be euthanized per the facility IACUC policy on humane care of animals. Depending on the timepoint of the experiment, animals were replaced if at all possible.
  • Tissue Collection and Preservation The right eye of each animal was immersed in 4% paraformaldeyhyde for 24-30 hours, after which they eye was transferred to a vial containing 70% ethanol and stored for potential histology and/or immunohistochemistry.
  • a model of concanavalin (ConA)-induced dry eye in rabbits can be used to induce dry eye syndrome.
  • the lacrimal gland was injected with ConA.
  • a self-limiting dry eye develops developed over about 2 weeks.
  • Injection of ConA into the lacrimal glands of rabbits produced a consistent and measureable decrease in both the Tear Break up Test (TBUT) and the Schirmer Tear Test (STT).
  • the Schirmer Tear Test included use of Schirmer tear test strips (Clement Clarke) according to the manufacturer's instructions. No topical anaesthetic was used. The rounded wick end of the test strip was folded at the indentation and then inserted into the lower fornix of the eye 1/3 of the distance from the lateral canthus of the lower eye lid. After 5 minutes, the strip was removed, and the wet part was measured up to the folded line. A reading of less than 5 mm can be judged to indicate dry eyes and less than 10 mm can indicate marginally dry eyes.
  • FIGs. 2, 3a and 3b present typical reduction and recovery of tear production in control and treated animals after ConA injection.
  • the study design included groups with at least 6 rabbits per group. Con A was injected into the lacrimal gland as a single dose on Day 0 and Day 3. Assessments included a Schirmer Tear Test (STT) and a Tear Break Up Test (TBUT) over a 2 week period.
  • STT Schirmer Tear Test
  • TBUT Tear Break Up Test
  • Histopathology was performed on the eye and adenexal structures.
  • a Schirmer Tear Test (STT) and a Tear Break Up Test (TBUT) can be performed.
  • STT Schirmer Tear Test
  • TBUT Tear Break Up Test
  • FIG. 2 the effects of DEX and CSA are shown in the TBUT test. Also, the effects of a test article (e.g., an adenosine A3 receptor antagonist) on the TBUT test are shown, demonstrating a statistically significant effect of both DEX and CSA on the TBUT Test. There was a clear dose related trend with a three doses of a test article, as well as with the high dose, demonstrating a statistically significant improvement in the TBUT.
  • a test article e.g., an adenosine A3 receptor antagonist
  • FIG. 3a the effects of ConA injection on the STT are presented. There was a decrease in the STT beginning about Day 2, continuing for about 3-4 days with recovery occurring between Days 8-10.
  • FIG. 3b DEX and CSA were used, and a trend towards improvement with DEX was seen, but it did not achieve statistical significance.
  • Uveitis is a swelling and irritation of the uvea, the middle layer of the eye.
  • Uveitis can be caused by autoimmune disorders such as rheumatoid arthritis or ankylosing spondylitis, infection or exposure to toxins.
  • autoimmune disorders such as rheumatoid arthritis or ankylosing spondylitis
  • the most common form of uveitis is anterior uveitis, which involves inflammation in the front part of the eye.
  • Symptoms of uveitis include, but are not limited to blurred vision, dark, floating spots in the vision, eye pain, redness of the eye, or sensitivity to light.
  • Acute anterior uveitis is a recurrent immune-mediated inflammatory condition involving the iris and ciliary region in man and occurs in about 4 out of 1000 individuals. Although it may be self-limiting, it may also be chronic and lead to irreversible eye damage including cataract and glaucoma. Photophobia and pain with hyperemia of the vessels, hypophyon, and iridiocyclitis is typical, and the condition is relatively responsive to steroids. However, in many instances, the disorder is unresponsive to treatment and may lead to blindness with glaucoma, cataract and retinopathy. In some cases, HLA B27 appears to be involved. Humans are usually treated with steroids; however, there are undesirable side effects. In the field of veterinary medicine, horses suffer from an analogous anterior recurrent uveitis which often leads to blindness as well and was known in the past as "Moon Blindness.”
  • a MAA-induced uveitis model in rats can be used.
  • a preparation of melanin- associated antigen (MAA) complexed with adjuvant was administered to Lewis Rats as an inoculation.
  • An acute to sub-acute recurrent immune-mediated iridiocyclitis developed and resolved over a 1-2 -month period. Both the ophthalmo logical presentation and the histologic features were analogous to the human condition.
  • the MAA antigen is an extract of bovine ocular melanin derived from the iris.
  • the MAA antigen induced uveitis by Day 15 in all treated rats, and the disease proceeded consistently between and among groups.
  • Both topical steroids and systemic cyclosporine markedly and consistently suppressed the inflammation.
  • Topical cyclosporine also suppressed inflammation, but not to the degree of steroid and systemic cyclosporine.
  • One study design included 10 Lewis rats per group, treated with a vehicle or test article (e.g., an adenosine A3 receptor antagonist) at 2-3 dose levels, and a positive control of topical dexamethasone (DEX) or systemic cyclosporine (CSA). Inflammation was induced with a MAA injection. Slit lamp scoring of inflammation was performed in the anterior segment of the eye thrice weekly for 4 weeks. The body weights of the animalswere measured weekly, and a necropsy with histopathologic examination was performed on the eye.
  • a vehicle or test article e.g., an adenosine A3 receptor antagonist
  • DEX topical dexamethasone
  • CSA systemic cyclosporine
  • a slit lamp examination was performed at least three times a week during the course of the study and the findings scored as per the scoring criteria.
  • Dexamethasone and to a lesser extent, CSA reduced the clinical ocular scoring in the anterior chamber as per direct slit lamp examination (topical Restasis has no effect). Ocular scoring results are presented below.
  • FIG. 4 shows representative photographs of the anterior area of the eye. Multiple hematoxylin-eosin stained sections of each eye were examined histopathologically and scored 0-4 for inflammation severity at Day 18 and Day 30. In MAA-treated eyes, there was a marked inflammation of the iris and ciliary areas. Both DEX and CSA significantly reduced the inflammation.
  • FIG. 4a shows a normal anterior chamber.
  • FIG. 4b shows the anterior chamber of a DEX and MAA treated rat, where there is hyperemia in the iris (Score 1).
  • FIG. 4c shows a photo of a MAA-treated rat, and the eye has an opaque anterior chamber and an obscured pupil (red reflex absent, proptosis and hypophyon; Score 4). Topical Restasis had no significant effect on inflammation.
  • FIG. 5 shows histopathology scores 18 days after MAA injection in untreated rats and rats treated with vehicle, DEX, CSA, or Restasis.
  • “a” indicates that the p- value was ⁇ 0.05 relative to the vehicle control group
  • “b” indicates that the p-value was ⁇ 0.05 relative to the no-treatment group.
  • FIGs. 6a-6c illustrate the photomicroscopy of histopathology findings in MAA treated rats.
  • Fig. 6a shows the normal ciliary process and sclera (HE lOOx; 0 score).
  • Fig. 6b shows a picture of a MAA-treated rat where there is marked inflammation of the ciliary process, iris, cornea, and uvea (200x; 4 score).
  • Fig. 6c shows a picture of a MAA-treated rat where there is there is marked inflammation of the ciliary process, iris, cornea, and uvea (400x; 4 score).
  • the MAA-induced model of anterior uveitis in Lewis Rats was characterized by a robust, uniform inflammation that peaks about Day 18 and underwent resolution by Day 30. The procedure was well tolerated, and there were marked inflammatory changes in the anterior segment that were visible via slit lamp examination clinically and were present histopathologically. Both DEX and CSA showed activity in inhibition of the inflammation, while topical Restasis is not particularly active.
  • neutrophils and/or mononuclear cells are neutrophils and/or mononuclear cells.
  • anterior chamber and posterior chamber anterior chamber and posterior chamber.
  • Inflammation may extend into cornea, anterior
  • Experimental autoimmune anterior uveitis can be induced by a self-antigen.
  • EAU Experimental autoimmune anterior uveitis
  • MA melanin-associated antigen isolated from bovine retinal pigment epithelium (RPE), iris, ciliary body, and choroid.
  • RPE retinal pigment epithelium
  • EAU can also be induced by adjuvant and a soluble protein fraction released by proteolytic digestion of bovine MAA with V8 protease.
  • MAA without the use of adjuvant. Bora, N.S. et al. 1997.
  • a murine model can be used to test the effects of compositions of the invention on experimental autoimmune uveitis (EAU).
  • EAU experimental autoimmune uveitis
  • C57BL/6 mice can be immunized with human interphotoreceptor retinoid-binding protein peptide (h-IRBP) to induce ocular inflammation.
  • h-IRBP human interphotoreceptor retinoid-binding protein peptide
  • the severity of EAU can be assessed clinically and histopatho logically, along with measures of T-cell activation and leukocyte adhesion in the retina.
  • Acute ocular inflammation can be induced using composition 48/80 (N-methyl-p- methoxyphenethylamine formaldehyde condensation product) in animal models.
  • Composition 48/80 can selectively degranulate mast cells to induce conjunctival eosinophilia in rabbits.
  • composition 48/80 topically on the eye in rabbits produced a brisk acute ocular inflammation that was responsive to dexamethasone treatment over a 1-3 day period.
  • the inflammation was characterized by an acute conjunctivitis
  • An allergic conjunctivitis can be induced by ovalbumin administration to guinea pigs and rabbits.
  • Guinea pigs are the preferred species due to the consistency of response. Animals were sensitized by ovalbumin and adjuvant injected intraperitoneally at two week intervals. One week later, one eye was challenged by topical administration of ovalbumin. In some embodiments, acute inflammation developed and persisted over about three days with a peak at about 24 hours.
  • a study design included groups with at least 6 guinea pigs or rabbits per group. Ovalbumin and adjuvant were injected intraperitoneally at two week intervals. One eye was challenged by topical application as a single dose of albumin in PBS to the eye. Topical dexamethasone was an appropriate positive control. Slit lamp and funduscopic examination was performed. A visual scoring of ocular clinical signs and/or a histopathology examination on the eye and adenexal structures was performed.
  • ovalbumin produced a brisk acute ophthalmitis that was responsive to dexamethasone treatment.
  • the inflammation was characterized clinically by redness, and inflammation of the eye.
  • the effects of Dexamethasone on cellularity of aqueous humor and ocular histopathology are shown in the tables below.
  • Example 1 Methods of Treatment of Dry Eye in Animal Models Using
  • Dry eye can be induced in animal models using one or more of the methods described above. As described in Sections LA through I.D, dry eye can be induced by botulinum toxin injections, administration of scopolamine, use of the CAE protocol, or injection of concanavalin A (Con A) into lacrimal glands. Suitable animal models include, but are not limited to, rodents, such as Sprague Dawley rats, or rabbits.
  • a composition or formulation comprising an adenosine A3 receptor antagonist described above is administered to the animal and is used to treat the dry eye condition.
  • Any of the compositions of the invention described above can be used for treatment of dry eye.
  • the adenosine A3 receptor antagonist used is ACN-1052, LJ1251, MRS 3820, MRS 3771, LJ 979, MRS 3826, or MRS 3827.
  • the adenosine A3 receptor antagonist used is MRS 1191 or MRS 1292.
  • the adenosine A3 receptor antagonist is ACN-1052.
  • the formulations of adenosine A3 receptor antagonist can be prepared for oral administration, injection, or non-invasive administration, such as topical administration.
  • the formulations can be prepared as (a) liquid solutions; (b) capsules, sachets, tablets, lozenges, and troches; (c) powders; (d) suspensions in an appropriate liquid; or (e) suitable emulsions. Examples include formulations administered as eye-drops, injectable liquids or solutions, or topical eye creams.
  • the dosage of the formulation can vary depending on the form of administration.
  • a suitable dose of a formulation of an adenosine A3 receptor antagonist will be in the range of 0.001 to 1000.0 milligrams per kilogram body weight of the animal per day, in the range of 0.01 to 200.0 milligrams per kilogram body weight of the animal per day, or in the range of 1 to 50 mg per kilogram body weight per day.
  • Optimal dosing schedules can be calculated by administering various doses of the adenosine A3 receptor antagonist to the animal and measuring efficacy of the different doses.
  • the optimized dose or formulation may be administered at various intervals of time: once a week, once every two days, once daily, BID (e.g., twice daily), TID, QID, or in multiple doses at appropriate intervals throughout the day, or even using continuous infusion or delivery through a controlled release formulation.
  • a dry eye study is performed using two experimental groups of mammals (e.g., a control group and a test group of 6 male NZW rabbits per group). The rabbits are assessed for inclusion in the study. Criteria include the entry criteria (scores of ⁇ 1 for conjunctival congestion and swelling; scores of 0 for all other observation variables) or exclusion criteria (no rabbits with gross signs of ocular irritation are used in this study).
  • ConA Prior to induction of dry eye (e.g., injection of Con A on day -4), one or more of the following assays can be performed: gross ocular observations; scoring of signs of irritation or discomfort; examination of corneal thickness by pachymetry; slit lamp ophthalmic examinations with fluorescein dye to examine the cornea surface; a Shirmer Tear Test; or a Tear Break Up Test.
  • ConA can be injected bilaterally into the lacrimal glands (on day -3 and day 0).
  • Each group is administered a vehicle or an adenosine A3 receptor antagonist (or a formulation of an adenosine A3 receptor antagonist).
  • Treatment can include administering a vehicle or an adenosine A3 receptor antagonist to both eyes three times daily (e.g., 9 am, 12 pm, 4 pm).
  • the following examinations are made (on days 1 to 14): daily gross ocular observations, signs of irritation or discomfort scored; daily corneal thickness measured by pachymetry; daily slit lamp ophthalmic examinations with fluorescein dye to examine cornea surface, scored according to McDonald Shadduck method; daily Shirmer Tear Test; or a daily Tear Break Up Test.
  • a terminal examination is performed based on a histopathology of the eye and adenexal structures.
  • the tear volume, tear function, and inflammatory cytokine levels in the lacrimal glands are assessed, along with a histological evaluation.
  • the density of mucus producing goblet cells, corneal inflammation, and corneal epithelial damage also are measured and compared to normal control animals. Evaluation of the results shows that treatment with a composition or formulation comprising an adenosine A3 receptor antagonist produces a significant improvement or amelioration in the symptoms and condition of dry eye syndrome in the animal compared to the control group.
  • Example 2 Methods of Treatment of Dry Eye Syndrome in Mice Using A
  • Dry eye syndrome was induced in mice using the CAE model and protocol described above in Section I.C.
  • the adenosine A3 receptor antagonist ACN- 1052 was selected and used for treatment of dry eye syndrome.
  • FIG. 7 shows an example of inhibition of corneal injury after treatment of ACN- 1052 in mice using the CAE model described above. Mice were exposed to 10 days of an adverse environment according to the CAE protocol and treated with ACN-1052, Restasis, or a vehicle control on the fifth day. The ACN-1052 antagonist resulted in approximately 30% reduction in corneal injury based on a measurement of corneal staining of the eye. Restasis produced a 20% change in corneal staining, whereas the vehicle control did not inhibit corneal injury.
  • FIG. 8 illustrates the effect of ACN- 1052 in a mouse model using the CAE protocol to induce dry eye syndrome.
  • Mice were exposed to 10 days of an adverse environment and treated with ACN-1052, Restasis or a vehicle control on the fifth day.
  • the percentage of eyes with an improvement in corneal staining was 88% in the group treated with ACN-1052, compared to 81% in the Restasis group and 63% in the vehicle control. .
  • Example 3 Methods of Treatment of Dry Eye Syndrome in Patients Using
  • compositions or formulations of adenosine A3 receptor antagonist can be used for treatment of patients with dry eye syndrome.
  • the composition or formulation can include a therapeutically effective amount of an adenosine A3 receptor antagonist and a
  • a subject e.g., a patient with dry eye syndrome or dry eye symptoms is suitable for treatment.
  • the symptoms of the patient Prior to treatment, the symptoms of the patient are assessed.
  • Symptoms can include, but are not limited to, a dry, gritty, scratchy, or filmy feeling in the eyes, a burning or itching in the eyes, redness of the eyes, intermittent excessive tearing, blurred vision, a sensation of having a foreign body in the eye, and light sensitivity.
  • a control group of patients without dry eye syndrome can be included in the study.
  • a composition or formulation comprising an adenosine A3 receptor antagonist described above is administered to the patient to treat the dry eye condition. Any of the compositions of the invention described above can be used for treatment of dry eye.
  • the adenosine A3 receptor antagonist used is ACN-1052, LJ1251, MRS 3820, MRS 3771, LJ 979, MRS 3826, or MRS 3827.
  • the adenosine A3 receptor antagonist used is MRS 1191 or MRS 1292.
  • the adenosine A3 receptor antagonist is ACN-1052.
  • the formulations can be suitable for oral administration, injection, or non-invasive administration, such as topical administration.
  • the formulations can be prepared as (a) liquid solutions; (b) capsules, sachets, tablets, lozenges, and troches; (c) powders; (d) suspensions in an appropriate liquid; or (e) suitable emulsions. Examples include formulations administered as eye-drops, injectable liquids or solutions, or topical eye creams.
  • Dosing is dependent on severity and responsiveness of the dry eye condition to be treated, with the course of treatment lasting hours, days, months, or until a cure is effected or an improvement of the dry eye is achieved. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Dosage will vary with the age, gender, weight and response of the particular patient, as well as the severity of the patient's symptoms. Optimum dosages may vary depending on the relative potency of individual compositions, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • a suitable dose of a formulation of an adenosine A3 receptor antagonist will be in the range of 0.001 to 1000.0 milligrams per kilogram body weight of the patient per day, in the range of 0.01 to 200.0 milligrams per kilogram body weight of the patient per day, or in the range of 1 to 50 mg per kilogram body weight per day.
  • the formulation may be administered at various intervals of time: once a week, once every two days, once daily, BID (e.g., twice daily), TID, QID, or in multiple doses at appropriate intervals throughout the day, or even using continuous infusion or delivery through a controlled release formulation.
  • BID e.g., twice daily
  • TID e.g., twice daily
  • QID e.g., QID
  • Improvement of symptoms can be determined by ocular examination and/or self-reporting of symptoms by the patient. For example, an improvement in redness of the eyes or a reduction in burning or itching in the eyes can be assessed. Tear volume, tear function, and corneal inflammation can also be analyzed. Treatment with a composition or formulation of an adenosine A3 receptor antagonist alleviates or improves the symptoms of dry eye syndrome in the patient.
  • Example 4 Methods of Treatment of Uveitis Using an Adenosine A3 Receptor Antagonist
  • Methods of the invention include treatment of uveitis using an adenosine A3 receptor antagonist.
  • Uveitis is induced by melanin-associated antigen, as described in methods above.
  • the compositions of the invention described above can be used in a formulation for treatment of uveitis.
  • the adenosine A3 receptor antagonist used for treatment of uveitis is ACN-1052, LJ1251 , or MRS3820.
  • the adenosine A3 receptor antagonist used is MRS 1 191 or MRS 1292.
  • the adenosine A3 receptor antagonist is ACN-1052.
  • a study is performed using two experimental groups (e.g., a control group and a test group of 6 male Lewis rats each).
  • uveitis is induced in all animals by an injection into the tail head of an emulsion containing melanin-associated antigen (MAA), Complete Freund's Adjuvant (CFA), and pertussis toxin.
  • MAA melanin-associated antigen
  • CFA Complete Freund's Adjuvant
  • pertussis toxin are used.
  • a vehicle and a composition comprising an adenosine A3 receptor antagonist is administered to both eyes in rats in Groups 1 and 2. Dosing occurs twice on day 0 and TID (three times per day) for the next 21 days.
  • the animals are housed in a well ventilated dry environment.
  • a treatment with a composition comprising an adenosine A3 receptor antagonist is shown to produce a significant improvement or amelioration in the symptoms and condition of uveitis in the animal subject, compared with the control group.
  • Example 5 Diabetic retinopathy and diabetic macular edema in the
  • Diabetic retinopathy refers to the damage to the eye's retina that occurs with long- term diabetes and that can cause blindness.
  • Diabetic macular edema is a swelling of the retina due to leaking of fluid from blood vessels within the macula, which is the central portion of the retina. Macular edema causes blurring in the middle of side of the central visual field, and visual loss can progress over time, making it impossible to focus clearly.
  • a streptozotocin-induced diabetic rat model is used to study the effects of administration of compositions of the invention.
  • Type I diabetes is induced in rats by administration of streptozotocin.
  • Retinopathy and diabetic macular edema in streptozotocin- induced diabetic rat model are described in Zheng et al. (2007) Diabetes. 56:337-345; Carmo, A. et al. (2000) Mediators of ' Inflammation. 9:243-248; Kusari, J. et al. (2007) Invest. Opthal. & Visual Sci. 48(11): 5152-5159.
  • the following assessments are performed: glycemia, angiography (HRA), electroretinography (ERG), histology and
  • the retinae are flat-mounted in a water-based fluorescence-anti-fading medium (Southern Biotechnology, Birmingham, Al) and imaged via fluorescence microscopy (Zeiss Axiovert FITC filter).
  • Leukocyte location is scored as either arteriolar, venular, or capillary and the total number of adherent leukocytes per retina are counted.
  • Blood-retinal barrier breakdown are counted by using Evans blue dye.
  • the retinae are carefully dissected and thoroughly dried in a Speed- Vac (Savant, St. Paul, MN) for 5 h. Evans blue is extracted by incubating each retina in 120 ⁇ formamide (Sigma) for 18 hours at 70°C. The supernatant is filtered through Ultrafree-MC tubes (30,000 NMWL, MiUipore, Bedford, MA) at 3,000 g, and the filtrate is used for triplicate spectrophotometric
  • the background- subtracted absorbance is determined by measuring each sample at both 620 nm, the absorbance maximum for Evans blue in formamide, and 740 nm, the absorbance minimum.
  • the concentration of dye in the extracts is calculated from a standard curve of Evans blue in formamide and is normalized for retina dry weight.
  • the blood-retinal barrier breakdown is calculated by using the following equation, with results expressed in microliters of plasma x g retina dry weight- 1 x hr-1 : [Evans blue ⁇ g)/ Retina dry weight (g)] / [Time-averaged Evans blue concentration ⁇ g /plasma ⁇ )/ circulation time (hr)].
  • a treatment using a composition comprising an adenosine A3 receptor antagonist is shown to produce a significant improvement or amelioration in the symptoms and condition of diabetic macular edema or diabetic retinopathy in the subject, compared with the control group.

Abstract

Disclosed are compositions comprising adenosine A3 receptor antagonists and methods of their use for the treatment of inflammatory eye diseases, such as uveitis and dry eye.

Description

TITLE
[0001] COMPOSITIONS AND METHODS USING ADENOSINE A3 RECEPTOR
ANTAGONISTS FOR THE TREATMENT OF INFLAMMATORY EYE DISEASES BACKGROUND OF THE INVENTION
Field of the invention
[0002] The invention relates to compositions of adenosine A3 receptor antagonists and methods of their use for the treatment of inflammatory eye diseases.
Description of the Related Art
[0003] Inflammatory eye diseases, such as dry eye and uveitis, are serious conditions that can cause eye pain, redness, discomfort, or sensitivity to light. In many cases, the inflammatory eye disease can be unresponsive to treatment and may lead to blindness with glaucoma, cataract or retinopathy. Treatment options for inflammatory eye disease are limited to steroids, non-steroid anti-inflammatory drugs (NSAIDs), immunosuppressive chemotherapy medications or immunomodulatory therapy (IMT). Conventional treatments may not reverse or stop progression of the eye disease. Generally, inflammatory eye diseases can be difficult to treat and may recur after treatment.
[0004] Adenosine receptors belong to the G protein-coupled family of proteins and are characterized by seven transmembrane helical domains. Receptor activation leads to its internalization and the subsequent inhibition of: (i) adenylyl cyclase activity, (ii) cAMP formation and (iii) protein kinase A (PKA) expression, resulting in the initiation of various signaling pathways. There are four types of adenosine receptors: Al, A2a, A2b, and A3. Adenosine A3 receptors are involved in a variety of important patho-physiological processes, including modulation of cerebral and cardiac ischemic damage, inflammation, modulation of intraocular pressure, regulation of normal and tumor cell growth, and immune suppression.
[0005] Several adenosine A3 receptor agonists and antagonists have been identified, such as adenosine derivatives, fiavonoids, and triazoloquinazolines, which specifically bind to the adenosine A3 receptor. See e.g., PCT/KR2007/001131; U.S. Patent No. 6,376,521; WO 2010/014921; U.S. Patent No. 6,199,127. Agonists of the adenosine A3 receptor have been shown to desensitize the adenosine A3 receptor. Palmer and Stiles. (1999) Molecular Pharmacology; Palmer, T.M. et al. (1995) J. Biol. Chem. In addition, the adenosine A3 receptor agonist, CF101, has been shown to improve corneal staining and tear break-up time (TBUT) and tear meniscus (TM) in severe dry eye syndrome. Avni, I. et al. (2010) Ophthamology. 117(7): 1287-93. However, their performance as effective drugs for treatment of uveitis, dry eyes, and other inflammatory eye diseases remains suboptimal.
[0006] Accordingly, there is a need for effective and suitable methods of treating
inflammatory eye diseases. The invention addresses these and other shortcomings of the prior art by providing methods for treating inflammatory eye disease using adenosine A3 receptor antagonist compositions.
SUMMARY OF THE INVENTION
[0007] Methods of the invention include treating an inflammatory eye disease by
administering to a mammalian subject in need of such treatment an effective amount of an adenosine A3 receptor antagonist. In some embodiments, the inflammatory eye disease is selected from the group consisting of: dry eye, uveitis, scleritis, blepharitis, keratitis, conjunctivitis, chorioretinital inflammation, choroiditis, retinitis, iridocyclitis, iritis, posterior cyclitis, diabetic retinopathy, a post-surgery condition, allergies, eye trauma, eye bruises, foreign body, toxin exposure, chemical exposure, food allergies, hives, rheumatoid arthritis, sarcoidosis, juvenile idiopathic arthritis, lupus, Behcet's disease, viral infection, fungal infection, mycobacterial infection, parasite infection, bacterial infection, acanthamoeba infection, spirochete infection, Crohn's disease, arthritis, ulcerative colitis, lymphoma, eye tumors, cancer of blood vessels, neuroretinitis, Sarcoidosis-related retinitis, Behcet' s-related retinitis, or acute retinal pigment epitheliitis.
[0008] In certain embodiments, the adenosine A3 receptor antagonist comprises any of the compositions described herein. In another embodiment, the adenosine A3 receptor antagonist is selected from the group consisting of: ACN-1052, LJ1251, MRS3820, MRS3771, LJ 979, MRS 3826, MRS 3827, MRS1191 or MRS 1292. In some embodiments, the inflammatory eye disease is uveitis, and the adenosine A3 receptor antagonist is selected from the group consisting of: ACN-1052, LJ1251, or MRS3820. In other embodiments, the inflammatory eye disease is dry eye and the adenosine A3 receptor antagonist is selected from the group consisting of: ACN-1052, LJ1251, MRS3820, MRS 3771, LJ 979, MRS 3826, or MRS3827. In another embodiment, the adenosine A3 receptor antagonist is ACN-1052.
[0009] In other embodiments, administering an effective amount of an adenosine A3 receptor antagonist reduces corneal inflammation. In one embodiment, administering an effective amount of an adenosine A3 receptor antagonist reduces inflammatory cytokine levels. [0010] In another embodiment, the mammalian subject is a human subject. In yet another embodiment, the adenosine A3 receptor antagonist is formulated for topical delivery to an eye of the mammalian subject. In other embodiments, the adenosine A3 receptor antagonist is formulated for topical delivery, intraocular injection, intravitreal injection, oral delivery, sublingual delivery, intranasal delivery, intravenous injection, intramuscular injection, intraperitoneal injection, or rectal delivery.
[0011] In some embodiments, the method includes co-administering to the mammalian subject an effective amount of a steroid, a non-steroidal anti-inflammatory, or an
immunosuppressant. In one embodiment, the immunosuppressant is cyclosporine A.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0012] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:
[0013] FIG. la shows a graph of tear reduction in mice after scopolamine patch application under dry environmental conditions.
[0014] FIG. lb shows histopathology scores 12 days after scopolamine administration in mice. In the figure, "a" indicates that the p-value is less than 0.05 relative to the vehicle control group, and "b" indicates that the p-value was less than 0.05 reltaive to the no- treatment group. N=10 female C57BL/6 mice per group.
[0015] FIG. 2 shows the effects of Dexamethasone (DEX) and cyclosporine (CSA, Restasis) in a Tear Break Up Test (TBUT) in concanavalin A (ConA)-induced dry eye in rabbits.
[0016] FIG. 3a shows the effects of ConA injection on the Schirmer Tear Test (STT) in rabbits.
[0017] FIG. 3b shows the use of DEX and CSA in the Schirmer Tear Test (STT) in rabbits.
[0018] FIGs. 4a-4c shows representative photographs of the anterior area of the eye of a rat with a melanin-associated antigen (MAA) induced anterior uveitis.
[0019] FIG. 5 shows histopathology scores 18 days after MAA injection in untreated rats and rats treated with vehicle, DEX, CSA, or Restasis.
[0020] FIGs. 6a-6c illustrates the photomicroscopy of histopathology findings in MAA- treated rats. [0021] FIG. 7 shows inhibition of corneal injury by ACN-1052 in a mouse model of dry eye syndrome measured by corneal staining.
[0022] FIG. 8 shows the effect of ACN-1052 on dry eye syndrome in a mouse model measured by corneal staining.
DETAILED DESCRIPTION OF THE INVENTION
Advantages and utility
[0023] Briefly, and as described in more detail below, described herein are methods and compositions for the treatment of inflammatory eye diseases using adenosine A3 receptor antagonists.
Definitions
[0024] Terms used in the claims and specification are defined as set forth below unless otherwise specified.
[0025] "Adenosine" refers to a purine nucleoside and a metabolite of adenosine triphosphate (ATP). Adenosine is a nucleoside composed of a molecule of adenine attached to a ribose sugar molecule (ribofuranose) moiety via a P-N9-glycosidic bond. Adenosine plays an important role in biochemical processes, such as energy transfer, as adenosine triphosphate (ATP) and adenosine diphosphate (ADP), as well as in signal transduction as cyclic adenosine monophosphate, cAMP. The IUPAC name is (2R,3R,4S,5R)-2-(6-amino-9H- purin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol. Adenosine mediates a wide variety of physiological functions by interacting with four cell surface receptors namely Al, A2a, A2b and A3.
[0026] "Adenosine receptor" refers to a class of purinergic receptors or G protein-coupled receptors that bind adenosine as an endogenous ligand. Adenosine receptors include four types: Al, A2a, A2b, and A3.
[0027] "Adenosine A3 receptor" or "A3AR" or "A3 Adenosine Receptor" refers to a G protein-coupled receptor that is involved in a variety of intracellular signaling pathways and physiological functions. Adenosine A3 receptors are ubiquitously expressed in a wide variety of tissues in human body, with high levels in peripheral organs and low levels in the brain. Adenosine A3 receptors are involved in a variety of important patho-physiological processes, including modulation of cerebral and cardiac ischemic damage, inflammation, modulation of intraocular pressure, regulation of normal and tumor cell growth, and immunosuppression. Adenosine A3 receptors are also involved in the inhibition of neutrophil degranulation in neutrophil-mediated tissue injury, have been implicated in both neuroprotective and neurodegenerative effects, and may also mediate both cell proliferation and cell death. In humans, the adenosine A3 receptor is encoded by the mR A with
GenBank Accession No. NM 000677 and has the protein sequence of GenBank Accession No. NP 000668. In mice, the adenosine A3 receptor is encoded by the mRNA with
GenBank Accession No. NM 009631. Citations in this specification to GenBank Accession Numbers refer to those sequence versions current as of the filing date of this specification.
[0028] "Adenosine A3 receptor antagonist" or "A3AR Ag" refers to a ligand, molecule, composition, or drug that binds to an adenosine A3 receptor and inhibits or dampens agonist- mediated responses. The A3AR Ag exerts its effect through binding and inactivation of the A3AR. Adenosine A3 receptor antagonists can have affinity but no efficacy for their cognate receptors, and binding can disrupt the interaction and inhibit the function of an agonist or inverse agonist at the receptors.
[0029] "Agonists" refer to receptor ligands, molecules, compositions or drugs that activate a cellular response by binding to a receptor.
[0030] "Antagonists" refer to receptor ligands, molecules, compositions or drugs that inhibit the receptor's agonist response, in some cases by blocking the receptor from the agonist. Competitive antagonists (or surmountable antagonists) reversibly bind to receptors at the same binding site (active site) as the endogenous ligand or agonist, but without activating the receptor. For competitive antagonists, the level of receptor inhibition is determined by the relative affinity of each molecule for the site and their relative concentrations. Noncompetitive antagonists (or non-surmountable antagonists) are allosteric antagonists. These antagonists bind to a distinctly separate binding site from the agonist, exerting their action to that receptor via the other binding site.
[0031] "Inflammatory eye disease" refers to disorders, diseases, or conditions that affect the eye, including, but not limited to, dry eye, uveitis, scleritis, blepharitis, keratitis,
conjunctivitis, chorioretinital inflammation, choroiditis, retinitis, iridocyclitis, iritis, posterior cyclitis, diabetic retinopathy, post-surgery, allergy, trauma, bruises, foreign body, exposure to toxins or chemicals, food allergies, hives, autoimmune disorders (such as rheumatoid arthritis, sarcoidosis, juvenile idiopathic arthritis, lupus, Behcet's disease, etc.), infection (such as viral, fungal, mycobacteria, parasite, bacteria, acanthamoeba, spirochete, etc.), inflammatory conditions (such as Crohn's disease, arthritis and ulcerative colitis) and cancers (such as lymphoma, eye tumors, cancer of blood vessels, etc.), or an inflammatory retinal disease (such as neuroretinitis, Sarcoidosis-related retinitis, Behcet' s-related retinitis, or acute retinal pigment epitheliitis).
[0032] "In vivo" refers to processes that occur in a living organism.
[0033] "Effective amount" or "pharmaceutically effective amount" refers to a sufficient amount of an agent to provide the desired biological result. The result can be a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic uses is the amount of the composition comprising an active composition herein required to provide a clinically significant decrease in inflammation in the eye, and thereby reducing symptoms of an eye disease, e.g. , uveitis, such as blurred vision or eye pain.
[0034] "Treat" or "treatment" are used interchangeably and are meant to indicate
administering one or more compositions in accordance with the methods of the invention to obtain a desired therapeutic objective. The terms further include ameliorating existing eye symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, and/or promoting resolution of the disease or condition.
[0035] "Ameliorating" refers to any therapeutically beneficial result in the treatment of a disease state, e.g., an eye disease, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.
[0036] By "pharmaceutically acceptable" or "pharmacologically acceptable" is meant a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual without causing any substantial undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
[0037] A "subject" encompasses mammals. A "mammal" includes any member of the Mammalia class: humans, non-human primates (chimpanzees, and other apes and monkey species), canines, felines, murines, bovines, equines, porcines, etc. Other examples of mammals include cattle, horses, sheep, goats, swine, rabbits, dogs, cats, and rodents, such as rats, mice and guinea pigs, and the like. The terms "subject" or "mammal" do not denote a particular age or gender.
[0038] "Aryl" refers to aromatic moieties such as phenyl, naphthyl, anthracenyl, and biphenyl. "Heterocyclyl" refers to 3-7 membered rings which can be saturated or unsaturated or heteroaromatic, comprising carbon and one or more heteroatoms such as O, N, and S, and optionally hydrogen; optionally in combination with one or more aromatic rings. Examples of heterocyclyl groups include pyridyl, piperidinyl, piperazinyl, pyrazinyl, pyrolyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, pyrrolidinyl, furanyl, tetrahydrofuranyl, thienyl, furyl, thiophenyl, tetrahydrothiophenyl, purinyl, pyrimidinyl, thiazolyl, thiazolidinyl, thiazolinyl, oxazolyl, tetrazolyl, tetrazinyl, benzoxazolyl, morpholinyl, thiomorpholinyl, quinolinyl, and isoquinolinyl. Examples of heteroaryl alkyl include heteroaryl methyl such as 2- or 3- methyl substituted groups, e.g., thienylmethyl, pyridylmethyl, and furylmethyl.
[0039] Alkyl, alkoxy, and alkylamino groups can be linear or branched. When an aryl group is substituted with a substituent, e.g., halo, amino, alkyl, hydroxyl, alkoxy, and others, the aromatic ring hydrogen is replaced with the substituent and this can take place in any of the available hydrogens, e.g., 2, 3, 4, 5, and/or 6-position wherein the 1 -position is the point of attachment of the aryl group in the composition of the present invention.
[0040] "Halo" refers to fluorine, chlorine, bromine, and iodine.
[0041] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.
Compositions of the invention
[0042] In some embodiments, the compositions of the invention comprise adenosine A3 receptor antagonists. In certain aspects, the compositions of the invention comprise adenosine derivatives, dihydropyridine derivatives, pyridine derivatives, or purine nucleosides.
[0043] In one embodiment, the composition of the invention comprises:
Figure imgf000009_0001
[0044] wherein R is 3-iodobenzyl. This composition is named ACN-1052 (also called LJ1251 or MRS3820) and has the chemical name (2R,3R,4S)-2-(6-(3-iodobenzylamino)-2- chloro-9H-purin-9-yl)-tetrahydrothiophene-3,4-diol. It is described in Jeong et al. (2007) Journal of Medicinal Chemistr . ACN-1052 is also shown below:
Figure imgf000009_0002
[0045] ACN-1052 has a molecular weight of 503, a melting point of 197-198°C, an optical rotation of [<x]D = -77.7, c= 0.13, DMSO, and a particle size of ~4. ACN-1052 is freely soluble in DMSO and various mixtures of organic solvents such as DCM and DMSO. It is not appreciably soluble in water-based vehicles with low concentrations of organics such as DMSO or ethanol.
[0046] ACN-1052 can be synthesized by the following steps:
Sch me L Svnthesis of the Glveosv. Donor 15"
Figure imgf000010_0001
10 n
Figure imgf000010_0002
14 1S s Reage,iit¾ aad condrfioiii,: (a) 2,2-tli et oxvpropaise. ca hoisulioiuc acid CHiCOCHj.. r 15 k <b) XaB¾, EtOH. rt, 2 ; (c) MsCI, E?.N. CMiCh, ft I fa; ( NaS, DMF, SO :C: 15 ; (e) 60% AcOH, tt, 2 Is; (f) Pb(OAc}4; EIQAe, it, overnight
Figure imgf000011_0001
a Reageats sod conditions: (a) ^-dic loK^iirine, MBmoaiam sulfate,
Figure imgf000011_0002
THF, rt, 15 li; (c) SN¾ EfeN, EtOH, it, 1-3 d.
[0047] In another embodiment, the composition of the invention is selected from the group comprising:
Figure imgf000011_0003
mm 3771 U 979
A.¾ antagonists
Figure imgf000011_0004
Figure imgf000012_0001
R^COCH3 R'-GOCBS -H
MRS 3826 MRS 3827 LJ 1251
A¾ antagonist prodrugs A antagonist {truncated}
(0-aoet lated)
[0048] These adenosine A3 receptor antagonists (MRS 3771, LJ 979, MRS 3826, MRS 3827, and LJ 1251) are described in Wang et al. (2010) Experimental Eye Research. Synthesis of the above compounds includes the following scheme, described by Gao et al. (2006)
Bioorg. Med. Chem. Lett. 16:596-601.
Figure imgf000012_0002
10
8 R = N{Ct¾& 4
Figure imgf000012_0003
[0049] Synthesis of the adenosine A3 receptor antagonists can also follow the following pathways, as described in Jeong et al. (2007) J. Med. Chem. :
Figure imgf000013_0001
Adenos ne (i mlw i i d I ( - H, fell ssgontet)
3 R « Gli, full agonM
Figure imgf000013_0002
5, M\ mg&nmi
[0050] In other embodiments, the composition of the invention comprises:
Figure imgf000013_0003
[0051] MRS 1292 is derived from the agonist IB-MECA (N6-(3-iodobenzyl)-adenosine-5'-N- methyluronamide). MRS 1292 is described in Yang et al. (2005) Current Eye Research. 30:747-754. MRS 1292 can be synthesized by the following steps, described in Besada et al. (2006) Collect. Czech. Chem. Commun. 71(6):912-928.
Figure imgf000014_0001
J; B ' « t CH^CH^ R'! ~ M
S nt esis of s¾ds« s of Α,ΛΙί. agonist l¾ag¾?«f:s and conditions: (i) Procedure ,4. HO . OMAF, Py; r.t; Pracedufs B. HO, DMAP,€.¾€½ r.t.
[0052] In another embodiment, the composition of the invention comprises:
Figure imgf000014_0002
MRS 1191
4osi oo/>i , woBi ,4(h)
[0053] MRS 1191 has the IUPAC name 03-ethyl 05-(phenylmethyl) 2-methyl-6-phenyl-4- (2-phenylethynyl)-l ,4-dihydropyridine-3,5-dicarboxylate. MRS 1191 is described in Jacobson et al. (1997) Neuropharmacology. 36(9):1 157-1165. MRS 1191 can be synthesized by the following steps, wherein R4 is phenylethynyl.
Figure imgf000015_0001
Figure imgf000015_0002
tow
<8o-i B5 » OCHjPh (su stj
[0054] In some embodiments, the composition of the invention comprises:
Figure imgf000015_0003
[0055] wherein R1 is selected from the group consisting of hydrogen, Ci-C6 alkyl, Ci-C6 alkoxy, hydroxyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl Ci-C6 alkyl, C3-C8 dicycloalkyl Ci-C6 alkyl, C7-C12 bicycloalkyl Ci-C6 alkyl, C7-C14 tricycloalkyl Ci-C6 alkyl, C6-Ci4 aryl, C6-Ci4 aryl Ci- C6 alkyl, C6-Ci4 diaryl Ci-C6 alkyl, C6-Ci4 aryl Ci-C6 alkoxy, Ci-C6 alkyl carbonyl, sulfonyl, Ci-C6 alkyl sulfonyl, C6-Cj4 aryl sulfonyl, heterocyclyl Ci-C6 alkyl, heterocyclyl, heteroaryl Ci-C6 alkyl, 4-[[[4-[[[(2-amino Ci-C6 alkyl) amino]-carbonyl]-Ci-C6 alkyl] aniline] carbonyl] Ci-C6 alkyl] C6-Ci4 aryl, and C6-Ci4 aryl C3-C8 cycloalkyl, wherein the aryl or heterocyclyl portion of R is optionally substituted with one or more substituents selected from the group consisting of halo, amino, hydroxyl, carboxy, Ci-C6 alkoxycarbonyl, aminocarbonyl, Ci-C6 alkylaminocarbonyl, Ci-C6 dialkyl aminocarbonyl, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 alkoxy, C6-Ci4 aryloxy, hydroxy Ci-C6 alkyl, hydroxy C2-C6 alkenyl, hydroxy C2-C6 alkynyl, carboxy Ci-C6 alkyl, carboxy C2-C6 alkenyl, carboxy C2-C6 alkynyl, aminocarbonyl Ci-C6 alkyl, aminocarbonyl C2-C6 alkenyl, aminocarbonyl C2-C6 alkynyl, and C≡C-(CH2)n-COR7, wherein R7 is selected from the group consisting of OH, OR8, and NR9R10, wherein R8 is selected from the group consisting of Ci-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl Ci-C6 alkyl, C3-C8 dicycloalkyl Ci-C6 alkyl, C7-C12 bicycloalkyl Ci-C6 alkyl, C7-C14 tricycloalkyl Ci-C6 alkyl, C6-Ci4 aryl, C6-Cu aryl Ci-C6 alkyl, C6-Ci4 and diaryl Ci-C6 alkyl; and R9 and R10 are independently selected from the group consisting of hydrogen, Ci-C6 alkyl, and (CH2)„Rn wherein R11 is NR12R13, wherein R12 and R13 are independently selected from the group consisting of hydrogen, Ci-C6 alkyl, and COR14 wherein R14 is hydrogen or Ci-C6 alkyl; wherein n is an integer from 1 to 10; and the alkyl or cycloalkyl portion of R1 is optionally substituted with one or more substituents selected from the group consisting of halo, amino, Ci-C6 alkyl, Ci-C6 alkoxy, C6-Ci4 aryloxy, Ci-C6 hydroxyalkyl, C2-C6 hydroxyalkenyl, C2-C6 hydroxy alkynyl, aminocarbonyl Ci-C6 alkoxy, and C6-Ci4 aryl Ci-C6 alkoxy;
[0056] wherein R2 is selected from the group consisting of hydrogen, halo, amino, hydrazido, mercapto, C1-C20 alkylamino, C6-Ci4 aryl amino, C6-Ci4 aryloxy, C1-C20 alkyl, C1-C20 alkoxy, C1-C20 thioalkoxy, pyridylthio, C7-C12 cycloalkyl C1-C20 alkyl, C7-C12 bicycloalkyl C1-C20 alkyl, C7-C2 bicycloalkenyl C1-C20 alkyl, C6-Ci4 aryl C1-C20 alkyl, C2-C20 alkenyl, C7- C12 cycloalkyl C2-C2o alkenyl, C7-C12 bicycloalkyl C2-C2o alkenyl, C7-C12 bicycloalkenyl C2- C20 alkenyl, C6-Ci4 aryl C2-C20 alkenyl, C2-C20 alkynyl, -C≡C-(CH2)m-C(=0)-0-Ci-C6 alkyl, - C≡C-(CH2)m-C(=0)-NH-(CH2)n-NH2, -C≡C-(CH2)m-C C6 alkyl, -C≡C-(CH2)m-aryl, wherein m and n are independently 1 to 10, C7-C12 cycloalkyl C2-C20 alkynyl, C7-C12 bicycloalkyl C2- C2o alkynyl, C7-C12 bicycloalkenyl C2-C2o alkynyl, C6-Ci4 aryl C2-C2o alkynyl, and the alkyl, cycloalkyl, or aryl portion of R2 is optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, amino, alkylamino, dialkylamino, sulfur, carboxy, alkoxycarbonyl, aminocarbonyl, alkylammocarbonyl, dialkyl aminocarbonyl, aminoalkyl aminocarbonyl, and trialkylsilyl;
[0057] and wherein R3 and R4 are independently selected from the group consisting of hydroxyl, amino, thiol, ureido, Ci-C6 alkyl carbonylamino, hydroxy Ci-C6 alkyl, and hydrazinyl; and R5 is selected from the group consisting of hydrogen, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, heteroaryl, and Ci-C6 aminoalkyl; or a pharmaceutically acceptable salt.
[0058] In one embodiment, R1 is selected from the group consisting of C6-Ci4 aryl Ci-C6 alkyl and C6-Ci4 aryl C3-C8 cycloalkyl, wherein the aryl portion of R1 is optionally substituted with one or more substituents selected from the group consisting of halo, amino, Ci-C6 alkyl, Ci-C6 alkoxy, C6-Ci4 aryloxy, hydroxy Ci-C6 alkyl, hydroxy C2-C6 alkenyl, hydroxy C2-C6 alkynyl, aminocarbonyl Ci-C6 alkoxy, and C6-Ci4 aryl Ci-C6 alkoxy; and in one embodiment, R1 is selected from the group consisting of benzyl, phenyl cyclopropyl, or 1-naphthyl methyl, wherein the phenyl or naphthyl portion of R1 is optionally substituted with one or more substituents selected from the group consisting of halo, amino, hydroxyl, carboxy, alkoxycarbonyl, aminocarbonyl, alkylammocarbonyl, dialkyl aminocarbonyl, Ci-C6 alkyl, Ci-C6 alkoxy, phenoxy, hydroxy Ci-C6 alkyl, hydroxy C2-C6 alkenyl, and hydroxy C2- C6 alkynyl.
[0059] In another embodiment, R1 is benzyl, phenyl cyclopropyl, or 1- naphthyl methyl, wherein the phenyl or naphthyl portion of R1 is optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, and alkoxy. Examples of R1 are benzyl and benzyl substituted with one or more substituents selected from the group consisting of halo and Ci-C6 alkoxy. In yet another embodiment, R1 is selected from the group consisting of 3-chlorobenzyl, 3-bromobenzyl, 3-iodobenzyl, 2-hydroxy-5-methoxy- benzyl, and 2,5-dimethoxybenzyl. In one embodiment, the phenyl cyclopropyl is traro-2- phenyl- 1 -cyclopropyl.
[0060] In other embodiments, R2 is halo, specifically chloro, bromo, or iodo, or R2 is -C≡C- (CH2)m-CH3, -C≡C-(CH2)m-aryl, -C≡C-(CH2)m-C(=0)-0-CH3, -C≡C-(CH2)m-C(=0)-NH- (CH2)n-NH2, wherein m and n are independently 1 to 10, where in certain embodiments, m and n are 2 to 6, and in certain other embodiments, m and n are 3 to 5, and wherein the CH3 or aryl group is optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, amino, alkylamino, dialkylamino, sulfur, carboxy, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkyl aminocarbonyl, aminoalkyl aminocarbonyl, and trialkylsilyl; or a pharmaceutically acceptable salt thereof. In some embodiments, R3 and R4 are particularly hydroxyl. In other embodiments, R5 is hydrogen. In other embodiments, adenosine A3 receptor antagonists include those described in WO 2010/014921 A2 (PCT/US2009/052439), which is incorporated by reference in its entirety.
[0061] In one embodiment, the composition comprises:
Figure imgf000018_0001
[0062] wherein A is O or S, R is a linear or branched Ci-C5 alkyl which is non-substituted or is independently or selectively substituted with one or more C6-Cio aryl groups, a benzyl which is non-substituted or is independently or selectively substituted with halogen or one or more linear or branched C1-C4 alkoxy groups, or a hydroxycarbonyl-substituted benzyl; and
Y is H or a halogen atom. In another embodiment, A is O or S, R is methyl, ethyl, propyl, naphthylmethyl, benzyl, benzyl independently or selectively substituted with a substituent selected from a group consisting of F, CI, Br, I, C1-C3 alkoxy and combinations thereof, or toluic acid, and Y is H or CI. In another embodiment, A is O or S, R is methyl, ethyl, 1- naphthylmethyl, benzyl, 2-chlorobenzyl, 3-fluorobenzyl, 3-chlorobenzyl, 3-bromobenzyl, 3- iodobenzyl, 2-methoxy-5-chlorobenzyl, 2-methoxybenzyl, or 3-toluic acid, and Y is H or CI.
These compositions are described in U.S. Publication No. 2010/0137577.
[0063] In some embodiments, the adenosine derivatives comprise: (2R,3R,4S)-2-(2-chloro-6-
(3-fluorobenzylamino)-9H-purin-9-yl)tetrahydrothiophen-3,4-diol; (2R,3R,4S)-2-(2-chloro-
6-(3-chlorobenzylamino)-9H-purin-9-yl)tetrahydrothiophen-3,4-diol; (2R,3R,4S)-2-(6-(3- bromobenzylamino)-2-chloro-9H-purin-9-yl)tetrahydrothiophen-3,4-diol; (2R,3R,4S)-2-(2- chloro-6-(3-iodobenzylamino)-9H-purin-9-yl)tetrahydrothiophen-3,4-diol; (2R,3R,4S)-2-(2- chloro-6-(2-chlorobenzylamino)-9H-purin-9-yl)tetrahydrothiophen-3,4-diol; (2R,3R,4S)-2- (2-chloro-6-(5-chloro-2-methoxybenzylamino)-9H-purin-9-yl)tetrahydrothiophen-3,4-diol; (2R,3R,4S)-2-(2-chloro-6-(2-methoxybenzylamino)-9H-purin-9-yl)tetrahydrothiophen-3,4- diol; (2R,3R,4S)-2-(2-chloro-6-(naphthalen- 1 -ylmethylamino)-9H-purin-9-yl)tetra- hydrothiophen-3,4-diol; 3-((2-chloro-9-((2R,3R,4S)-3,4-dihydroxytetrahydrothiophen-2-yl)- 9H-purine-6-ylamino)methyl)benzoic acid; 2-(2-chloro-6-methylamino-purin-9- yl)tetrahydrothiophen-3,4-diol; (2R,3R,4S)-2-(6-(3-fluorobenzylamino)-9H-purin-9- yl)tetrahydrot- hiophen-3,4-diol; (2R,3R,4S)-2-(6-(3-chlorobenzylamino)-9H-purin-9- yl)tetrahydrothiophen-3,- 4-diol; (2R,3R,4S)-2-(6-(3-bromobenzylamino)-9H-purin-9- yl)tetrahydrothiophen-3,4- diol; (2R,3R,4S)-2-(6-(3-iodobenzylamino)-9H-purin-9- yl)tetrahydrothiophen-3,4- diol; (2R,3R,4R)-2-(6-(3-bromobenzylamino)-2-chloro-9H-purin- 9-yl)tetrahydrofur- an-3,4-diol; or (2R,3R,4R)-2-(2-chloro-6-(3-iodobenzylamino)-9H-purin- 9-yl)tetrahydrofura- n-3, 4-diol.
[0064] Other embodiments of the adenosine derivatives for use as adenosine A3 receptor antagonists and synthesis are described in U.S. Publication No. 2010/0137577, which is incorporated by reference in its entirety.
[0065] In some embodiments, the composition comprises:
Figure imgf000019_0001
[0066] wherein Ri and R3 are selected from the group consisting of hydrogen, hydroxy, Ci- C6 alkyloxy, and Ci-C6 alkylcarbonyloxy; R2 is selected from the group consisting of:
hydrogen, hydroxy, Ci-C6 alkyloxy, and C2-C6 alkenyloxy, said alkenyloxy together with the carbon atom of the phenyl ring forming an oxygen heterocycle; and R4 is selected from the group consisting of: phenyl, styryl, phenylbutadienyl, phenylacetylenyl, and -CH=N-phenyl, and substituted phenyl, styryl, phenylacetylenyl, and phenylbutadienyl, wherein the phenyl ring is substituted with 1 to 5 Ci-C6 alkyloxy groups; such that when R3 is hydrogen, Ri and R2 are neither hydroxy nor alkyloxy; when Rls R2, and R3 are hydrogen, R4 is neither phenyl nor alkyloxyphenyl; when R3 is hydrogen and R4 is phenyl, neither Ri nor R2 is alkylcarbonyloxy; and when R3 is hydroxy or alkyloxy, Ri and R2 are not dihydroxy. These compositions are described in U.S. Patent No. 6,066,642.
[0067] In other embodiments, the composition comprises:
Figure imgf000020_0001
[0068] wherein Ri is selected from the group consisting of hydroxyl and Ci-C6 alkoxy, and M is a divalent radical selected from the group consisting of— CH(OH)— CH(R2)— and—
C(OH)=C(R2)~, wherein R2 is selected from the group consisting of styryl and
phenylacetylenyl. These compositions are described in U.S. Patent No. 6,066,642.
[0069] In another embodiment, the composition comprises:
Figure imgf000020_0002
[0070] or a pharmaceutically acceptable salt, wherein R2 is a Ci -C6 alkyl; R<5 is selected from the group consisting Ci-C6 alkyl, Ci-C6 haloalkyl, and phenyl which may be further substituted with Ci-C6 alkyl, halo, nitro, furyl, or thienyl; R3 is selected from the group consisting of Ci-C6 alkyl, Ci-C6 alkyloxycarbonyl, aryl Ci-C6 alkyloxycarbonyl, Ci-C6 alkylthiocarbonyl, Ci-C6 alkylaminocarbonyl, and Ci-C6 alkyloxy Ci-C6 alkylcarbonyl, or R3 together with R2 forms a ring having 2-4 methylene groups, and Ci-C6 alkenyloxycarbonyl; R4 is selected from the group consisting of Ci-C6 alkyl, aryl Ci-C6 alkenyl, Ci-C6 alkylamino, Ci-C6 alkyl silyl Ci-C6 alkyloxy, aryl, heterocyclic, aryl Ci-C6 alkyl, phenylacetylenyl which may be further substituted with nitro, Ci-C6 alkyl, hydroxy, halo, amino, carboxy, Ci-C6 alkoxy, Ci-C6 haloalkyl, or Ci-C6 alkylamino, and styryl whose phenyl ring may be further substituted with one or more substituents selected from the group consisting of halo, nitro, amino, hydroxy, Ci-C6 alkyl, cyano, Ci-C6 alkyloxy, Ci-C6 alkyloxycarbonyl, Ci-C6 alkylcarbonyl, hydroxy Ci-C6 alkyl, Ci-C6 haloalkyl, carboxy, aminocarbonyl, Ci-C6 alkylamino, amino Ci-C6 alkyl, and Ci-C6 dialkylamino; and R5 is selected from the group consisting of Ci-C6 alkyloxycarbonyl, aryl Ci-C6 alkyloxycarbonyl, Ci-C6 alkyloxy Ci-C6 alkyloxycarbonyl, aryloxy Ci-C6 alkyloxycarbonyl, Ci-C6 alkyloxycarbonyl, aryl Ci-C6 alkyloxy Ci-C6 alkyloxycarbonyl, silyl Ci-C6 alkyloxycarbonyl, Ci-C6 alkylthio, hydroxy, and Ci-C6 alkylamino, wherein said aryl may be further substituted with Ci-C6 alkyl, Ci-C6 halo alkyl, trifluoromethyl, halo, nitro, Ci-C6 amino alkyl, Ci-C6 amino alkylamino, or Ci-C6 amino alkylamino carbonyl; wherein said aryl is phenyl or naphthyl. These compositions are described in U.S. Patent No. 6,066,642.
[0071] In other embodiments, the composition comprises:
Figure imgf000021_0001
[0072] or pharmaceutically acceptable salts, wherein R2 is selected from the group consisting of hydrogen and Ci-C6 alkyl; R3 is selected from the group consisting of hydrogen and Ci-C6 alkyloxycarbonyl; R4 is selected from the group consisting of Ci-C6 alkyl, phenyl C2-C6 alkenyl, phenyl C2-C6 alkynyl, aryl, and aryl substituted with one or more substituents selected from the group consisting of nitro and Ci-C6 alkyloxy; R5 is selected from the group consisting of hydrogen, Ci-C6 alkyloxycarbonyl, and aryl Ci-C6 alkyloxy carbonyl; 5 is selected from the group consisting of hydrogen, aryl, and Ci-C6 alkyl; with the proviso that when
Figure imgf000021_0002
R4 is not alkyl. These compositions are described in U.S. Patent No. 6,066,642.
[0073] In another embodiments, the composition comprises:
Figure imgf000022_0001
[0074] wherein Ri is selected from the group consisting of Ci-C6 alkylcarbonyl, Ci-C6 alkyloxycarbonyl, amino Ci-C6 alkylcarbonyl, and arylcarbonyl wherein the aryl may be further substituted with halo, nitro, hydroxy, amino or cyano; and R2 is hydrogen or halogen. This composition is described in U.S. Patent No. 6,066,642. Other embodiments of compositions including dihydronepyridine-, pyridine-, benzopyran-4-one- and
triazoloquinazoline derivatives are described in U.S. Patent No. 6,066,642, which is incorporated by reference in its entirety.
[0075] In another embodiment, the composition comprises:
Figure imgf000022_0002
[0076] wherein R2 is selected from the group consisting of Ci-C6 alkyl, C3-C7 cycloalkyl, and Ci-C6 alkoxy Ci-C6 alkyl; R3 is selected from the group consisting of Ci-C6 alkoxy, Ci-C6 alkylsulfanyl, hydroxy, Ci-C6 alkoxy Ci-C6 alkylsulfanyl, hydroxy Ci-C6 alkylsulfanyl, and halo Ci-C6 alkylsulfanyl, or R3 together with R4 forms a 3-7 membered heterocyclic ring containing O, N, or S; R4 is selected from the group consisting of Ci-C6 alkyl, halo Ci-C6 alkyl, hydroxy Ci-C6 alkyl, Ci-C6 alkoxy, Ci-C6 alkylsulfanyl, Ci-C6 alkylamino, Ci-C6 alkylcarbonyl sulfanyl Ci-C6 alkyl, aryl C2 -C6 alkenyl, aryl C2 -C6 alkynyl, formyl, and acetal; R5 is selected from the group consisting of Ci-C6 alkyl, aryl Ci-C6 alkyl, hydroxy Ci- C6 alkyl, and halo Ci-C6 alkyl; and 5 is selected from the group consisting of aryl, C3 -C7 cycloalkyl, and haloaryl; wherein the aryl is a phenyl or naphthyl; or a pharmaceutically acceptable salt. These compositions are described in U.S. Patent No. 6,376,521.
[0077] In one embodiment, the composition comprises:
Figure imgf000023_0001
[0078] wherein R2 is a Ci-C6 alkyl; R3 is selected from the group consisting of Ci -C6 alkoxy, Ci-C6 alkoxy Ci-C6 alkylsulfanyl, and Ci -C6 alkylsulfanyl; R4 is selected from the group consisting of Ci -C6 alkyl, acetal, formyl, aryl C2-C6 alkenyl, and aryl C2 -C6 alkynyl; R5 is selected from the group consisting of Ci-C6 alkyl and aryl Ci-C6 alkyl; and ¾ is selected from the group consisting of aryl and C3 -C6 cycloalkyl; wherein said aryl is a phenyl or naphthyl; or a pharmaceutically acceptable salt thereof. This composition is described in U.S. Patent No. 6,376,521. Other embodiments are described in U.S. Patent No. 6,376,521, which is incorporated by reference in its entirety.
[0079] In another embodiment, the composition comprises:
Figure imgf000023_0002
[0080] wherein X is sulfur or oxygen; Ri is hydrogen, C1-C5 alkyl, benzyl, halobenzyl, or phenyl C1-C5 alkyl; R2 is hydrogen, halogen, C1-C5 alkoxy group, C2-C6 alkenyl, C2-C6 alkynyl, C1-C5 alkylthio, or thio; R3 and R3' may be the same or different and are hydrogen, hydroxy C1-C5 alkyl, C1-C5 alkoxycarbonyl, or C1-C5 alkylaminocarbonyl; R4 is hydrogen or C1-C5 alkyl; or a pharmaceutically acceptable salt, or isomer. In an embodiment, R3 and R3' are not the same. In one embodiment, X is sulfur. In other embodiments, Ri is 3-iodobenzyl, R2 is chloride, R3 is methylaminocarbonyl, R3> and R4 are hydrogen, and X is sulfur. These compositions are described in U.S. Patent No. 7,199,127.
[0081] In another embodiment, the composition comprises:
Figure imgf000024_0001
[0082] wherein Rls R2 and R3 are as defined by the preceding formula. In one embodiment, the adenosine A3 receptor antagonist is (2R,3S,4R)-2-[2-chloro-6-(3-iodobenzylamino)purin- 9-yl]-3,4-dihydroxy-tet- rahydrothiophene-2-carboxylic acid methyl amide. These compositions are described in U.S. Patent No. 7,199,127.
[0083] In another embodiment, the composition comprises:
Figure imgf000024_0002
[0084] wherein Rls R2 and R3 are as defined above for the preceding formula, and specifically, Ri is hydrogen, methyl group, or 3-iodobenzyl group, R2 is chloride, R3 and R4 are hydrogen, R3' is methylaminocarbonyl group or hydroxymethyl group, and X is sulfur. [0085] Some examples of compositions include (2R,3R,4S,5R)-2-[2-chloro-6- (3iodobenzylamino)purin-9-yl]-5-hydroxymethyl- tetrahydrothiophene-3,4-diol,
(2R,3R,4S,5R) -2-(2-chloro-6-methylaminopurin-9-yl]-5hydroxymethyl- tetrahydrothiophene-3,4-diol, (2R,3R,4S,5R)-2-(2chloro-6-aminopurin-9-yl]-5- hydroxymethyltetra- hydrothiophene-3,4-diol, (2S,3S,4R,5R)-5-(6-amino-2-chloro-purin-9- yl]-3,4-dihydroxytetrahydrothio- phene-2-carboxylic acid methyl amide, (2S,3S,4R,5R)-5-(2- chloro-6-methylaminopurin-9-yl]-3 ,4-dihydroxytetrahydr- othiophene-2-carboxylic acid methyl amide, and (2S,3S,4R,5R)-5-[2-chloro-6-(3-iodobenzylamino)purin-9-yl]-3,4- dihydroxyt- etrahydrothiophene-2-carboxylic acid methyl amide. These compositions are described in U.S. Patent No. 7,199,127.
[0086] In some embodiments, the composition comprises:
Figure imgf000025_0001
[0087] wherein Rls R2 and R3 are as defined by the preceding formula. An example of the composition is (2S,3R,4S,5R)-2-[2-chloro-6-(3-iodobenzylamino)purin-9-yl]-5- hydroxymethy- ltetrahydrothiophene-3,4-diol. This composition is described in U.S. Patent No. 7,199,127. Other embodiments of compositions and their synthesis are described in U.S. Patent No. 7,199,127, which is incorporated by reference in its entirety.
[0088] In some embodiments, the composition of the invention comprises:
Figure imgf000025_0002
[0089] wherein, A is O or S, R is a linear or branched Ci-C5 alkyl which is non-substituted or is independently or selectively substituted with one or more C6-Cio aryl groups, a benzyl which is non-substituted or is independently or selectively substituted with halogen or one or more linear or branched C1-C4 alkoxy groups, or a hydroxycarbonyl-substituted benzyl; and Y is H or a halogen atom. In one embodiment, A is O or S, R is methyl, ethyl, propyl, naphthylmethyl, benzyl, benzyl independently or selectively substituted with a substituent selected from a group consisting of F, CI, Br, I, C1-C3 alkoxy and combinations thereof, or toluic acid, and Y is H or CI. In another embodiment, A is O or S, R is methyl, ethyl, 1- naphthylmethyl, benzyl, 2-chlorobenzyl, 3-fluorobenzyl, 3-chlorobenzyl, 3-bromobenzyl, 3- iodobenzyl, 2-methoxy-5-chlorobenzyl, 2-methoxybenzyl, or 3-toluic acid, and Y is H or CI.
[0090] Some examples of the adenosine derivatives include: (2R, 3R, 4S)-2-(2-chloro-6-(3- fluorobenzylamino)-9H-purin-9-yl) tetrahydrothiophen-3, 4-diol; (2R, 3R,4S)-2-(2-chloro-6- (3-chlorobenzylamino)-9H-purin-9-yl) tetrahydrothiophen-3, 4-diol; (2R, 3R,4S)-2-(6- (3- bromobenzylamino)-2-chloro-9H-purin-9-yl) tetrahydrothiophen-3, 4-diol; (2R, 3R, 4S)-2- (2-chloro-P-(3-iodobenzylamino)-9H-purin-9-yl) tetrahydrothiophen-3, 4-diol; (2R, 3R,4S)- 2- (2-chloro-6-(2-chlorobenzylamino)-9H-purin-9-yl) tetrahydrothiophen-3, 4-diol; (2R 3R 4S)-2-(2-chloro-6-(5-chloro-2-methoxybenzylamino)-9H-purin-9-yl) tetrahydrothiophen-3, 4-diol; (2R, 3R, 4S)-2-(2-chloro-6-(2-methoxybenzylamino)-9H-purin-9-yl)
tetrahydrothiophen-3, 4-diol; (2R, 3R, 4S)-2-(2-chloro-6-(naphthalen-l-ylmethylamino)-9H- purin-9-yl) tetrahydrothiophen-3, 4-diol; 3-((2-chloro-9((2R 3R 4S)-3, A- dihydroxytetrahydrothiophen-2-yl)-9H-purine-6-ylamino) methyl) benzoic acid; 2-(2-chloro- 6-methylamino-purin-9-yl) tetrahydrothiophen-3, 4-diol; (2R, 3R, 4S)-2-(6-(3- fluorobenzylamino)-9H-purin-9-yl) tetrahydrothiophen-3, 4-diol; (2R, 3R, 4S)-2-(6-(3- chlorobenzylamino)-9H-purin-9-yl) tetrahydrothiophen-3, 4-diol; (2R, 3R, 4S)-2-(6-(3- bromobenzylamino)-9H-purin-9-yl) tetrahydrothiophen-3, 4-diol; (2R, 3R, 4S)-2-(6- (3- iodobenzylamino)-9H-purin-9-yl) tetrahydrothiophen-3, 4-diol;(2R, 3R, 4R)-2-(6- (3- bromobenzylamino)-2-chloro-9H-purin-9-yl) tetrahydrofuran-3, 4-diol; or (2R, 3R, 4R) -2- (2-chloro-6-(3-iodobenzylamino)-9H-purin-9-yl) tetrahydrofuran-3, 4-diol. These compositions are described in PCT/KR2007/001131. Other embodiments of adenosine derivatives for use as adenosine A3 receptor antagonists and their synthesis are described in PCT/KR2007/001131.
[0091] In other embodiments, the compositions of the invention comprise 2-(6-Cyano-l- yl)adenosine, 2-(6-cyano-l-hexyn-l-yl)adenosine 5 '-monophosphate, or salts thereof, as described in U.S. Patent No. 7,132,409 and U.S. Patent No. 6,936,596, which are incorporated by reference in their entireties.
[0092] In one embodiment, the composition of the invention comprises 2-(6-cyano-l-hexyn-
1- yl)adenosine, which can be produced by the following steps. Under argon, into a solution of 2-iodoadenosine (420 mg, 1.07 mmol) and bis(triphenylphosphine)palladium dichloride (75 mg, 10 mol %) dissolved in N,N-dimethylformamide (10 mL), diisopropylamine (0.18 mL, 1.28 mmol, 1.2 eq.) and 6-cyano-l-hexyne (137 mg, 1.28 mmol, 1.2 eq.) are added. Under ice-cooling conditions, cuprous iodide (10 mg, 5 mol %) is added, followed by stirring at 50°C for 24 hours. The reaction mixture is cooled to room temperature, and the solvent is removed under reduced pressure. The residue is purified by dry packed silica gel column chromatography (ethyl acetate:methanol=10: l), to yield a compound as a pale yellow powder (yield: 217 mg, percent yield: 55%). Other conditions include: mp: 91-94°C; ESI-MS m/z: 373(M+H)+; 1H-NMR(DMSO-d6 +D2 O, 300 ΜΗζ)δ: 1.65-1.81 (m, 4H), 2.45-2.57 (m, 4H), 3.58 (dd, IH, J=3.1 Hz, 12.3 Hz), 3.70 (dd, IH, J=2.9 Hz, 12.3 Hz), 4.00 (ddd, IH, J=2.8 Hz, 2.9 Hz, 3.1 Hz), 4.16 (dd, IH, J=2.8 Hz, 4.9 Hz), 4.54 (dd, IH, J=4.9 Hz, 6.1 Hz), 5.88 (d, IH, J=6.1 Hz), 8.35 (s, IH); and IR omax(KBr): 3332, 2931, 2240, 1646, 1589, 1454, 1388, 1330, 1272, 1214, 1126, 1083 cm"1.
[0093] In other embodiments, the composition of the invention comprises triethylammonium
2- (6-cyano-l-hexyn-l-yl)adenosine 5 '-monophosphate, which can be produced by the following steps. Under argon, a 2-(6-cyano-l-hexyn-l-yl)adenosine (2.41 g) can be dissolved in triethyl phosphate (15 mL), and under ice-cooling conditions, phosphorous oxychloride (25.2 mL, 3.9 eq.) is added, followed by stirring at the same temperature for 5 hours. The reaction mixture is added drop-wise to a 10% sodium hydroxide solution (300 mL), and the resultant mixture is washed with ether. The aqueous layer is separated and water is removed under reduced pressure, and the residue was purified by C18 silica gel column chromatography (0.1M triethylammonium acetate buffer:acetonitrile=100:0 to 60:40). The obtained fraction is concentrated, and the concentrate is subjected to C18 silica gel column chromatography (water: acetonitrile= 100:0 to 60:40), to remove the
triethylammonium acetate buffer. The residue is freeze-dried to yield 1.14 g of the above- titled compound as a colorless amorphous substance. Other conditions include: 1H- NMR(D20)5: 1.10 (t, 9H, J=7.3 Hz), 1.57-1.74 (m, 4H), 2.34-2.42 (m, 4H), 3.02 (q, 6H, J=7.3 Hz), 3.91-4.03 (m, 2H), 4.20-4.22 (m, 1H), 4.32 (dd, 1H, J=4.4 Hz, 4.9 Hz), 4.52 (dd, 1H, J=4.9 Hz, 5.3 Hz), 5.89 (d, 1H, J=5.3 Hz), 8.28 (s, 1H); 31P-NMR(D20)5: -2.25(s);
IR(KBr)omax: 3337, 3179, 2938, 2678, 2243, 1654, 1637, 1594, 1457, 1380, 1068, 919 cm"1.
[0094] In another embodiment, the composition of the invention comprises 2-(6-cyano-l- hexyn-l-yl)adenosine 5 '-monophosphate , which can be synthesized by the following steps. A triethylammonium 2-(6-cyano-l-hexyn-l-yl)adenosine 5 '-monophosphate (1.1 g) is dissolved in water (20 mL), and, under ice-cooling conditions, IN HC1 solution (2 mL) was added thereto, followed by standing for 10 minutes. After having been diluted with water (20 mL), the resultant mixture is filtered. The residue is washed with water (20 mL) and then with ethanol (20 mLx2), to yield 617 mg of the compound as a colorless amorphous substance. Other conditions include: 1H-NMR(D20+NaOD)5: 1.59-1.75 (m, 4H), 2.36-2.42 (m, 4H), 3.44-3.51 (m, 2H), 4.09-4.10 (m, 1H), 4.49 (dd, 1H, J=4.4 Hz, 4.9 Hz), 4.65 (dd, 1H, J=4.9 Hz, 5.5 Hz), 5.73 (d, 1H, J=5.5 Hz), 8.41 (s, 1H); 31P-NMR (D20+NaOD)5:
1.44(s); IR(KBr)omax: 3330, 3125, 2934, 2241, 1690, 1592, 1398, 1227, 1081, 967 cm"1.
[0095] In yet another embodiment, the composition of the invention comprises monosodium 2-(6-cyano-l-hexyn-l-yl)adenosine 5 '-monophosphate or disodium 2-(6-cyano-l -hexyn- 1- yl)adenosine 5 ' -monophosphate. A 2-(6-Cyano- 1 -hexyn- 1 -yl)adenosine 5 ' -monophosphate (800 mg) is dispersed in water (8 mL), and IN sodium hydroxide solution (1.8 mL) is added thereto, followed by stirring for 0.5 hours. After having been diluted with water (10 mL), the resultant mixture is purified by C18 silica gel column chromatography
(water:acetonitrile=100:0 to 40:60), and the residue is freeze-dried, to thereby yield 130 mg of monosodium 2-(6-cyano-l -hexyn- l-yl)adenosine 5 '-monophosphate and 709 mg of disodium 2-(6-cyano-l -hexyn- l-yl)adenosine 5 '-monophosphate both as a colorless amorphous substance, a: monosodium 2-(6-cyano- 1 -hexyn- 1 -yl)adenosine 5 ' - monophosphate. Other conditions include: MS(ESI-)m/z: 451(M+)-H-Na; 1H-NMR(D20)5: 1.60-1.76 (m, 4H), 2.37-2.46 (m, 4H), 3.95-4.09 (m, 2H), 4.23-4.26 (m, 1H), 4.35 (dd, 1H, J=4.2 Hz, 4.9 Hz), 4.55 (dd, 1H, J=4.9 Hz, 5.3 Hz), 5.92 (d, 1H, J=5.3 Hz), 8.31 (s, 1H); 31P- NMR(D20)5: 0.56(s); IR(KBr)omax: 3338, 3190, 2942, 2244, 1647, 1594, 1383, 1070, 923 967 cm"1. UV(H20) max: 270, 232 nm; b: disodium 2-(6-cyano-l -hexyn- l-yl)adenosine 5'- monophosphate. MS(ESI-)m/z: 451(M+)-H-2Na; 1H-NMR(D20)5: 1.58-1.77 (m, 4H), 2.38-2.45 (m, 4H), 3.86-3.88 (m, 2H), 4.20-4.25 (m, 1H), 4.36 (dd, 1H, J=4.2 Hz, 4.9 Hz), 4.62 (dd, 1H, J=4.9 Hz, 5.5 Hz), 5.94 (d, 1H, J=5.5 Hz), 8.48 (s, 1H); 31P-NMR(D20)5: 1.33(s); IR(KBr)omax: 3368, 2244, 1654, 1593, 1368, 1089, 978 cm.sup.-l; UV(H20) 270, 232 nm.
[0096] In certain embodiments, the compositions of the invention include purine derivatives described in U.S. Patent No. 7,465,715, which is incorporated by reference in its entirety. In one embodiment, the composition of the invention comprises the purine derivative of the formula:
Figure imgf000029_0001
[0097] wherein Rl represents an alkyl, hydroxyalkyl, carboxyalkyl or cyanoalkyl or a group of the following general formula:
Figure imgf000029_0002
[0098] wherein Y represents oxygen, sulfur or CH2; Xi represents H, alkyl, RaRbNC(=0)~ or
HORc~, wherein Ra and Rb may be the same or different and are selected from the group consisting of hydrogen, alkyl, amino, haloalkyl, aminoalkyl, BOC-aminoalkyl, and cycloalkyl or are joined together to form a heterocyclic ring containing two to five carbon atoms; and Rc is selected from the group consisting of alkyl, amino, haloalkyl, aminoalkyl,
BOC-aminoalkyl, and cycloalkyl; X2 is H, hydroxyl, alkylamino, alkylamido or
hydroxyalkyl; X3 and X4 represent independently hydrogen, hydroxyl, amino, amido, azido, halo, alkyl, alkoxy, carboxy, nitrilo, nitro, trifluoro, aryl, alkaryl, thio, thioester, thioether,—
OCOPh,— OC(=S)OPh or both X3 and X4 are oxygens connected to >C=S to form a 5- membered ring, or X2 and X3 form the ring of formula (III):
Figure imgf000030_0001
[0099] where R and R" represent independently an alkyl group; R2 is selected from the group consisting of hydrogen, halo, alkylether, amino, hydrazido, alkylamino, alkoxy, thioalkoxy, pyridylthio, alkenyl; alkynyl, thio, and alkylthio; and R3 is a group of the formula --NR4R5 wherein R4 is a hydrogen atom or a group selected from alkyl, substituted alkyl or aryl-NH— C(Z)~, with Z being O, S, or NRa with Ra having the above meanings; wherein when R4 is hydrogen, then R5 is selected from the group consisting of R- and S-l-phenylethyl, benzyl, phenylethyl or anilide groups unsubstituted or substituted in one or more positions with a substituent selected from the group consisting of alkyl, amino, halo, haloalkyl, nitro, hydroxyl, acetoamido, alkoxy, and sulfonic acid or a salt thereof; benzodioxanemethyl, furfuryl, L-propylalanyl-aminobenzyl, β-alanylamino-benzyl, T-BOC-P-alanylaminobenzyl, phenylamino, carbamoyl, phenoxy or cycloalkyl; or R5 is a group of the following formula:
Figure imgf000030_0002
[00100] or when R4 is an alkyl or aryl-NN— C(Z)— , then, R5 is selected from the group consisting of heteroaryl-NRa-C(Z)-, heteroaryl-C(Z)-, alkaryl-NRa~C(Z)-, alkaryl-C(Z)--, aryl-NR--C(Z)-- and aryl-C(Z)— ; Z representing an oxygen, sulfur or imine; or a
physiologically acceptable salt of the above compound.
[00101] In one embodiment, the composition of the invention comprises the nucleoside derivative, described in U.S. Patent No. 7,465,715:
Figure imgf000031_0001
[00102] wherein Xls R2 and R5 are as defined above, and physiologically acceptable salts of said compound. In other embodiments, the composition of the invention comprises N6-2- (4-aminophenyl)ethyladenosine (APNEA), N6-(4-amino-3-iodobenzyl) adenosine-5'-(N- methyluronanide) (AB-MECA), N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB- MECA) and 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), which are described in U.S. Patent No. 7,465,715.
[00103] In some embodiments, the compositions of the invention include adenosine A3 receptor antagonists described in U.S. Patent No. 7,064,112, which is incorporated by reference in its entirety. In one embodiment, the composition of the invention comprises:
Figure imgf000031_0002
[00104] wherein X is S; R6 is RaRbNC(=0)~ or HORc~, wherein Ra and Rb may be the same or different and are selected from the group consisting of hydrogen, CiCio alkyl, amino, CiCio haloalkyl, CiCio aminoalkyl, and C3C10 cycloalkyl, or are joined together to form a heterocyclic ring containing two to five carbon atoms; and Rc is selected from C1C10 alkyl, amino, C1C10 haloalkyl, C1C10 aminoalkyl, C1C10 BOC-aminoalkyl and C3C10 cycloalkyl; R7 and Rg may be the same or different and are selected from the group consisting of CiCio alkyl, CiCio cycloalkyl, R- or S-l-phenylethyl, an unsubstituted benzyl or anilide group, and a phenylethyl or benzyl group substituted in one or more positions with a substituent selected from the group consisting of CiCio alkyl, amino, halo, CiCio haloalkyl, nitro, hydroxyl, acetamido, CiCio alkoxy, and sulfonic acid; R9 is selected from the group consisting of halo, benzyl, phenyl, C3C10 cylcycloalkyl, and C1C10 alkoxy; or a salt of such a compound, for example, a triethylammonium salt thereof. Synthesis and description of these compounds are found in U.S. Patent Nos. 5,688,774, 5,774,423, 6,048,865, WO 95/02604, WO 99/20284, and WO 99/06053, which are incorporated in reference in their entireties.
[00105] In some embodiments, the compositions of the invention include adenosine A3 receptor inhibitors described in U.S. Patent No. 6,673,802, which is incorporated by reference in its entirety. In one embodiment, the composition of the invention comprises:
Figure imgf000032_0001
[00106] or pharmaceutically acceptable salts thereof. In some embodiments, Ri and R2 are each independently a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety or together form a substituted or unsubstituted heterocyclic ring. R3 is a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety. R4 is a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety. R5 and R6 are each independently a halogen atom, e.g., chlorine, fluorine, or bromine, a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety or R4 and R5 or R5 and R6 together form a substituted or unsubstituted heterocyclic or carbocyclic ring. In certain embodiments, Ri and R2 can each independently be a substituted or unsubstituted cycloalkyl or heteroarylalkyl moieties. In other embodiments, R3 is a hydrogen atom or a substituted or unsubstituted heteroaryl moiety. In still other embodiments, R4, R5 and R6 can each be independently a heteroaryl moieties. In another embodiment, Ri is a hydrogen atom, R2 is a cyclohexanol, e.g., trans- cyclohexanol, R3 is phenyl, R4 is a hydrogen atom, R5 is a methyl group and 5 is a methyl group. In still another embodiment, Ri is a hydrogen atom, R2 is:
Figure imgf000033_0001
[00107] R3 is phenyl, R4 is a hydrogen atom, and R5 and R6 are methyl groups.
[00108] In other embodiments, the composition of the invention comprises:
Figure imgf000033_0002
[00109] which is described in U.S. Patent No. 6,673,802, wherein Ri is hydrogen; R2 is substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkyl, or Ri and R2 together form a substituted or unsubstituted heterocyclic ring; R3 is unsubstituted or substituted aryl; R4 is hydrogen; and R5 and R6 are each independently hydrogen or alkyl, and pharmaceutically acceptable salts thereof.
[00110] In another embodiment, the composition of the invention comprises:
Figure imgf000033_0003
[00111] which is described in U.S. Patent No. 6,673,802, wherein X is N or CRe; Ri and R2 are each independently hydrogen, or substituted or unsubstituted alkoxy, aminoalkyl, alkyl, aryl, or alkylaryl, or together form a substituted or unsubstituted heterocyclic ring, provided that both Ri and R2 are both not hydrogen; R3 is substituted or unsubstituted alkyl, arylalkyl, or aryl; R4 is hydrogen or substituted or unsubstituted Ci-C6 alkyl; L is hydrogen, substituted or unsubstituted alkyl, or R4 and L together form a substituted or unsubstituted heterocyclic or carbocyclic ring; R6 is hydrogen, substituted or unsubstituted alkyl, or halogen; Q is CH2, O, S, or NRg, wherein R8 is hydrogen or substituted or unsubstituted Ci- C6 alkyl; and W is unsubstituted or substituted alkyl, cycloalkyl, aryl, arylalkyl, biaryl, heteroaryl, substituted carbonyl, substituted thiocarbonyl, or substituted sulfonyl; provided that if R3 is pyrrolidino, then R4 is not methyl.
[00112] In one embodiment, the composition of the invention comprises:
Figure imgf000034_0001
[00113] which is described in U.S. Patent No. 6,673,802, wherein Ri is H and R2 is cyclopropyl methylamino carbonylethyl, cis-3 -hydroxy cyclopentyl, acetamido butyl, methylamino carbonylamino butyl, ethylamino carbonylamino propyl, methylamino carbonylamino propyl, 2-acetyl amino-3 -methyl butyl, Ν,Ν-diethylamino carbonylamino ethyl, thioacetamido ethyl, 3 -amino acetyloxy cyclopentyl, 3 -hydroxy cyclopentyl, 2-pyrrolyl carbonyl aminoethyl, 2-imidazolidinone ethyl, l-aminocarbonyl-2-methyl propyl, 1- aminocarbonyl-2-phenyl ethyl, 3 -hydroxy azetidino, 2-imidazolyl ethyl, acetamido ethyl, 1- (R)-phenyl-2-hydroxyethyl, N-methylamino carbonyl pyridyl-2-methyl, or Rl s R2 and the nitrogen together are 3-acetamido piperadino, 3-hydroxy pyrrolidino, 3-methyloxy carbonylmethyl pyrrolidino, 3-aminocarbonylmethyl pyrrolidino, or 3-hydroxymethyl piperadino. wherein R3 is a substituted or unsubstituted four to six membered ring, pyrrole, thiophene, furan, thiazole, imidazole, pyrazole, 1 ,2,4-triazole, pyridine, 2(lH)-pyridone, 4(lH)-pyridone, pyrazine, pyrimidine, pyridazine, isothiazole, isoxazole, oxazole, tetrazole, naphthalene, tetralin, naphthyridine, benzofuran, benzothiophene, indole, 2,3-dihydroindole, lH-indole, indoline, benzopyrazole, 1 ,3-benzodioxole, benzoxazole, purine, coumarin, chromone, quinoline, tetrahydroquinoline, isoquinoline, benzimidazole, quinazoline, pyrido[2,3-b]pyrazine, pyrido[3,4-b]pyrazine, pyrido[3,2-c]pyridazine, purido[3,4-b]- pyridine, lH-pyrazole[3,4-d]pyrimidine, pteridine, 2(lH)-quinolone, l(2H)-isoquinolone, 1 ,4-benzisoxazine, benzothiazole, quinoxaline, quinoline-N-oxide, isoquinoline-N-oxide, quinoxaline-N-oxide, quinazoline-N-oxide, benzoxazine, phthalazine, or cinnoline. wherein Its is H, alkyl, substituted alkyl, aryl, or substituted aryl. wherein R6 is H, alkyl, substituted alkyl, or cycloalkyl.
[00114] In yet another embodiment, the compositions of the invention include nucleoside 5 '-pyrophosphate pyranosides and analogues described in U.S. Patent No. 6,897,201 , which is incorporated by reference in its entirety. In one embodiment, the composition of the invention comprises:
[00115]
Figure imgf000035_0001
R4=H; E=0; El and E2 independently are H or F; YiMi and Y2M2 are independently OH, F, or H; Z=0, NH, CH2, CHF, CF2, CC12, or CHC1; Zx and Z2 are independently O or S; Gi=0 or S; G2 =CH;
G3=CH2, CHF, CF2, CH(OH) or CH(NHJ7); G4=CH2, CHF, CF2, CH(OH) or CH(NHJi3); G5 =CH2, CHF, CF2, CH(OH) or CH(NHJi7); G6=CH2, CH(CH2OH); the number of hydrogen atoms bonded to the Gi-G6 ring atoms is limited to a maximum of 8; also with the provision that the number of nitrogen atoms bonded to the Gi-G6 ring atoms are limited to a maximum of 2; J7, Ji3, and Ji7 are independently H, C(0)H, or C(0)alkyl.
[00116] In one embodiment, the compositions of the invention include triazolopurine derivatives described in U.S. Patent Nos. 6,288,070 and 6,686,343, which are incorporated by reference in their entireties. In one embodiment, the composition of the invention comprises:
Figure imgf000036_0001
[00117] wherein Ri represents an alkyl group, or a phenyl group which is optionally substituted with a lower alkyl group; R2 represents a pyridyl group, a furyl group, a thienyl group, a lower alkyl group, a phenyl lower alkyl group which optionally has 1 to 3 lower alkoxy groups as a substituent, a styryl group which optionally has 1 to 3 lower alkoxy groups as a substituent, a naphthyl group which optionally has a hydroxy groups as a substituent, or a phenyl group which optionally has 1 to 3 groups selected from lower alkyl group, lower alkoxy group, nitro group, hydroxyl group, amino group, N-lower alkylamino group, N,N-di lower alkylamino group, N-phenyl lower alkylamino group, N,N-bisphenyl lower alkylamino group, phenyl group, phenoxy group, phenyl lower alkoxy group, halogen- substituted lower alkyl group, halogen-substituted lower alkoxy group, di lower
alkylphosphorylmethyl group, lower alkylthio group, lower alkoxy lower alkyl group, phenyl lower alkoxy lower alkyl group and halogen atom as a substituent; and A represents a group:
\
HN- N- N- or
N N N
RJ
[00118] wherein R3 represents a lower alkyl group or a phenyl lower alkyl group.
[00119] In another embodiment, the compositions of the invention include compositions described by Okamura et al. (2004) Bioorg. & Med. Chem. Lett. 14:2443-2446. In one embodiment, the composition of the invention is selected from the group consisting of: Monocyclic
Figure imgf000037_0001
Figure imgf000037_0002
[00120] Pyrazolo[4,3-e]-l,2,4-triazolo[l,5-c]pyrimidine and l ,2,4-triazolo[l,5- c]quinazoline rings can be synthesized bearing various substituents at the 2 and 5 positions of the ring to generate the A3 receptor antagonists of the invention. The fused 1,2,4- triazolo[l,5-c]pyrimidine 9 can be synthesized by condensation of iminoester 8 and acylhydrazine based on the synthetic strategy, as shown in the Scheme below:
Se
Figure imgf000037_0003
teifSi- 1, Sj'isthesi: ¾ϊ¾ΐί¾;ί for oae-peri. of the f¾sed 1,2,4-
[00121] The iminoester 8 can be prepared by the reaction of primary amine analog 7 with a large excess of substituted orthoester. Iminoesterification of various aromatic or heterocyclic amines 7a-c were accomplished by condensation of trimethyl orthovalerate under the three different conditions, as shown in the table below.
Figure imgf000038_0001
f hht «52
8 8*
¾ ¾ CSA 85 8a
?b t itt 0 8i
?b ! ::¾; r 98 ¾ife
7h :¾ CSA 98 8b
Ϊ sf (f
Figure imgf000038_0002
/! /fi,S ( w ¥) f ¾ CSA %
[00122] Chemical yields of the iminoesters can be estimated from the ratio of amines 7 and iminoesters 8 in the reaction mixture with the integral values of 1H NMR spectral data.
5-Amino-4- cyanoimidazole 7a can be converted to iminoester 8a in good yields (62-86%) at room temperature, at 90°C, or in the presence of a catalytic amount of dl-camphorsulfonic acid (CSA) at room temperature. 3-Amino-4-cyanopyrazole 7b can be converted to 8b quantitatively by heating at 90°C or in the presence of a catalytic amount of CSA at room temperature, but there is no yield at room temperature without CSA. Iminoesterification of
2-aminobenzonitrile 7c does not afford 8c at room temperature or by heating at 90°C. The reaction can proceed in the presence of CSA at room temperature. The reaction conditions using CSA can be chosen for the one-pot synthesis of fused l,2,4-triazolo[l,5-c]pyrimidine derivatives 9a-c. Amines 7a-c can be transformed into the fused l,2,4-triazolo[l,5- c]pyrimidine derivatives 9a-c via intermediates 8a-c in situ by treatment with l . lmol equiv. of the corresponding orthoesters in the presence of 1% of CSA in DMF at room temperature.
1.2 mol equiv of the corresponding benzoylhydrazines can be added in each reaction vessels and heated at reflux temperature. In addition to unsubstituted phenyl compounds, 4-methoxy and 4-trifluoromethylphenyl analogs can be synthesized due to the high potency and selectivity for human adenosine A3 receptor. The isolation yields of l,2,4-triazolo[5,l- i]purines (9a), pyrazolo[4,3-e]-l,2,4-triazolo[l,5-c]pyrimidines (9b), and l,2,4-triazolo[l,5- c] quinazolines (9c) can be 62-82%, 60-76%), and 48-49%), respectively. [00123] In another embodiment, the compositions of the invention include compositions described by Okamura et al. (2004) Bioorg. & Med. Chem. Lett. 14:3775-3779. Okamura et al. describe l,2,4-triazolo[5,l-i]purine derivatives as human adenosine A3 ligands. A facile synthetic method of fused l,2,4-triazolo[l,5-c]pyrimidine was developed and applied to find new hA3 ligands, such as pyrazolo[4,3-e]-l,2,4-triazolo-[l,5-c]pyrimidine 2 and l,2,4-triazolo[l,5-c]quinazoline 3 scaffolds, which have strong and selective affinity for the hA3 receptor. In one embodiment, the composition of the invention comprises an adenosine A3 receptor antagonist selected from the group of:
Figure imgf000039_0001
1 2 3
[00124] wherein R1 and R2 are selected from the table below
la Ph
Figure imgf000039_0002
U 4-Bspheti?]
CH 5OC3¾
tt H ;iCCi¾
M<¾€Q-¾
HOsCC5 ¾ 4-Ci-Pf*
ii
Compd
2a Ph
2!> Ph
2c Ph
24 2-O-Pb
2 3-O-Pb
M 4d-P
% 4-F-l¾
Hi 4-Βί-ϊ¾
2
2§
2k
21
1m 4 FrFb
2» »-€A 4-B:s heayi
2fc 4 ·<Ή
2p 4-Cs-¾0"Ph .3
3a II
3fe H 2-Furyl
¾ e €«%
3i H 4-F-Ph
& H 4~B*~JPk
a
¾ H 4 F ¾
Ji H -Bipheay!
e 4-HO-Ph
¾ H <1¾0··ϊ»¾
3k a 4 Vf¾0--l%
31 S-CI :§¾
3m S-Cl 4-α-~ι%
3» B-CI 4-C! O f>h
¾ ¾-0 Fb
3p -0 C!-F¾
3q 9~€I
Jr 9~0 4-C¾0~**h
3s ιο-α l¾
M l¾
[00125] In some embodiments, the compositions of the invention include isoquinolines or quinazolines described by van Muijlwijk-Koezen, J.E. et al. (2000) J. Med. Chem. 43(11):2227-2238. In one embodiment, the composition of the invention comprises an adenosine A3 receptor antagonist selected from the group of:
Figure imgf000041_0001
ssoquirtotme fnazoitne spacer
Figure imgf000041_0002
[00126] These compositions of isoquinolines or quinazolines described by van Muijlwijk- Koezen, J.E. et al. can be synthesized by the following steps.
Sctxeine V
Figure imgf000041_0003
2
s Si ss s tai is Tabic I ,
[00127] The preparation of compounds 5a-k can be performed following the general synthetic strategy depicted the scheme shown above. The intermediates 4a-k are synthesized initially based on a method described by Linschoten et al. with some modifications (Eur. J. Med. Chem.-Chim. Ther. 1984, 19, 137-142). Treatment of 2-aminobenzonitrile (1) with strong base yielded a relatively stable anion as intermediate. This nucleophile reacts with nitriles 3a-k, and after hydrolysis the quinazoline derivatives 4a-k are obtained. The yield of the derivatives is dependent on the bulkiness of substituents Rl of nitriles 3a-k due to steric hindrance in the nucleophilic attack of the anion. In such a case, the anion can react with the starting nitrile 1 which, after hydrolysis, yields the dimeric side product 2-(2-aminophenyl)- 4-quinazolineamine (2). The course of the condensation reaction's dependence on the accessibility of the participating nitrile group to undergo nucleophilic attack is demonstrated by a mixture of 1 and trimethylacetonitrile (3c), which yields only dimer 2. The derivatives 5a-k are prepared by reaction of phenyl isocyanate (7i) with 4-aminoquinazolines 4. The substituted phenylurea derivatives 9a- h as well as compounds 8, 10, and 11 are prepared as depicted in the scheme shown below. Reaction of quinazolines 4 and isoquinolines 6 in dry acetonitrile at 30-50 °C with the appropriate isocyanate 7a-i affords the products 8-11 in good overall yields. The low solubility of the products in acetonitrile simplified the isolation and purification.
Figure imgf000042_0001
X
8A- . ft OH 11
s- ¾ and 1½ are ύ defined in Table L
[00128] A list of example compounds with the following structures of Isoquinoline and Quinazoline Analogues are found in the various tables below (Tables taken from van Muijlwijk-Koezen, J.E. et al. (2000) J. Med. Chem. 43(11):2227-2238.):
Figure imgf000042_0002
Compel X formula
4 a N H C.HJN,
4 c N C(C!I3), C i2H3 ,
4d N Ph
4e N 3-pyridyl C H
4f N 4-pyridyl
Figure imgf000043_0001
4j f Ct2Ht6N4
Figure imgf000043_0002
C mpd X , K2 formula
5a N H H CiS3Eil2 «0
5 N 2-pyridyl B
5 c N C(Oy3 H Ί«ι *4ν .0.3 CH,OH
Sd N Ph H
5e N 3-pyridyl H .0.2 CH3OH
Sf N 4- rid l H .0,2 CH.OH
2 , 0.1 CHjOIi
Figure imgf000044_0001
Conipd X JRj * formula
CH H H
8b CH H .3 CILOH
✓v
u CH Ph H C„HnN,0.0.2 CH-
9a N Ph 4 CI C2j¾ ClN40
9b Ph -Mc
9 c N Ph 3s4-diC C,,HI4C1, ,0.03 CH.OH
9d N Ph 4-OMe
CaHieN402. CUCHpH
9e N Ph 30Με
C,,H5gN0., 0.2 ILO
formula
9f N Pii 2- e Ca2JH ] 9N402 . 0.2 H.
n P 2~Ci C21H C1N.¾0 . 0.2 CH,G h N Ph 2-Me C2,ii^Ni40 , 0.2 CH H
10a N 3-pyridyl 2- Me C )H N 0,
10b N 3-pyridyl 2-Me C}, H .,K,0
I I N 2-pyridyl 2-OMe
[00129] In other embodiments, the compositions of the invention include derivatives of triazoloquinazoline and pyrazolotriazolopyrimidine described in Canadian Patent Application No. 2437437. In one embodiment, the composition of the invention comprises:
Figure imgf000046_0001
[00130] described in Canadian Patent Application No. 2437437, wherein Rl represents a lower alkyl group, a phenyl group, a lower alkoxy carbonyllower alkyl group, or a carboxy lower alkyl group; R2 represents a pyridyl group, a furyl group, a thienyl group, or a phenyl group which optionally has 1 to 3 groups selected from lower alkyl group, halogen atom, phenyl group, halogen-substituted lower alkyl group, hydroxyl group, lower alkoxy group, N,N-di lower alkylamino group, lower alkylthio group, lower alkanoyloxy group and nitro group as a substituent; A represents a pyrazole ring which is optionally substituted with a group selected from lower alkyl group, phenyl lower alkyl group, lower alkoxycarbonyllower alkyl group, carboxy lower alkyl group and hydroxyl lower alkyl group as a substituent, or a benzene ring which is optionally substituted with 1 to 2 groups selected from halogen atom, lower alkyl group, nitro group and lower alkoxy group as a substituent; with the exception that A is a benzene ring, R2 is a pyridyl group or a phenyl group, and Rl is a methyl group, an ethyl group or a phenyl group.
[00131] In another embodiment the composition of the invention comprises:
Figure imgf000047_0001
[00132] described in Canadian Patent Application No. 2437437, wherein R1 represents a lower alkyl group, a phenyl group, a lower alkoxycarbonyllower alkyl group, or a carboxy lower alkyl group; R2 represents a pyridyl group, a furyl group, a thienyl group, or a phenyl group which optionally has 1 to 3 groups selected from lower alkyl group, halogen atom, phenyl group, halogen-substituted lower alkyl group, hydroxyl group, lower alkoxy group, N,N-di lower alkylamino group, lower alkylthio group, lower alkanoyloxy group and nitro group as a substituent.
[00133] The compositions described in Canadian Patent Application No. 2437437 can be synthesized by the following steps:
R C(OZ)3
Figure imgf000048_0001
(2) (4)
Figure imgf000048_0002
( 1 a)
[00134] wherein R2 is as defined above; Rla represents a lower alkyl group or a phenyl group; Al represents a non-substituted pyrazole ring, or a benzene ring which is optionally substituted with 1 to 2 groups selected from halogen atom, lower alkyl group, nitro group and lower alkoxy group as a substituent; and Z represents a lower alkyl group. Next, the compound (la') is converted into the compound (lb) by the hydrolysis reaction, the reaction with an acid chloride (7) or the cyclization reaction, as shown in the following reaction scheme.
Figure imgf000049_0001
Figure imgf000049_0002
[00135] wherein R2 and Al are as defined above; Rla represents a lower alkyl group; R1 represents a lower alkyl group, a phenyl group or a lower alkoxy carbonyllower alkyl group, R2b represents a pyridyl group, a furyl group, a thienyl group, or a phenyl group which optionally have 1 to 3 groups selected from lower alkyl group, halogen atom, phenyl group, halogen-substituted lower alkyl group, hydroxy group, lower alkoxy group, N ,N-di lower alkylamino group, lower alkylthio group and nitro group as a substituent; and R2c represents a pyridyl group, a furyl group, a thienyl group, or a phenyl group which optionally have 1 to 3 groups selected from lower alkyl group, halogen atom, phenyl group, halogensubstituted lower alkyl group, lower alkoxy group, N,N-di lower alkyl amino group, lower alkylthio group, lower alkanoyloxy group and nitro group as a substituent. [00136] The compound (lc) of the present invention can be converted into compounds (ld-1) and (ld-2) wherein a substituent is introduced into the pyrazole ring by treating with a halide (9), as shown in the following reaction scheme.
Figure imgf000050_0001
[00137] wherein Rlb is as defined above; R2a represents a pyridyl group, a furyl group, a thienyl group, or a phenyl group which optionally has 1 to 3 groups selected from lower alkyl group, halogen atom, phenyl group, halogen-substituted lower alkyl group, lower alkoxy group, N,N-di lower alkylamino group, lower alkylthio group, lower alkanoyloxy group and nitro group as a substituent; R3 represents a lower alkyl group, a phenyl lower alkyl group, a lower alkoxycarbonyllower alkyl group, or a hydroxy lower alkyl group, R3a represents a lower alkyl group, a phenyl lower alkyl group, a lower alkoxy carbonyl lower alkyl group, or a trimethylsilyloxy-lower alkyl group; and X represents a halogen atom. Various other compositions for use as adenosine A3 receptor antagonists and synthesis of compounds thereof are described in Canadian Patent Application No. 2437437.
[00138] In other embodiments, the compositions of the invention include 2,6,9- trisubstituted purines. The synthesis of 2,6,9-trisubstituted purines is described by
McKeveney, D. (2004) The Solid-Phase Combinatorial Synthesis of 2,6,9 -Trisubstituted Purines as Potential Adenosine A 3 Receptor Antagonists. Ph.D. Thesis.
Pharmaceutical compositions of the invention
Formulations
[00139] The compositions of the invention can be formulated in pharmaceutical compositions. These compositions can comprise pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g., topical or intraocular injection.
[00140] Administration is preferably in a "therapeutically effective amount" or
"prophylactically effective amount" (as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the inflammatory eye disease being treated. Prescription of treatment, e.g. , decisions on dosage, etc. , is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington 's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack
Publishing Company, 1990).
[00141] The methods described herein use pharmaceutical compositions comprising the molecules described above, together with one or more pharmaceutically-acceptable excipients or vehicles, and optionally other therapeutic and/or prophylactic ingredients. Such excipients include liquids such as water, saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol, cyclodextrins, modified cyclodextrins {i.e., sufobutyl ether cyclodextrins), etc. Suitable excipients for non-liquid formulations are also known to those of skill in the art. Pharmaceutically acceptable salts can be used in the compositions of the present invention and include, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients and salts is available in Remington 's Pharmaceutical Sciences. [00142] Additionally, auxiliary substances, such as wetting or emulsifying agents, biological buffering substances, surfactants, and the like, may be present in such vehicles. A biological buffer can be virtually any solution which is pharmacologically acceptable and which provides the formulation with the desired pH, i.e., a pH in the physiologically- acceptable range. Examples of buffer solutions include saline, phosphate buffered saline (PBS), Tris buffered saline, Hank's buffered saline, and the like.
[00143] In general, compositions of this invention will be administered as pharmaceutical formulations including those suitable for oral (including buccal and sublingual), topical, intravitreal administration, intraocular injection, intravenous injection, intramuscular injection, rectal, intranasal administration, intraperitoneal administration, administration by inhalation or insufflation, or in other forms suitable for administration to an eye. The preferred modes of administration are topical or intraocular administration, preferably using a convenient daily dosage regimen which can be adjusted according to the degree of the condition or disease.
[00144] Depending on the intended mode of administration, the pharmaceutical compositions may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, creams, ointments, lotions or the like, preferably in unit dosage form suitable for single
administration of a precise dosage. The compositions will include an effective amount of the selected compound in combination with a pharmaceutically acceptable carrier and, in addition, may include other pharmaceutical agents, adjuvants, diluents, buffers, etc.
Formulations also include compositions of the invention administered as eye-drops, injectable liquids or solutions, or topical eye creams.
[00145] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and cornstarch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid,
micro crystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.
[00146] The invention includes a pharmaceutical composition comprising a composition of the present invention including isomers, racemic or non-racemic mixtures of isomers, or pharmaceutically acceptable salts or solvates thereof together with one or more
pharmaceutically acceptable carriers, and optionally other therapeutic and/or prophylactic ingredients.
[00147] In other embodiments, the compositions can be presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. In certain embodiments, the composition that is administered is in powder or particulate form. In other embodiments, the active compounds may be in powder form for constitution with a suitable vehicle, e.g. , a buffered saline, before use.
[00148] Effective amounts of the compositions of the invention are administered to a subject in need of such treatment. Effective amounts are those amounts, which will result in a desired improvement in the condition, disease or disorder or symptoms of the condition, disease or disorder. In general, a suitable dose of a formulation of an adenosine A3 receptor antagonist will be in the range of 0.001 to 1000.0 milligrams per kilogram body weight of the recipient per day, in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, or in the range of 1 to 50 mg per kilogram body weight per day. For example, the formulation of an adenosine A3 receptor antagonist can be administered at 0.001 mg/kg, 0.01 mg.kg, 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg per single dose. The
pharmaceutical composition may be administered once daily, or the adenosine A3 receptor antagonist may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the adenosine A3 receptor antagonist contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days. The absolute amount will depend upon a variety of factors (including whether the administration is in conjunction with other methods of treatment, the number of doses and individual patient parameters including age, physical condition, size and weight) and can be determined with routine experimentation. In some embodiments, a maximum dose can be used, that is, the highest safe dose according to sound medical judgment.
[00149] In another embodiment, the pharmaceutical composition is formulated for administration of a dosage regimen described herein, e.g., not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week. In another embodiment, the administration of the
pharmaceutical composition can be maintained for a month or longer, e.g., one, two, three, or six months, or one year or longer.
Methods of the invention
[00150] Methods for the use of adenosine A3 receptor antagonists and methods of treatment of various inflammatory eye diseases using adenosine A3 receptor antagonists are encompassed by the present invention. Said methods of the invention include administering an effective amount of a composition comprising an adenosine A3 receptor antagonist to a subject that has an inflammatory eye disease. Any of the adenosine A3 receptor antagonists described above may be used in the methods of use and methods of treatment of the invention.
[00151] Inflammatory eye diseases include, but are but not limited to, dry eye, uveitis, scleritis, blepharitis, keratitis, conjunctivitis, chorioretinital inflammation, choroiditis, retinitis, iridocyclitis, iritis, posterior cyclitis, diabetic retinopathy, post-surgery, allergy, trauma, bruises, foreign body, exposure to toxins or chemicals, food allergies, hives, autoimmune disorders (such as rheumatoid arthritis, sarcoidosis, juvenile idiopathic arthritis, lupus, Behcet's disease, etc.), infection (such as viral, fungal, mycobacteria, parasite, bacteria, acanthamoeba, spirochete, etc), inflammatory conditions (such as Crohn's disease, arthritis and ulcerative colitis) and cancers (such as lymphoma, eye tumors, cancer of blood vessels, etc.), or an inflammatory retinal disease (such as neuroretinitis, Sarcoidosis-related retinitis, Behcet's-related retinitis, or acute retinal pigment epitheliitis). In some
embodiments, animal models are used to induce inflammatory eye disease. See Barabino, S. et al. (2004) Investigative Opthalmo logy & Visual Science. 45(6): 1641-1646.
L Induction of Dry Eye Syndrome in Animal Models
[00152] Dry eye syndrome is a prevalent inflammatory disorder of the lacrimal functional unit of multifactorial origin leading to chronic ocular surface disease, impaired quality of vision, and a wide range of complications, eventually causing a reduction in quality of life. Some dry eye animal models have been developed in mice, rats, and rabbits, e.g., for lacrimal inflammation or human Sjogren's syndrome. Animal models for dry eye syndrome are discussed herein and in detail in Barabino, S. et al. (2004) Investig. Opthal. & Visual Sci.
A. Botulinum-Induced Model of Dry Eye Syndrome in Mice
[00153] A mouse model of botulinum (BTX)-induced dry eye was used to induce dry eye. Botulinum toxin was injected into the lacrimal gland of mice. Dry eye developed and persisted over about 3-4 weeks with resultant decreases in tear production and
histopathologic damage to the cornea. The study included groups with at least 10 CBA/J female mice per group. BTX was injected into the lacrimal gland of the mouse.
Dexamethasone was used as an appropriate positive control (cyclosporine is not as effective, but is still active). A Phenol Red Thread Test for tear production was performed, and a fluorescein slit lamp examination was used to assess corneal damage. A histopathology exam was performed on the eye and adenexal structures.
[00154] In one embodiment, the Phenol Red Thread Test was used. The Phenol Red Thread Test is a commercially available test (Zone-Quick, Showa Yakuhin Kako Co., Ltd). A cotton thread impregnated with phenol red dye was used. Phenol red is pH sensitive and changes from yellow to red when wetted by tears. The crimped end of a 70mm long thread was placed in the lower conjunctival fornix. Forceps were used to insert the folded portion of the thread into the palpebral conjunctiva of the eye 1/3 of the distance from the lateral canthus of the lower eye lid. After 15 seconds, the length of the color change on the thread (which indicated the length of the thread wetted by the tears) was measured in millimeters. Wetting lengths should normally be between 9mm and 20mm. Patients with dry eyes have wetting values of less than 9 mm. A reading of less than 10 mm was used to indicate dry eyes, less than 20 mm indicates marginally dry eyes, and more than 20 mm indicated normal tear volume.
[00155] Administration of botulinum toxin into the lacrimal gland in mice produced a consistent and measureable decrease in the Phenol Red Thread Test for tear production and increases in fluorescein stippling of the cornea over a period of weeks. Dexamethasone (DEX) and cyclosporine (CSA) improved tear production and offered some protection to the cornea.
[00156] Data tables showing control data for tear test and corneal staining are presented below. Dexamethasone, and to a lesser extent, CSA, offered some protection to the corneal histopathologically and reduced the mild inflammation that occurs in the lacrimal gland secondarily to injection with botulinum toxicity.
Table 1. Effects of Dexamethasone on Phenol Red Tear Test (3 week study)
Figure imgf000056_0001
Botulinum injected and treatment started on Day 0 and for all groups. ANOVA followed by post hoc.
Dunnett's: Statistically significant (**) p value less than 0.01; (*) p value less than 0.05.
Table 2. Effects of Dexamethasone on Corneal Fluorescein Test (3 week study)
Dex 0± 0 1.4± 1.1± 0.9 1.5± 0.9* 1.8±
1.2 0.9*
CSA 0± 0 1.7± 1.9± 0.9 2.1± 0.9 1.9±
1.0 0.8*
Normal 0± 0 0± 0 0± 0 0± 0 0± 0
Eye
Botulinum injected and treatment started on Day 0 for all groups. ANOVA followed by post hoc Dunnett's: Statistically significant (**) p value less than 0.01 ; (*) p value less than 0.05. 50 μΐ of 1% sodium fluorescein applied to corneal for slit lamp examination with cobalt light.
Table 3. Effects of Dexamethasone on Corneal Histopathology (3 week study)
Figure imgf000057_0001
Botulinum injected and treatment started on Day 0 for all groups. ANOVA followed by post hoc Dunnett's: Statistically significant (**) p value less than 0.01 ; (*) p value less than 0.05.
Grading Criteria Used:
Clinical Ocular Grading System for Corneal Damage
Figure imgf000057_0002
Histopathology Grading System for Corneal Damage Grade Criteria Criteria
Cornea Lacrimal Gland
0 Normal, cornea, no edema, normal Normal, no damage to glands, no
epithelial thickness, no cellular inflammation
infiltration.
1 Slight increase in epithelial and Trace numbers of inflammatory
stroma edema cells in gland
2 Mild edema of cornea and stroma, Minimal to mild numbers of
increased thickness of epithelium, inflammatory cells in gland low number of inflammatory cells
3 Moderate edema of cornea and Moderate numbers of
stroma, increased thickness of inflammatory cells in gland epithelium, moderate number of
inflammatory cells
4 Severe edema of cornea and Severe increase in numbers of
stroma, increased thickness of inflammatory cells in gland epithelium, inflammatory cell
infiltration
[00157] Botulinum-Induced Model of Dry Eye in mice produced a consistent uniform decrease in tear production with concomitant increases in corneal punctate stippling and histopathologic changes. DEX and to a lesser extent, CSA provided protection to the cornea in the Phenol Red Tear Test, fluorescein staining and histopathology.
B. Scopolamine-Induced Model of Dry Eye Syndrome in Mice
[00158] A mouse model of Scopolamine -induced dry eye syndromecan be used.
Scopolamine patches were applied to the tails of mice, and the mice were maintained in an environment with blowing air and low humidity. Dry eye developed over about 2 weeks with resultant decreases in tear production and histopathologic damage to the cornea. The dry eye continued as long as the patches were applied.
[00159] The study design included experimental groups with at least 10 mice per group. A scopolamine patch was applied, and a dry environment was initiated. A topical
dexamethasone (DEX) was administered as an appropriate positive control (or cyclosporine (CSA) (Restasis) offers some protection as well). Regular assessments of the Phenol Red Thread Test for tear production was performed. A fluorescein slit lamp assessment was performed to determine corneal changes. A histopathology was performed on eye and adenexal structures.
[00160] Scopolamine and a dry environment in mice produced a consistent and measureable dry eye condition in mice. There was a significant and consistent decrease in the Phenol Red Thread Test for tear production. FIG. la shows a graph of typical tear reduction after scopolamine patch application and a dry environment.
[00161] Clinical corneal damage as indicated by punctate corneal stippling was present and reduced by DEX, and to a lesser extent by CSA treatment. Histopathologic changes in the cornea were reduced by DEX, but not by CSA. Histopathologically, there can be thickening of the corneal epithelium and corneal stroma with some edema and inflammation. Dexamethasone significantly improved tear production and offered some histopathologic protection. Cyclosporine produced a similar trend, but is generally not statistically significant.
[00162] Data tables showing control data for tear test and histopathology are presented below. FIG. lb also shows histopathology scores 12 days after scopolamine administration. Four groups are shown for no-treatment, vehicle, DEX, and Restasis. For a, the p-value is less than 0.05 relative to the vehicle control group. For b, the p-value was less than 0.05 reltaive to the no-treatment group. N=10 female C57BL/6 mice per group.
Table 4. Effects of Dexamethasone on Phenol Red Thread Tear Test
Figure imgf000059_0001
Scopolamine and air draft started on Day 0 for all groups. ANOVA followed by post hoc Dunnett's: Statistically significant (**) p value less than 0.01; (*) p value less than 0.05.
Table 5. Effects of Dexamethasone on Corneal Fluorescein Test Group Day (severity score#)
-2 2 5 7 10
Vehicle 0± 0 2.1± 0.9 2.1± 1.1 2.2± 1.1 2.4± 0.9
Dex 0± 0 1.4± 1.2 1.7± 0.9 1.5± 0.9* 1.8± 0.9*
CSA 0± 0 1.9± 1.0 1.9± 0.9 2.1± 0.9 2.2± 0.8*
Normal Eye 0± 0 0± 0 0± 0 0± 0 0± 0
Patches applied and treatment started on Day 0 for all groups. ANOVA followed by post hoc Dunnett's: Statistically significant (**) p value less than 0.01; (*) p value less than 0.05. 50 μΐ of 1% sodium fluorescein applied to corneal for slit lamp examination with cobalt light.
Table 6. Effects of Dexamethasone on Histopathology
Figure imgf000060_0001
Scored 0-4 with 0=normal and 4=severe* statistically significant (P <0.05) compared to Group 1.
Severity scoring: 0= no lesions; l=trace, 2=mild, 3=moderate, 4=severe.
[00163] A corneal edema refers to the presence of edema in the corneal stroma as indicated by separation and spreading of the fibers. Epithelial edema refers to the presence of intracellular or extracellular edema in the corneal epithelium. Corneal thickness refers to the number of epithelial cell layers in the corneal epithelium, normally about 2-3 cell layers thick. In cases of injury to the corneal epithelium, the number of cell layers may be increased, and there may be other indications of epithelial proliferation, such as increased mitosis on the basal layer or cellular hypertrophy or hyperplasia.
[00164] Scopolamine and a dry environment in mice produced a consistent and
measureable decrease in the Phenol Red Thread Test for tear production with clinical evidence of corneal damage. Histopathologically, there was thickening of the corneal epithelium and corneal stroma with some edema and inflammation. Dexamethasone significantly improved tear production and offered some histopathologic protection.
Cyclosporine (CSA) produced a similar trend, but was generally not statistically significant. C. Controlled Adverse Environment (CAE) Model for
Induction of Dry Eye Syndrome
[00165] A controlled adverse environment (CAE) model combines multiple
administrations of the anti-cholinergic agent, scopolamine, and exposure to a low-humidity, high-airflow environment. The CAE model is based on a clinical model, which has been widely used in human clinical dry eye studies and has been pivotal in determining treatment efficacy in various dry eye development programs.
[00166] Rodents are known to be excellent models for understanding the role of specific molecules in disease processes. For example, C57BL/6 mice are accepted as valid models in this field of study. The number of animals used in this study was the minimum number needed to perform statistical analysis of the results. See Pflugfelder, S.C. et al. A Mouse Model of Keratoconjunctivitis Sicca. Investigative Ophthalmology and Visual Science. 2002; 43: 632-638; Pflugfelder, S.C. et al. Expresson of Th-1 Chemokines and Chemokin Receptors on the Ocular Surface of C57BL/6 Mice: Effects of Desiccating Stress. Investigative
Ophthalmology and Visual Science. 2007; 48: 2561-2569; Dana, R. et al. Amelioration of Murine Dry Eye Disease by Topical Antagonist to Chemokine Receptor 2. Archives of Ophthalmology. 2009; 127: 882-887.
[00167] 1. Housing and Husbandry
[00168] The mice were housed in polycarbonate cages with direct contact bedding. The cages conformed to standards set forth in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences). Space recommendations for animals were in accordance with PHS policy and the Animal Welfare Act. Litter or bedding in animal cages was changed as often as necessary to keep animals dry and clean and to minimize offensive odors.
[00169] Animals were fed food that is fresh, palatable and nutritionally adequate ad libitum. Clean, potable, and uncontaminated water was provided ad libitum. Transgel was added as a supplement to keep mice hydrated during the period when scopolamine was administered. Approximately 30 grams was added to each cage on days of injection.
[00170] 2. Environmental Conditions
[00171] Animals in the experimental group were housed in a custom-made mouse chamber. Environmental controls were set to maintain temperatures 20- 28°C with high airflow and relative humidity less than 25%. A 14-hour light and 10-hour dark cycle was maintained.
[00172] 3. Acclimation
[00173] The animals were acclimated for at least 7 days after arrival at the facility prior to Baseline Evaluations. An adequate number of extra animals were purchased so that no animal in obviously poor health was placed in test.
[00174] 4. Veterinary Care
[00175] A staff veterinarian was available as needed throughout the study.
[00176] 5. Assignment to Groups
[00177] Animals were randomly be assigned to test groups, based on the severity of staining after CAE exposure, prior to dosing, such that all groups were approximately equivalent.
[00178] 6. Experimental Design
[00179] The following chart provides an example of an experimental design for the CAE model:
Table 7.
Figure imgf000062_0001
n ma s were ran om ze a ter uoresce n sta n ng on ay . os ng egan at least 1 hour after staining. [00181] ** Scopolamine injections began after staining evaluations.
[00182] Evaluations on Baseline, Days 5, 10, and 15, and 19 were performed as follows:
On dosing/injection days, staining evaluations were performed 1 hour following
dosing/injection.
[00183] 7. Administration of Test/ Control Articles
[00184] Appropriate equipment was used for all procedures and documented thoroughly in the final report.
[00185] Baseline: On day 0, eyes were examined for any abnormalities such as ocular discharge or opacities, and eye lid deformation. Eyes were evaluated at baseline Day 0 prior to any other procedures. Mice with scratches or scrapes to the corneal surface were excluded from the study. Baseline staining evaluations were performed, and mice that had a score of 3 or greater in any region in either eye, or a total score of 6 or greater in either eye were used in the study.
[00186] Randomization on Day 5 : After four full days of CAE exposure, mice were evaluated on the morning of day 5. Mice were assigned to treatment groups such that the average total staining between the groups was approximately equivalent (±2).
[00187] Treatment and Dosing: Topical treatment was administered according to the Guidance document: Topical Administration of Ocular Treatments in Preclinical Studies. On days 5 through 19, topical treatment with test article, positive control, or the control article (vehicle control) was administered as outlined in the table below. Group 1 received ocular treatment four times daily at approximately 7am, 1 lam, 2pm, and 6pm. All other groups were dosed twice daily at approximately 7am and 6pm. All times listed were ±1 hour, and the exact time of dosing was recorded in the datasheets. Mice were dosed topically to the central cornea using a calibrated micropipette, with a 3 drop of treatment (test or vehicle control article) in each eye. For example, the mice were dosed topically with either ACN- 1052 or a vehicle control.
[00188] The test article tubes were masked throughout the experiment (labeled A-D) such that personnel dosing or performing the examinations did know the identity of the treatment groups.
[00189] Below is a table illustrating topical treatment and dosing. Table 8.
Figure imgf000064_0001
[00190] Scopolamine Hydrobromide Injection: Subcutaneous injection of scopolamine was administered according to the Technical Procedure: Injection of Scopolamine in a Murine Model of Dry Eye. Dursun D, Wang M, Monroy D, et al. "A mouse model of keratoconjunctivitis sicca." Invest Ophthalmol Vis Sci. 2002/43. -632-638.
[00191] Route: Subcutaneous injection was performed between the shoulder blades or on the back.
[00192] Frequency: Treatment groups 1-4 received scopolamine injections twice daily at approximately 7am (± 1 hour) and 6pm (± 1 hour) on days 10-19. On the last day of injections, mice only received the morning injection prior to evaluation.
[00193] Procedure: Scopolamine hydrobromide (Sigma/ Aldrich) was prepared aseptically in PBS at a concentration of 2.5mg/ml. Subcutaneous injection of 0.75mg/0.3ml will be administered on the appropriate days and times.
[00194] Corneal Fluorescein Staining: Corneal staining was assessed according to the Guidance: Fluorescein Staining and Grading Procedure. 0.6 μΐ^ οΐ \% sodium fluorescein was applied via a micro-pipette to the central cornea. Eight to ten minutes following application, corneal fluorescein staining was evaluated under magnification. The degree of fluorescein staining was assessed using a modified 0-4 grading scale (Ora Scale, modified from NEI), similar to that used in clinical dry eye studies, where five distinct areas of the cornea were graded individually. The sum of scores and the differences in the different regions of the cornea was assessed.
[00195] 8. Additional Experimental Procedures
[00196] Mortality and Morbidity Checks [00197] All animals were checked daily for general health and mortality (or moribundity). Skin turgor on all mice was checked twice daily during the scopolamine administration period, prior to the first and last injections to assess dehydration. Mice that were
significantly dehydrated received 0.5-1.0 mL of physiological saline (0.9%) subcutaneously.
[00198] Clinical Observations
[00199] All animals were observed daily by laboratory personnel, and observations were recorded. Special attention was given to ocular findings such as redness, swelling, discharge, etc. The study director was notified of any unusual findings.
[00200] Body weight was measured at baseline, at pre-scopolamine injection, and at sacrifice.
[00201] Ophthalmology
[00202] Eyes were monitored at baseline as well as during dosing for any abnormalities (e.g., conjunctival hyperemia, chemosis, discharge, lid edema and ulceration).
[00203] Photography
[00204] On evaluation days, immediately after corneal staining, representative
photographs were taken using a digital camera with a macro lens and Wratten filter.
[00205] 9. Terminal Procedures and Anatomic Pathology
[00206] Unscheduled Deaths: If an animal appears in poor condition or in extremis, it may be euthanized per the facility IACUC policy on humane care of animals. Depending on the timepoint of the experiment, animals were replaced if at all possible.
[00207] Scheduled Euthanasia: On the final day of the study, all animals were euthanized via C02 inhalation followed by surgical dislocation for verification of death. Euthanasia was performed in accordance with accepted American Veterinary Medical Association (AVMA) guidelines.
[00208] Tissue Collection and Preservation: The right eye of each animal was immersed in 4% paraformaldeyhyde for 24-30 hours, after which they eye was transferred to a vial containing 70% ethanol and stored for potential histology and/or immunohistochemistry.
[00209] 10. Statistical Analysis: The Student's two-sample t-test was used to compare the difference in fluorescein staining scores between both individual eyes and an average of both eyes of the same animal in the experimental and control groups, p-values < 0.05 was considered statistically significant. [00210] The CAE protocol induced dry eye-related ocular surface signs, caused an increase in inflammatory markers that have been implicated in the pathogenesis of dry eye disease, and reflected the clinical state of patients with dry eye syndrome.
D. Concanavalin A-Induced Dry Eye Syndrome in Rabbits
[00211] A model of concanavalin (ConA)-induced dry eye in rabbits can be used to induce dry eye syndrome. The lacrimal gland was injected with ConA. A self-limiting dry eye develops developed over about 2 weeks. Injection of ConA into the lacrimal glands of rabbits produced a consistent and measureable decrease in both the Tear Break up Test (TBUT) and the Schirmer Tear Test (STT).
[00212] The Schirmer Tear Test (STT) included use of Schirmer tear test strips (Clement Clarke) according to the manufacturer's instructions. No topical anaesthetic was used. The rounded wick end of the test strip was folded at the indentation and then inserted into the lower fornix of the eye 1/3 of the distance from the lateral canthus of the lower eye lid. After 5 minutes, the strip was removed, and the wet part was measured up to the folded line. A reading of less than 5 mm can be judged to indicate dry eyes and less than 10 mm can indicate marginally dry eyes.
[00213] In the Tear Break up Test (TBUT), a fluorescein strip as moistened with one drop of distilled water and applied to the inferior temporal bulbar conjunctiva. Blinking several times facilitated an even distribution of fluorescein. The eye is positioned for slit-lamp examination. The tear film was scanned through a cobalt blue, filtered light at a
magnification of xl6 and a 3 -mm wide vertical beam. A stop-watch was used to measure the interval between the last complete blink and the first appearance of a randomly distributed dry spot, the BUT. The BUT can be measured five times in succession for each eye during a single test period. See Interpretation of Tear Film Breakup, Gregory T. Vanley, BA; Irving H. Leopold, MD; Terrance H. Gregg, (Arch Ophthalmol (1977) 95:445-448).
[00214] Histopathologically, there was brisk inflammation within the lacrimal gland. Dexamethasone significantly reduced inflammation in the eye and improved both the TBUT and the STT.
[00215] FIGs. 2, 3a and 3b present typical reduction and recovery of tear production in control and treated animals after ConA injection. [00216] The study design included groups with at least 6 rabbits per group. Con A was injected into the lacrimal gland as a single dose on Day 0 and Day 3. Assessments included a Schirmer Tear Test (STT) and a Tear Break Up Test (TBUT) over a 2 week period.
Histopathology was performed on the eye and adenexal structures.
[00217] A Schirmer Tear Test (STT) and a Tear Break Up Test (TBUT) can be performed. Within 2-3 days of ConA injection, a decrease in the SST and TBUT was shown, consistent with a reduction in tear production as a result of inflammation of the lacrimal gland induced by the ConA injection. Dexamethasone (DEX) and cyclosporine (CSA, Restasis) both offer protection in this model, with statistically significant increases in the TBUT and trends demonstrated in the Schirmer Tear Test. Generally, significant changes are not seen in slit lamp examination of the cornea. The study did not result in mortality or decreases of body weight, and the procedure was well tolerated.
[00218] In FIG. 2, the effects of DEX and CSA are shown in the TBUT test. Also, the effects of a test article (e.g., an adenosine A3 receptor antagonist) on the TBUT test are shown, demonstrating a statistically significant effect of both DEX and CSA on the TBUT Test. There was a clear dose related trend with a three doses of a test article, as well as with the high dose, demonstrating a statistically significant improvement in the TBUT.
[00219] In FIG. 3a, the effects of ConA injection on the STT are presented. There was a decrease in the STT beginning about Day 2, continuing for about 3-4 days with recovery occurring between Days 8-10. In FIG. 3b, DEX and CSA were used, and a trend towards improvement with DEX was seen, but it did not achieve statistical significance.
[00220] The Model of ConA-Induced Dry Eye in the Rabbit was characterized by a consistent, uniform decrease in TBUT and to a lesser extent, Schirmer Tear Test, beginning about Day 3 and continuing until about Day 10-12, when recovery began for TBUT and Days 6-8, when recovery began for the Schirmer Tear Test. The procedure was well tolerated. Both DEX and CSA showed activity in inhibition of reduction of tear production in the STT and TBUT test.
II. Induction of Uveitis in Animal Models
[00221] Uveitis is a swelling and irritation of the uvea, the middle layer of the eye.
Uveitis can be caused by autoimmune disorders such as rheumatoid arthritis or ankylosing spondylitis, infection or exposure to toxins. The most common form of uveitis is anterior uveitis, which involves inflammation in the front part of the eye. Symptoms of uveitis include, but are not limited to blurred vision, dark, floating spots in the vision, eye pain, redness of the eye, or sensitivity to light.
[00222] Acute anterior uveitis is a recurrent immune-mediated inflammatory condition involving the iris and ciliary region in man and occurs in about 4 out of 1000 individuals. Although it may be self-limiting, it may also be chronic and lead to irreversible eye damage including cataract and glaucoma. Photophobia and pain with hyperemia of the vessels, hypophyon, and iridiocyclitis is typical, and the condition is relatively responsive to steroids. However, in many instances, the disorder is unresponsive to treatment and may lead to blindness with glaucoma, cataract and retinopathy. In some cases, HLA B27 appears to be involved. Humans are usually treated with steroids; however, there are undesirable side effects. In the field of veterinary medicine, horses suffer from an analogous anterior recurrent uveitis which often leads to blindness as well and was known in the past as "Moon Blindness."
A. Melanin- Associated Antigen Induced Anterior Uveitis in Rats
[00223] A MAA-induced uveitis model in rats can be used. A preparation of melanin- associated antigen (MAA) complexed with adjuvant was administered to Lewis Rats as an inoculation. An acute to sub-acute recurrent immune-mediated iridiocyclitis developed and resolved over a 1-2 -month period. Both the ophthalmo logical presentation and the histologic features were analogous to the human condition.
[00224] The MAA antigen is an extract of bovine ocular melanin derived from the iris. The MAA antigen induced uveitis by Day 15 in all treated rats, and the disease proceeded consistently between and among groups. Both topical steroids and systemic cyclosporine markedly and consistently suppressed the inflammation. Topical cyclosporine also suppressed inflammation, but not to the degree of steroid and systemic cyclosporine.
[00225] One study design included 10 Lewis rats per group, treated with a vehicle or test article (e.g., an adenosine A3 receptor antagonist) at 2-3 dose levels, and a positive control of topical dexamethasone (DEX) or systemic cyclosporine (CSA). Inflammation was induced with a MAA injection. Slit lamp scoring of inflammation was performed in the anterior segment of the eye thrice weekly for 4 weeks. The body weights of the animalswere measured weekly, and a necropsy with histopathologic examination was performed on the eye.
[00226] A slit lamp examination was performed at least three times a week during the course of the study and the findings scored as per the scoring criteria. Dexamethasone and to a lesser extent, CSA, reduced the clinical ocular scoring in the anterior chamber as per direct slit lamp examination (topical Restasis has no effect). Ocular scoring results are presented below.
Table 9. Ocular Clinical Scoring.
Figure imgf000069_0001
[00227] FIG. 4 shows representative photographs of the anterior area of the eye. Multiple hematoxylin-eosin stained sections of each eye were examined histopathologically and scored 0-4 for inflammation severity at Day 18 and Day 30. In MAA-treated eyes, there was a marked inflammation of the iris and ciliary areas. Both DEX and CSA significantly reduced the inflammation. FIG. 4a shows a normal anterior chamber. FIG. 4b shows the anterior chamber of a DEX and MAA treated rat, where there is hyperemia in the iris (Score 1). FIG. 4c shows a photo of a MAA-treated rat, and the eye has an opaque anterior chamber and an obscured pupil (red reflex absent, proptosis and hypophyon; Score 4). Topical Restasis had no significant effect on inflammation.
[00228] FIG. 5 shows histopathology scores 18 days after MAA injection in untreated rats and rats treated with vehicle, DEX, CSA, or Restasis. In the graph, "a" indicates that the p- value was < 0.05 relative to the vehicle control group, and "b" indicates that the p-value was < 0.05 relative to the no-treatment group.
[00229] FIGs. 6a-6c illustrate the photomicroscopy of histopathology findings in MAA treated rats. Fig. 6a. shows the normal ciliary process and sclera (HE lOOx; 0 score). Fig. 6b shows a picture of a MAA-treated rat where there is marked inflammation of the ciliary process, iris, cornea, and uvea (200x; 4 score). Fig. 6c shows a picture of a MAA-treated rat where there is there is marked inflammation of the ciliary process, iris, cornea, and uvea (400x; 4 score).
Table 10. Histopathology Findings
Figure imgf000070_0001
6 Normal eye 0 ± 0 Normal eye, no
lesions
* statistically significant (p-value <0.05) relative to vehicle control.
** all animals except Group 6 received MAA.
Group Treatment** Mean Score Histologic
Day 30 comment
1 Vehicle 2.3 ± 0.7 Moderate
inflammation of the
ciliary process,
anterior chamber,
cornea and vitreous
was present
2 DEX (0.1 %) 1.6* ± 0.4 Minimal to mild
inflammation of
primarily the ciliary
process was
present
3 CsA (15 mg/kg IP) 1 .4* ± 0.5 Minimal to mild
inflammation of
primarily the ciliary
process was
present
4 No treatment 2.6 ± 0.8 Moderate
inflammation of the
ciliary process was
present
5 Normal Eye 0 ± 0 Normal eye, no
lesions
'statistically significant (p-value <0.05) relative to vehicle control.
** all animals except Group 5 received MAA.
[00230] The MAA-induced model of anterior uveitis in Lewis Rats was characterized by a robust, uniform inflammation that peaks about Day 18 and underwent resolution by Day 30. The procedure was well tolerated, and there were marked inflammatory changes in the anterior segment that were visible via slit lamp examination clinically and were present histopathologically. Both DEX and CSA showed activity in inhibition of the inflammation, while topical Restasis is not particularly active.
Table 11. Clinical Ocular Grading for MAA-Induced Inflammation
Figure imgf000072_0001
*Each higher grade includes criteria of the preceding one
Table 12. Histopathology Grading for MAA-Induced Inflammation
Figure imgf000072_0002
extension of inflammation into cornea, anterior
chamber and posterior chamber.
3 Moderate multifocal to diffuse, often expansile,
infiltration of the iris and/or ciliary area with
neutrophils and/or mononuclear cells.
Hemorrhage, congestion and edema. No to
moderate extension of inflammation into cornea,
anterior chamber and posterior chamber.
4 Severe, expansile infiltration of the iris and/or
ciliary area with neutrophils and/or mononuclear
cells. Hemorrhage, congestion and edema.
Inflammation may extend into cornea, anterior
chamber and posterior chamber.
B. Induction of Experimental Autoimmune Anterior Uveitis
[00231] Experimental autoimmune anterior uveitis can be induced by a self-antigen.
Experimental autoimmune anterior uveitis (EAU) is an organ-specific autoimmune disease of the eye. EAU can be induced in the Lewis rate by a single injection of adjuvant and detergent-insoluble melanin-associated antigen (MA) isolated from bovine retinal pigment epithelium (RPE), iris, ciliary body, and choroid. EAU can also be induced by adjuvant and a soluble protein fraction released by proteolytic digestion of bovine MAA with V8 protease. EAU can be induced by MAA without the use of adjuvant. Bora, N.S. et al. 1997.
Investigative Opthalmology & Visual Science. 38(10): 2171-2175. Rodent models used to study experimental autoimmune uveitis in rodents are also described in Agarwal R.K. et al. (2004) Methods Mol Med. 102:395-419
[00232] A murine model can be used to test the effects of compositions of the invention on experimental autoimmune uveitis (EAU). For example, C57BL/6 mice can be immunized with human interphotoreceptor retinoid-binding protein peptide (h-IRBP) to induce ocular inflammation. The severity of EAU can be assessed clinically and histopatho logically, along with measures of T-cell activation and leukocyte adhesion in the retina. III. Other Inflammatory Eye Diseases
A. Composition 48/80-Induced Ocular Inflammation in Rabbits
[00233] Acute ocular inflammation can be induced using composition 48/80 (N-methyl-p- methoxyphenethylamine formaldehyde condensation product) in animal models.
Composition 48/80 can selectively degranulate mast cells to induce conjunctival eosinophilia in rabbits.
[00234] In this animal model, 48/80 was applied topically in the eye of rabbits. Acute inflammation developed and persisted over about 3 days with a peak at about 8 hours. The study design included groups with at least 6 NZW rabbits per group. Composition 48/80 (50 mg/ml) was applied topically to the eye. One eye was left untreated or treated with a vehicle. Topical dexamethasone was an appropriate positive control.
[00235] Slit lamp examination was performed with particular attention to the anterior segment and conjunctiva at multiple time points on the first day of dosing and then twice or three times daily for 3 days. Histopathology was performed on the eye and adnexal structures.
[00236] Administration of composition 48/80 topically on the eye in rabbits produced a brisk acute ocular inflammation that was responsive to dexamethasone treatment over a 1-3 day period. The inflammation was characterized by an acute conjunctivitis
histopathologically characterized by edema, and inflammatory infiltration with eosinophils and mast cells.
Table 13. Ocular Inflammation Scoring
Figure imgf000075_0001
B. Ovalbumin-Induced Allergic Conjunctivitis in Guinea Pigs or Rabbits
[00237] An allergic conjunctivitis can be induced by ovalbumin administration to guinea pigs and rabbits. Guinea pigs are the preferred species due to the consistency of response. Animals were sensitized by ovalbumin and adjuvant injected intraperitoneally at two week intervals. One week later, one eye was challenged by topical administration of ovalbumin. In some embodiments, acute inflammation developed and persisted over about three days with a peak at about 24 hours.
[00238] A study design included groups with at least 6 guinea pigs or rabbits per group. Ovalbumin and adjuvant were injected intraperitoneally at two week intervals. One eye was challenged by topical application as a single dose of albumin in PBS to the eye. Topical dexamethasone was an appropriate positive control. Slit lamp and funduscopic examination was performed. A visual scoring of ocular clinical signs and/or a histopathology examination on the eye and adenexal structures was performed.
[00239] Administration of ovalbumin produced a brisk acute ophthalmitis that was responsive to dexamethasone treatment. The inflammation was characterized clinically by redness, and inflammation of the eye. There was a neutrophilic and proteinic effusion into the aqueous humor and an acute ophthalmitis, primarily eosinophilic, histopathologically. The effects of Dexamethasone on cellularity of aqueous humor and ocular histopathology are shown in the tables below.
Table 14. Effects of Dexamethasone on Cellularity of Aqueous Humor
Figure imgf000076_0001
Table 15. Effects of Dexamethasone on Ocular Histopathology
Figure imgf000076_0002
Severity Score: 0=normal, l=trace; 2=mild; 3=moderate; 4=severe inflammation, consisting neutrophilic infiltration, congestion, edema and fibrin formation
EXAMPLES
[00240] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be allowed.
[00241] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al, Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);
Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg, Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols. A and B (1992).
Example 1: Methods of Treatment of Dry Eye in Animal Models Using
Adenosine A3 Antagonists
[00242] Dry eye can be induced in animal models using one or more of the methods described above. As described in Sections LA through I.D, dry eye can be induced by botulinum toxin injections, administration of scopolamine, use of the CAE protocol, or injection of concanavalin A (Con A) into lacrimal glands. Suitable animal models include, but are not limited to, rodents, such as Sprague Dawley rats, or rabbits.
[00243] Following dry eye induction in the animal, a composition or formulation comprising an adenosine A3 receptor antagonist described above is administered to the animal and is used to treat the dry eye condition. Any of the compositions of the invention described above can be used for treatment of dry eye. In various examples, the adenosine A3 receptor antagonist used is ACN-1052, LJ1251, MRS 3820, MRS 3771, LJ 979, MRS 3826, or MRS 3827. In other examples, the adenosine A3 receptor antagonist used is MRS 1191 or MRS 1292. In one example, the adenosine A3 receptor antagonist is ACN-1052.
[00244] As described above, the formulations of adenosine A3 receptor antagonist can be prepared for oral administration, injection, or non-invasive administration, such as topical administration. The formulations can be prepared as (a) liquid solutions; (b) capsules, sachets, tablets, lozenges, and troches; (c) powders; (d) suspensions in an appropriate liquid; or (e) suitable emulsions. Examples include formulations administered as eye-drops, injectable liquids or solutions, or topical eye creams.
[00245] The dosage of the formulation can vary depending on the form of administration. A suitable dose of a formulation of an adenosine A3 receptor antagonist will be in the range of 0.001 to 1000.0 milligrams per kilogram body weight of the animal per day, in the range of 0.01 to 200.0 milligrams per kilogram body weight of the animal per day, or in the range of 1 to 50 mg per kilogram body weight per day. Optimal dosing schedules can be calculated by administering various doses of the adenosine A3 receptor antagonist to the animal and measuring efficacy of the different doses. The optimized dose or formulation may be administered at various intervals of time: once a week, once every two days, once daily, BID (e.g., twice daily), TID, QID, or in multiple doses at appropriate intervals throughout the day, or even using continuous infusion or delivery through a controlled release formulation.
[00246] In one example, a dry eye study is performed using two experimental groups of mammals (e.g., a control group and a test group of 6 male NZW rabbits per group). The rabbits are assessed for inclusion in the study. Criteria include the entry criteria (scores of < 1 for conjunctival congestion and swelling; scores of 0 for all other observation variables) or exclusion criteria (no rabbits with gross signs of ocular irritation are used in this study).
[00247] Prior to induction of dry eye (e.g., injection of Con A on day -4), one or more of the following assays can be performed: gross ocular observations; scoring of signs of irritation or discomfort; examination of corneal thickness by pachymetry; slit lamp ophthalmic examinations with fluorescein dye to examine the cornea surface; a Shirmer Tear Test; or a Tear Break Up Test. ConA can be injected bilaterally into the lacrimal glands (on day -3 and day 0).
[00248] Each group is administered a vehicle or an adenosine A3 receptor antagonist (or a formulation of an adenosine A3 receptor antagonist). Treatment (on days 1 to 14) can include administering a vehicle or an adenosine A3 receptor antagonist to both eyes three times daily (e.g., 9 am, 12 pm, 4 pm). During treatment, the following examinations are made (on days 1 to 14): daily gross ocular observations, signs of irritation or discomfort scored; daily corneal thickness measured by pachymetry; daily slit lamp ophthalmic examinations with fluorescein dye to examine cornea surface, scored according to McDonald Shadduck method; daily Shirmer Tear Test; or a daily Tear Break Up Test. A terminal examination is performed based on a histopathology of the eye and adenexal structures.
[00249] The tear volume, tear function, and inflammatory cytokine levels in the lacrimal glands are assessed, along with a histological evaluation. The density of mucus producing goblet cells, corneal inflammation, and corneal epithelial damage also are measured and compared to normal control animals. Evaluation of the results shows that treatment with a composition or formulation comprising an adenosine A3 receptor antagonist produces a significant improvement or amelioration in the symptoms and condition of dry eye syndrome in the animal compared to the control group.
Example 2: Methods of Treatment of Dry Eye Syndrome in Mice Using A
Controlled Adverse Environment (CAE) Model
[00250] Dry eye syndrome was induced in mice using the CAE model and protocol described above in Section I.C. In one example, the adenosine A3 receptor antagonist ACN- 1052 was selected and used for treatment of dry eye syndrome.
[00251] FIG. 7 shows an example of inhibition of corneal injury after treatment of ACN- 1052 in mice using the CAE model described above. Mice were exposed to 10 days of an adverse environment according to the CAE protocol and treated with ACN-1052, Restasis, or a vehicle control on the fifth day. The ACN-1052 antagonist resulted in approximately 30% reduction in corneal injury based on a measurement of corneal staining of the eye. Restasis produced a 20% change in corneal staining, whereas the vehicle control did not inhibit corneal injury.
[00252] FIG. 8 illustrates the effect of ACN- 1052 in a mouse model using the CAE protocol to induce dry eye syndrome. Mice were exposed to 10 days of an adverse environment and treated with ACN-1052, Restasis or a vehicle control on the fifth day. The percentage of eyes with an improvement in corneal staining was 88% in the group treated with ACN-1052, compared to 81% in the Restasis group and 63% in the vehicle control. .
Example 3: Methods of Treatment of Dry Eye Syndrome in Patients Using
Adenosine A3 Receptor Antagonists
[00253] Compositions or formulations of adenosine A3 receptor antagonist can be used for treatment of patients with dry eye syndrome. The composition or formulation can include a therapeutically effective amount of an adenosine A3 receptor antagonist and a
pharmaceutically acceptable carrier or excipient.
[00254] A subject (e.g., a patient) with dry eye syndrome or dry eye symptoms is suitable for treatment. Prior to treatment, the symptoms of the patient are assessed. Symptoms can include, but are not limited to, a dry, gritty, scratchy, or filmy feeling in the eyes, a burning or itching in the eyes, redness of the eyes, intermittent excessive tearing, blurred vision, a sensation of having a foreign body in the eye, and light sensitivity. A control group of patients without dry eye syndrome can be included in the study.
[00255] A composition or formulation comprising an adenosine A3 receptor antagonist described above is administered to the patient to treat the dry eye condition. Any of the compositions of the invention described above can be used for treatment of dry eye. In various examples, the adenosine A3 receptor antagonist used is ACN-1052, LJ1251, MRS 3820, MRS 3771, LJ 979, MRS 3826, or MRS 3827. In other examples, the adenosine A3 receptor antagonist used is MRS 1191 or MRS 1292. In one example, the adenosine A3 receptor antagonist is ACN-1052.
[00256] As described above, the formulations can be suitable for oral administration, injection, or non-invasive administration, such as topical administration. The formulations can be prepared as (a) liquid solutions; (b) capsules, sachets, tablets, lozenges, and troches; (c) powders; (d) suspensions in an appropriate liquid; or (e) suitable emulsions. Examples include formulations administered as eye-drops, injectable liquids or solutions, or topical eye creams.
[00257] Dosing is dependent on severity and responsiveness of the dry eye condition to be treated, with the course of treatment lasting hours, days, months, or until a cure is effected or an improvement of the dry eye is achieved. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Dosage will vary with the age, gender, weight and response of the particular patient, as well as the severity of the patient's symptoms. Optimum dosages may vary depending on the relative potency of individual compositions, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
[00258] A suitable dose of a formulation of an adenosine A3 receptor antagonist will be in the range of 0.001 to 1000.0 milligrams per kilogram body weight of the patient per day, in the range of 0.01 to 200.0 milligrams per kilogram body weight of the patient per day, or in the range of 1 to 50 mg per kilogram body weight per day. The formulation may be administered at various intervals of time: once a week, once every two days, once daily, BID (e.g., twice daily), TID, QID, or in multiple doses at appropriate intervals throughout the day, or even using continuous infusion or delivery through a controlled release formulation. [00259] During treatment, the patient's symptoms are monitored and assessed.
Improvement of symptoms can be determined by ocular examination and/or self-reporting of symptoms by the patient. For example, an improvement in redness of the eyes or a reduction in burning or itching in the eyes can be assessed. Tear volume, tear function, and corneal inflammation can also be analyzed. Treatment with a composition or formulation of an adenosine A3 receptor antagonist alleviates or improves the symptoms of dry eye syndrome in the patient.
Example 4: Methods of Treatment of Uveitis Using an Adenosine A3 Receptor Antagonist
[00260] Methods of the invention include treatment of uveitis using an adenosine A3 receptor antagonist. Uveitis is induced by melanin-associated antigen, as described in methods above. The compositions of the invention described above can be used in a formulation for treatment of uveitis. In various examples, the adenosine A3 receptor antagonist used for treatment of uveitis is ACN-1052, LJ1251 , or MRS3820. In other examples, the adenosine A3 receptor antagonist used is MRS 1 191 or MRS 1292. In one example, the adenosine A3 receptor antagonist is ACN-1052.
[00261] A study is performed using two experimental groups (e.g., a control group and a test group of 6 male Lewis rats each). On Day 0, uveitis is induced in all animals by an injection into the tail head of an emulsion containing melanin-associated antigen (MAA), Complete Freund's Adjuvant (CFA), and pertussis toxin. In related examples, other methods described above for inducing uveitis are used. Starting in the afternoon of the same day, a vehicle and a composition comprising an adenosine A3 receptor antagonist is administered to both eyes in rats in Groups 1 and 2. Dosing occurs twice on day 0 and TID (three times per day) for the next 21 days. The animals are housed in a well ventilated dry environment.
[00262] Clinical observations are performed prior to the start of the study and daily starting from Day 0. Opthalmology evaluations are performed, and slit lamp scoring of inflammation is assessed in the anterior segment prior to start of study and then three times weekly. A Schirmer tear (or Phenol Red) test is performed three times weekly. Tear break up time is assessed three times weekly. The intraocular pressure (IOP) is tested three times weekly. Body weight of the rodent is assessed, prior to start of the study, once weekly thereafter, and at sacrifice. Necropsies are performed on approximately Day 21. Tissues are collected from both eyes at necropsy and histopathology performed. Sections of each eye are examined histopathologically and scored 0-4 each for inflammation severity, including the iris and ciliary areas, anterior chamber, cornea and vitreous.
[00263] Based on these assessments, a treatment with a composition comprising an adenosine A3 receptor antagonist is shown to produce a significant improvement or amelioration in the symptoms and condition of uveitis in the animal subject, compared with the control group.
Example 5: Diabetic retinopathy and diabetic macular edema in the
streptozotocin-induced diabetic rat model
[00264] Diabetic retinopathy refers to the damage to the eye's retina that occurs with long- term diabetes and that can cause blindness. Diabetic macular edema is a swelling of the retina due to leaking of fluid from blood vessels within the macula, which is the central portion of the retina. Macular edema causes blurring in the middle of side of the central visual field, and visual loss can progress over time, making it impossible to focus clearly.
[00265] A streptozotocin-induced diabetic rat model is used to study the effects of administration of compositions of the invention. Type I diabetes is induced in rats by administration of streptozotocin. Retinopathy and diabetic macular edema in streptozotocin- induced diabetic rat model are described in Zheng et al. (2007) Diabetes. 56:337-345; Carmo, A. et al. (2000) Mediators of 'Inflammation. 9:243-248; Kusari, J. et al. (2007) Invest. Opthal. & Visual Sci. 48(11): 5152-5159. The following assessments are performed: glycemia, angiography (HRA), electroretinography (ERG), histology and
immunohistochemistry, or Fundus (slit lamp, HRT).
[00266] The retinae are flat-mounted in a water-based fluorescence-anti-fading medium (Southern Biotechnology, Birmingham, Al) and imaged via fluorescence microscopy (Zeiss Axiovert FITC filter). Leukocyte location is scored as either arteriolar, venular, or capillary and the total number of adherent leukocytes per retina are counted. Blood-retinal barrier breakdown are counted by using Evans blue dye. On day 8, after induction of diabetes, confirmed diabetic animals are anesthetized and Evans blue is injected through the tail vein over 10 seconds at a dosage of 45 mg/kg. Immediately following Evans blue infusion, the rats turn visibly blue, confirming their uptake and distribution of the dye. The retinae are carefully dissected and thoroughly dried in a Speed- Vac (Savant, St. Paul, MN) for 5 h. Evans blue is extracted by incubating each retina in 120 μΐ formamide (Sigma) for 18 hours at 70°C. The supernatant is filtered through Ultrafree-MC tubes (30,000 NMWL, MiUipore, Bedford, MA) at 3,000 g, and the filtrate is used for triplicate spectrophotometric
measurements (Beckman Du-640, Fullerton, CA). Each measurement occurs over a 5 second interval, and all sets of measurements are preceded by known standards. The background- subtracted absorbance is determined by measuring each sample at both 620 nm, the absorbance maximum for Evans blue in formamide, and 740 nm, the absorbance minimum. The concentration of dye in the extracts is calculated from a standard curve of Evans blue in formamide and is normalized for retina dry weight. The blood-retinal barrier breakdown is calculated by using the following equation, with results expressed in microliters of plasma x g retina dry weight- 1 x hr-1 : [Evans blue ^g)/ Retina dry weight (g)] / [Time-averaged Evans blue concentration ^g /plasma μΐ)/ circulation time (hr)].
[00267] Based on these assays, a treatment using a composition comprising an adenosine A3 receptor antagonist is shown to produce a significant improvement or amelioration in the symptoms and condition of diabetic macular edema or diabetic retinopathy in the subject, compared with the control group.
[00268] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.
[00269] All references, issued patents and patent applications cited within the body of the instant specification, are hereby incorporated by reference in their entirety for all purposes.
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Claims

CLAIMS What is claimed is:
1. A method for treating an inflammatory eye disease, comprising administering to a mammalian subject in need of such treatment an effective amount of an adenosine A3 receptor antagonist.
2. The method of claim 1 , wherein said inflammatory eye disease is selected from the group consisting of: dry eye, uveitis, scleritis, blepharitis, keratitis, conjunctivitis, chorioretinital inflammation, choroiditis, retinitis, iridocyclitis, iritis, posterior cyclitis, diabetic retinopathy, a post-surgery condition, allergies, eye trauma, eye bruises, foreign body, toxin exposure, chemical exposure, food allergies, hives, rheumatoid arthritis, sarcoidosis, juvenile idiopathic arthritis, lupus, Behcet's disease, viral infection, fungal infection, mycobacterial infection, parasite infection, bacterial infection, acanthamoeba infection, spirochete infection, Crohn's disease, arthritis, ulcerative colitis, lymphoma, eye tumors, cancer of blood vessels, neuroretinitis, Sarcoidosis-related retinitis, Behcet' s-related retinitis, or acute retinal pigment epitheliitis.
3. The method of claim 1, wherein said adenosine A3 receptor antagonist comprises any of the compositions described above.
4. The method of claim 1 , wherein said adenosine A3 receptor antagonist is selected from the group consisting of: ACN-1052, LJ1251, MRS 3820, MRS 3771, LJ 979, MRS 3826, MRS 3827, MRS 1191 or MRS 1292.
5. The method of claim 2, wherein said inflammatory eye disease is uveitis, and wherein said adenosine A3 receptor antagonist is selected from the group consisting of: ACN-1052, LJ1251, or MRS3820.
6. The method of claim 2, wherein said inflammatory eye disease is dry eye and wherein said adenosine A3 receptor antagonist is selected from the group consisting of: ACN-1052, LJ1251, MRS3820, MRS 3771, LJ 979, MRS 3826, or MRS 3827.
7. The method of claim 1 , wherein said administering reduces corneal inflammation.
8. The method of claim 1 , wherein said administering reduces inflammatory cytokine levels.
9. The method of claim 1, wherein said mammalian subject is a human subject.
10. The method of claim 1, wherein said adenosine A3 receptor antagonist is formulated for topical delivery to an eye of said mammalian subject.
11. The method of claim 1, wherein said adenosine A3 receptor antagonist is formulated for topical delivery, intraocular injection, intravitreal injection, oral delivery, sublingual delivery, intranasal delivery, intravenous injection, intramuscular injection, intraperitoneal injection, or rectal delivery .
12. The method of claim 1, further comprising co-administering to said mammalian subject an effective amount of a steroid, a non-steroidal anti-inflammatory, or an
immunosuppressant.
13. The method of claim 12, wherein the immunosuppressant is cyclosporine A.
14. The method of claim 1, wherein said adenosine A3 receptor antagonist is
administered topically to an eye and penetrates the intact cornea of the eye.
15. The method of claim 1, wherein said adenosine A3 receptor antagonist is ACN-1052.
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