WO2011133696A2 - Methods of suppressing atherosclerosis - Google Patents

Abstract

The present invention is directed to methods for treating atherosclerosis in a mammal.

Description

METHODS OF SUPPRESSING ATHEROSCLEROSIS

Related Application

This application claims priority to U.S. Provisional Patent Application No. 61/326,028, filed April 20, 2010, the entirety of which is incorporated herein by reference.

U.S. GOVERNMENT RIGHTS

This work was supported by National Institutes of Health Grants HL78679 and

HL080569-01. The United States Government has certain rights to this invention.

BACKGROUND OF THE INVENTION

Atherosclerosis is a chronic inflammatory disease of the arterial vessel wall that involves endothelial cells, vascular smooth muscle cells, mononuclear cells, platelets, growth factors, and inflammatory cytokines. Conditions that increase inflammation also exacerbate atherosclerosis in vivo, and most drugs that improve the clinical outcome of atherosclerosis also inhibit inflammation. Therefore, inflarnmation is considered a therapeutic target in

atherosclerosis.

Adenosine is an endogenous regulator of inflammation and tissue injury, and most of its anti-inflammatory effects are elicited via the A₂A receptor (A₂AR)- A₂AR exists on many inflammatory cells, including neutrophils, monocytes, lymphocytes, macrophages, and platelets, and loss of A₂A increases inflammatory responses and tissue damage in vivo. In contrast, occupancy of A₂AR reduces inflammation and protects tissues from injury.

SUMMARY OF THE INVENTION

The present invention provides a method of inhibiting formation of atherosclerotic lesions by administering an effective dose of a compound to inactivate A₂AR on cells in a patient in need thereof. As used herein, the term "inactivate" means that the A₂AR activity is decreased by at least 10% as compared to an A₂AR to which no agent is bound, such as, for example by means of an antagonist or blocking agent. In certain embodiments, the A₂AR activity is decreased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% 99% or even 100%. In certain embodiments, the cells are bone-marrow-derived cells (BMDC).

The present invention provides a method of inducing apoptosis of foam cells by administering an effective dose of a compound to inactivate A₂AR on cells in a patient in need thereof. In certain embodiments of the present invention, the compound is caffeine, ZM241385 (a high affinity antagonist ligand selective for the adenosine A₂A receptor), istradefylline (KW- 6002), or a compound of Formula (I). In certain embodiments of the present invention, the compound is caffeine or ZM241385.

The present invention provides a compound to inactivate A₂AR for use in the treatment of atherosclerosis, wherein the compound to inactivate A_2AR is to be administered to a patient that has atherosclerotic lesions or is at risk for developing atherosclerotic lesions. In certain embodiments of the present invention, the compound is caffeine, ZM241385, or istradefylline (KW-6002)). In certain embodiments of the present invention, the compound is caffeine or ZM241385.

A substance to inactivate A₂A receptors (A₂AR) on cells for the treatment or to inhibit the formation of atherosclerotic lesions. In certain embodiments, the cells are bone-marrow-derived cells (BMDC).

The present invention provides a substance to inactivate A₂AR on cells to induce apoptosis of foam cells. In certain embodiments, the substance is caffeine, ZM241385, or istradefylline (KW-6002). In certain embodiments, the substance further comprises one or more additional anti-atherosclerotic compounds. In certain embodiments, the one or more additional anti-atherosclerotic compounds are caffeine, ZM241385, istradefylline (KW-6002), or a compound of Formula (I) or a pharmaceutically acceptable salt thereof or prodrug thereof.

In certain embodiments, the substance is a compound of formula (I):

wherein: X is O or S;

Ri and R₂ are independently selected from hydrogen, alkyl, aryl, hydroxy, alkoxy, aryloxy, cyano, nitro, C0₂R₇, COR₇, OCOR₇, CONR₇Rs, CONR₇NRgR₉, OCONR^, NR₇R₉, NR₇CORs, NR₇CONRsR₉, NR₇C0₂Rs, NR₇S0₂Rs, NR₇CONR8NR₉R₁₀, NR₇NRsC0₂R₉, NR₇NR8CONR₉R₁₀, NR₇S0₂NRs R₉, S0₂R₇, SOR₇, SR₇ and S0₂NR₇Rs, or R_t and R₂ together form a carbonyl group (C=0), an oxime group (C=NORn), an imine group (C=NRn) or a hydrazone group (C= NRnR₁₂), or Ri and R₂ together form a 5, 6 or 7 membered carbocyclic or heterocyclic ring;

R₃ is alkyl or aryl;

R4, R₅ and R^ are independently selected from hydrogen, alkyl, aryl, halogen, hydroxy, nitro, cyano, alkoxy, aryloxy, COR₇, OCOR₇, C0₂R₇, SR₇, SOR₇, S0₂R₇, S0₂NR₇R8,

CONR₇Rg, CONR₇NRgR₉, OCONR^, NR₇Rg, NR₇CORg, NR₇CON 8R₉, NR₇C0₂R₈,

NR₇S0₂R8, CR₇=NOR8, NR₇CONR8NR₉R₁₀, NR₇NR₈C0₂R₉, NR₇NR8CONR₉R₁₀,

S0₂NR₇NR8R₉, NR₇S0₂NR8R₉, NR-zNRsSC^Rg, NR₇NR ₈COR₉, NR₇NRs R₉ and NR₇CSNRsR₉, or R₅ and Re together form a 5, 6 or 7 membered carbocyclic or heterocyclic ring; and

R₇, R%, R₉, R₁₀, Rn and R₁₂ are independently selected from hydrogen, alkyl and aryl, or a pharmaceutically acceptable salt thereof or prodrug thereof.

In certain embodiments, the substance further comprises one or more additional anti- atherosclerotic compounds. In certain embodiments, the one or more additional anti- atherosclerotic compounds are caffeine, ZM241385, or istradefylline (KW-6002).

The present invention further provides a composition comprising a compound to inactivate A₂AR for use in the treatment of atherosclerosis, wherein the compound to inactivate A₂AR is to be administered to a patient that has atherosclerotic lesions or is at risk for developing atherosclerotic lesions. In certain embodiments, the compound to inactivate A₂AR is caffeine, ZM241385, or istradefylline (KW-6002). In certain embodiments, the compound to inactivate A₂AR is the compound of Formula (I) or a pharmaceutically acceptable salt thereof or prodrug thereof.

The present invention provides a method of inhibiting formation of atherosclerotic lesions by administering an effective dose of a compound to inactivate A₂AR in cells in a patient in need thereof. In certain embodiments, the cells are bone-marrow-derived cells (BMDC).

The present invention provides a method of inducing apoptosis of foam cells by administering an effective dose of a compound to inactivate A₂AR on cells in a patient in need thereof. In certain embodiments, the compound is caffeine, ZM241385, or istradefylline (KW- 6002). In certain embodiments, the compound is the compound Formula (I) or a

pharmaceutically acceptable salt thereof or prodrug thereof. In certain embodiments, the method further comprises administering one or more additional anti-atherosclerotic compounds. In certain embodiments, the one or more additional anti-atherosclerotic compounds are caffeine, ZM241385, or istradefylline (KW-6002). BRIEF DESCRIPTION OF THE FIGURES

Figures la-lg. Atherosclerosis in Apoe /A_2AR mice and in chimeric mice lacking A₂AR on BMDCs Figs, la and lb, Representative micrographs and quantitative data for oil red O en face staining of aortas of mice fed a Western diet for three (Fig. l ) or six (Fig. lb) months. Each data point represents a value obtained from a single mouse. The flattened diamond indicates mean lesion size. One-way ANOVA followed by a Bonferroni test was used for statistical analysis. Fig. lc, Representative micrographs and quantitative data for cross-sections of aortic sinuses stained with oil red O. Aortic sinuses were obtained from mice fed a Western diet for three months. Samples from ten mice were analyzed per group. Fig. Id, Anti-F4/80 staining of infiltrated macrophages in cross-sections of aortic sinuses. The macrophage area was quantified by averaging the percent area of F4/80 positive staining relative to the total lesion area in ten cross-sections from ten mice per group. Aortic sinuses were obtained from mice fed a Western diet for three months. Fig. le, Quantitative data for the mRNA level of CD68 in lesions of aortic sinuses. Lesions were obtained from mice fed a Western diet for three months. The student's t-test was used to analyze data in Fig. lc, Fig. Id, and Fig. 1 e. Fig. If, Representative micrographs and quantitative data for oil red O staining of cross-sections of aortic sinuses from chimeric mice. Samples from eight chimeric mice were analyzed per group. Fig. Ig, Anti-F4/80 staining of infiltrated macrophages in cross-sections of aortic sinuses from chimeric mice. The macrophage area was quantified by averaging the percent area demonstrating F4/80 positive staining relative to the total lesion area in eight cross-sections from eight chimeric mice per group. Aortic sinuses were from chimeric mice fed a Western diet for three months. The student's t-test was used for statistical analysis.

Figures 2a-2c. Elevated inflammatory status of atherosclerotic lesions in Apoe^_/7A₂AR^{~ _} mice. Fig. 2a, Electrophoretic mobility shift assay to assess NF-κΒ activation. Ox-LDL was injected into the peritoneal cavities of wt and A₂AR-deficient mice on day 3 after thioglycollate- induced peritonitis. The working concentration of ox-LDL was 100 μg/mL. Macrophages were collected 30 minutes after ox-LDL injection. Arrows indicate nuclear p65/p50 binding to the NF-KB consensus sequence. In the cold probe lane, a 200-fold excess of unlabeled probe was added during the binding reaction. Data are representative of three independent experiments. Fig. 2b, Immunostaining of phospho-p65 (pP65) and F4/80 on sections of aortic sinuses from Apoe^{_ ~} and Apoe^_7A₂AR^_/~ mice. Brown positive staining for pP65 is clearly seen on the sections with 60 magnification, c, mRNA expression of TNF-a, IL-lb, IL-6, and IL-10 in atherosclerotic lesions from Apoe^~/_ and Apoe^/A^R^-7- mice. Data present means ± SEM (n = 6), Student's t-test.

Figures 3a-3b. Homing ability of A_2AR-deficient Ly-6C^hl monocytes. Fig. 3a,

Representative histograms showing the expression of PSGL-1, L-selectin, LFA-1, VLA-4, and CCR2 on wt and A_2AR-deficient Ly-6C^hl monocytes. Solid line, wt monocytes; broken line, A₂AR-deficient monocytes. Fig. 3b, Numbers of wt and A₂AR-deficient Ly-6C^hl monocytes that migrated toward the chemokine RANTES and MCP-1. HPF, high power magnification field. Data represent means ± SEM of three independent experiments. The Student's t-test was used.

Figures 4a-4d. Apoptosis of foam cells in lesions or formed in an in vivo foam cell formation model. Fig. 4a, Representative images and quantitative data on foam cell apoptosis demonstrated with TUNEL-staining in atherosclerotic lesions. Cells positive for TUNEL staining (arrows) occur in the F4/80-stained area. Fig. 4b and Fig. 4c, Percentages of apoptotic foam cells detected with annexin V staining (Fig. 4b) and TUNEL staining (Fig. 4c). Foam cells were isolated from the peritoneal cavities of Apoe^-/~ and Apoe^_ A₂AR^~/_ mice on day 3 after thioglycollate-induced peritonitis, d, Western blot showing the level of caspase-3 fragment, pi 7, in wt and A₂AR-deficient foam cells. GAPDH served as the internal control, and pi 7

quantification in each lane was scaled to the GAPDH level. Data represent means ± SEM (n = 4), Student's t-test.

Figures 5a-5c. The role of p38 activation in apoptosis of A₂AR-deficient macrophage. Fig. 5a, Western blot showing levels of p38 and phosphorylated p38 (pP38) in wt and A₂AR- deficient macrophages at different time points after stimulation with ox-LDL (100 μg/mL). n = 3; Student's t-test. Fig. 5b, Western blot showing the level of caspase-3 fragment, pi 7, in wt and A₂AR-deflcient macrophages following incubation with ox-LDL (100 mg/mL) for 20 hours. Data represent means ± SEM (n = 4), Student's t-test. Fig. 5c, Representative images showing the effect of p38 activation on ox-LDL-mediated macrophage apoptosis. Wt and A₂AR-deficient cells were pretreated with vehicle or the p38 inhibitor SB203580 (20 μΜ), followed by incubation with ox-LDL (100 μg/mL) for 24 hours. Apoptosis was assessed by TUNEL-staining. Data represent means ± SEM (n = 4), Student's t-test.

Figures 6a-6b. Evaluation of the size of atherosclerotic lesions in the aortas of male Apoe^~/_ mice treated with caffeine or ZM241385 as compared to Apoe^{- ~} mice treated with the vehicle (Fig. 6a). Evaluation of the percentage of apoptotic cells in lesions of Apoe^~/_ treated with caffeine or ZM241385 as compared to lesions of Apoe^~/_ mice treated with the vehicle (Fig. 6b). DETAILED DESCRIPTION OF THE INVENTION

Compounds to Inactivate A_2AR

Any compound to inactivate an A₂A receptor (A₂AR) is useful in the present invention to treat or prevent atherosclerotic lesions. As used herein, the term "treat" means to prevent or ameliorate symptoms associated with atherosclerosis or atherosclerotic lesions. As used herein, the term "agonist" is used to include chemicals that are able to bind/interacts to/with the receptors and cause signals. As used herein, the term "inactivate" means that the A₂AR activity is decreased by at least 10% as compared to an A₂AR to which no agent is bound, such as, for example by means of an antagonist or blocking agent. In certain embodiments, the A₂AR activity is decreased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% 99% or even 100%. A receptor "antagonist" is a chemical that is able to bind/interact to/with the cognate receptor. In certain embodiments, an antagonist does not provoke biological response upon binding to the receptor. In certain embodiments, the receptor antagonist blocks or dampens an agonist-mediated response. In certain embodiments, an antagonist has affinity but no efficacy for its cognate receptor, and binding disrupts the interaction and inhibits the function of an agonist or inverse agonist at a receptor. In certain embodiments, an antagonist generates a novel signaling cascade in a cell, and in other embodiments an antagonist blocks the receptor from binding its agonist. "Occupancy" refers, in most cases, to the binding of agonists to the receptors. "Blocking/blockade" in most cases, refers to the binding of antagonists to the receptors.

Examples of appropriate compounds to inactivate A₂AR include the compounds of Formula (I) as described below (U.S. Patent No. 6,787,541), caffeine, ZM241385, or istradefylline (KW-6002).

The compounds to inactivate A₂AR can be formulated as pharmaceutical compositions and admimstered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.

According to the present invention there is provided a compound of formula (I):

R₅ wherein: X is O or S;

Ri and R₂ are independently selected from hydrogen, alkyl, aryl, hydroxy, alkoxy, aryloxy, cyano, nitro, C0₂R₇, COR₇, OCOR₇, CONR₇Rg, CONR₇NR8R₉, OCONR₇Rg, NR₇R9, NR₇COR8, NR₇CONR8R₉, NR₇C0₂Rg, NR₇S0₂R8, NR₇CONR8NR₉R₁₀, NR₇NR8C0_{2 9}, NR₇NR8CONR₉R₁₀, NR₇S0₂NR8 R₉, S0₂R₇, SOR₇, SR₇ and S0₂NR₇R8, or R_t and R₂ together form a carbonyl group (C=0), an oxime group (C=NORn), an imine group (C=NRn) or a hydrazone group (C=NNRnR₁₂), or Ri and R₂ together form a 5, 6 or 7 membered carbocyclic or heterocyclic ring;

R₃ is alkyl or aryl;

R4, R5 and R_$ are independently selected from hydrogen, alkyl, aryl, halogen, hydroxy, nitro, cyano, alkoxy, aryloxy, COR₇, OCOR₇, C0₂R₇, SR₇, SOR₇, S0₂R₇, S0₂NR₇R8,

CONR₇R8, CONR₇NR8R₉, OCONR₇¾, NR₇Rs, R₇CORs, NR₇CONR8R₉, NR₇C0₂R8, NR₇S0₂R8, CR₇-NOR8, NR₇CONR8NR₉R₁₀, NR₇NR8C0₂R₉, NR₇NR8CONR₉R₁₀,

S0₂NR₇NR«R₉, NR₇S0₂NR8R₉, NR₇NR8S0₂R₉, NR₇NR ₈COR₉, NR₇NRg R₉ and NR₇CSNR₈R₉, or R₅ and together form a 5, 6 or 7 membered carbocyclic or heterocyclic ring; and

R₇, Re, R₉, R₁₀, Rn and R₁₂ are independently selected from hydrogen, alkyl and aryl, or a pharmaceutically acceptable salt thereof or prodrug thereof.

As used herein, the term "alkyl" means a branched or unbranched, cyclic or acyclic, saturated or unsaturated (e.g. alkenyl or alkynyl) hydrocarbyl radical which may be substituted or unsubstituted. Where cyclic, the alkyl group is preferably C₃ to C₁₂, more preferably C₅ to C₁₀, more preferably C₅, C₆ or C₇. Where acyclic, the alkyl group is preferably Ci to C₁₀, more preferably Ci to C₆, more preferably methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n- butyl, isobutyl or tertiary-butyl) or pentyl (including n-pentyl and iso-pentyl), more preferably methyl. It will be appreciated therefore that the term "alkyl" as used herein includes alkyl (branched or unbranched), alkenyl (branched or unbranched), alkynyl (branched or unbranched), cycloalkyl, cycloalkenyl and cycloalkynyl.

As used herein, the term "lower alkyl" means methyl, ethyl, propyl (n-propyl or isopropyl) or butyl (n-butyl, isobutyl or tertiary-butyl).

As used herein, the term "aryl" means an aromatic group, such as phenyl or naphthyl, or a heteroaromatic group containing one or more heteroatom, such as pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl isoxazolyl, pyrazolyl, triazolyl, imidazolyl or pyrimidinyl. As used herein, the term "alkoxy" means alkyl-O-. As used herein, the term "aryloxy" means aryl-O-

As used herein, the term "halogen" means a fluorine, chlorine, bromine or iodine radical.

As used herein the term "Ri and R₂ together form a carbonyl group, an oxime group, an imine group or a hydrazone group" means that Ri and R₂ in combination with the carbon atom to which they are bound together form a carbonyl group, an oxime group, an imine group or a hydrazone group, i.e. the carbon atom to which R_\ and R₂ are bound in formula (I) is attached via a double bond to an oxygen atom (for compounds wherein R_\ and R₂ together form a carbonyl group) or to a nitrogen atom (for compounds wherein R_\ and R₂ together form an oxime, imine or hydrazone group).

As used herein the term "oxime group" means a group of formula C=N-OR₁₁ where Rn is selected from hydrogen, alkyl and aryl.

As used herein the term "imine group" means a group of formula C=N-R₁] where Rn is selected from hydrogen, alkyl and amyl.

As used herein the term "hydrazone group" means a group of formula C=N-NRi _\ R₁₂ where Rn and R₁₂ arm independently selected from hydrogen, alkyl and aryl.

As used herein, the term "prodrug" means any pharmaceutically acceptable prodrug of a compound of the present invention.

According to one embodiment of the invention, alkyl and aryl groups may be substituted or unsubstituted. Where substituted, there will generally be 1 to 3 substituents present, preferably 1 substituent. According to this embodiment of the invention, substituents may include carbon-containing groups such as alkyl aryl, (e.g. substituted and unsubstituted phenyl), arylalkyl; (e.g. substituted and unsubstituted benzyl); halogen atoms and halogen containing groups such as haloalkyl (e.g. tnfluoromethyl), haloaryl (e.g. chlorophenyl); oxygen containing groups such as alcohols (e.g. hydroxy, hydroxyalkyl, hydroxyaryl, (aryl)hydroxy)alkyl), ethers (e.g. alkoxy, aryloxy, alkoxyalkyl, aryloxyalkyl, alkoxyaryl aryloxyaryl), aldehydes (e.g.

carboxaldehyde), ketones (e.g. alkylcarbonyl, arylcarbonyl, alkylcarbonylalkyl,

alkylcarbonylaryl, arylcarbonylalkyl, arylcarbonylaryl, arylalkylcarbonyl,

arylalkylcarbonylalkyl, arylalkylcarbonylaryl) acids (e.g. carboxy, carboxyalkyl, carboxyaryl), acid derivatives such as esters (e.g. alkoxycarbonyl, aryloxycarbonyl, alkoxycarbonylalkyl, aryloxycarbonylalkyl, alkoxycarbonylaryl, aryloxycarbonylaryl, alkylcarbonyloxy,

alkylcartonyloxyalkyl), amides (e.g. aminocarbonyl, mono- or di-alkylaminocarbonyl, aminocarbonylalkyl, mono- or di-alkylaminocarbonylalkyl, arylaminocarbonyl or

arylalkylaminocarbonyl, alkylcarbonylamino, arylcarbonylamino or arylalkylcarbonylamino), carbamates (eg. alkoxycarbonylamino, aryloxycarbonylamino, arylalkyloxycarbonylamino, aminocarbonyloxy, mono- or di-alkylaminocarbonyloxy, arylaminocarbonyloxy or

arylalkylaminocarbonyloxy) and ureas (eg. mono- or di-alkylaminocarbonylamino,

arylaminocarbonylamino or arylalkylaminocarbonylamino); nitrogen containing groups such as amines (e.g. amino, mono- or dialkylamino, arylamino, aminoalkyl, mono- or

dialkylaminoalkyl), azides, nitriles (e.g. cyano, cyanoalkyl), nitro; sulfur containing groups such as thiols, thioethers, sulfoxides, and sulfones (e.g. alkylthio, alkylsulfmyl, alkylsulfonyl, alkylthioalkyl, alkylsulfinylalkyl, alkylsulfonylalkyl, arylthio, arylsulfinyl, arylsulfonyl, arylthioalkyl, arylsulfinylalkyl, arylsulfonylalkyl) and heterocyclic groups containing one or more, preferably one, heteroatom, (e.g. thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, aziridinyl, azetidinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl, pyridazinyl, piperidyl, hexahydroazepinyl, piperazinyl, morpholinyl, thianaphthyl, benzofuranyl, isobenzofuranyl, indolyl, oxyindolyl, isoindolyl, indazolyl, indolinyl, 7-azaindolyl, benzopyranyl, coumarinyl, isocoumarinyl, quinolinyl, isoquinolinyl, naphthridinyl, cinnolinyl, quinazolinyl, pyridopyridyl, benzoxazinyl, quinoxalinyl, chromenyl, chromanyl, isochromanyl, phthalazinyl and carbolinyl).

According to another embodiment of the invention, alkyl and aryl groups may be substituted or unsubstituted. Where substituted, there will generally be 1 to 3 substituents present, preferably 1 substituent. According to this embodiment of the invention, the substituent groups are selected from carbon containing groups such as alkyl, aryl, arylalkyl (e.g. substituted and unsubstituted phenyl, substituted and unsubstituted benzyl); halogen atoms and halogen containing groups such as haloalkyl (e.g. trifluoromethyl); oxygen containing groups such as alcohols (e.g. hydroxy, hydroxyalkyl, aryl(hydroxy)alkyl), ethers (e.g. alkoxy, alkoxyalkyl, aryloxyalkyl), aldehydes (e.g. carboxaldehyde), ketones (e.g. alkylcarbonyl, alkylcarbonylalkyl, arylcarbonyl, arylalkylcarbonyl, arylcarbonylalkyl) acids (e.g. carboxy, carboxyalkyl), acid derivatives such as esters (e.g. alkoxycarbonyl, alkoxycarbonylalkyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl) and amides (e.g. aminocarbonyl, mono- or dialkylaminocarbonyl, aminocarbonylalkyl, mono- or dialkylaminocarbonylalkyl, arylaminocarbonyl); nitrogen containing groups such as amines (e.g. amino, mono- or dialkylamino, aminoalkyl, mono- or dialkylaminoalkyl), azides, nitriles (e.g. cyano, cyanoalkyl), nitro; sulfur containing groups such as thiols, thioethers, sulfoxides, and sulfones (e.g. alkylthio, alkylsulfmyl, alkylsulfonyl, alkylthioalkyl, alkylsulfinylalkyl, alkylsulfonylalkyl, arylthio, arylsulfinyl, arylsulfonyl, arylthioalkyl, arylsulfinylalkyl, arylsulfonylalkyl); and heterocyclic groups containing one or more, preferably one, heteroatom (e.g. thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl, pyridazinyl, piperidyl, piperazinyl, morpholinyl, thianaphthyl, benzofuranyl, isobenzofuranyl, indolyl, oxyindolyl, isoindolyl, indazolyl, indolinyl, 7-azaindolyl, benzopyranyl, coumarinyl, isocoumarinyl, quinolinyl, isoquinolinyl, naphthridinyl, cinnolinyl, quinazolinyl, pyridopyridyl, benzoxazinyl,

quinoxalinyl, chromenyl, chromanyl, isochromanyl, phthalazinyl and carbolinyl).

According to another embodiment of the invention, where any of Ri to R₁₂ is selected from alkyl and alkoxy, in accordance with formula (I) as defined above, then that alkyl group, or the alkyl group of the alkoxy group, may be substituted or unsubstituted. In certain embodiment where any of R_t to R₁₂ are selected from aryl and aryloxy, in accordance with formula (I) as defined above, then the aryl group, or the aryl group of the aryloxy group, is substituted or unsubstituted. Where R] and R₂ together form a carbocyclic or heterocyclic ring, or R and R6 together form a carbocyclic or heterocyclic ring, in accordance with formula (I) as defined above, then that carbocyclic or heterocyclic ring may be substituted or unsubstituted. Where substituted, there will generally be 1 to 3 substituents present, preferably 1 substituent. In the third embodiment of the invention, the substituents are those defined in respect of the second embodiment of the invention described above.

According to another embodiment of the invention, where any of Ri to R₁₂ is selected from alkyl and alkoxy, in accordance with formula (I) as defined above, then that alkyl group, or the alkyl group of the alkoxy group, may be substituted or unsubstituted. In certain embodiment where any of Ri to R₁₂ are selected from aryl and aryloxy, in accordance with formula (I) as defined above, then the aryl group, or the aryl group of the aryloxy group, is substituted or unsubstituted. Where Ri and R₂ together form a carbocyclic or heterocyclic ring, or R₅ and R<5 together form a carbocyclic or heterocyclic ring, in accordance with formula (I) as defined above, then that carbocyclic or heterocyclic ring may be substituted or unsubstituted. Where substituted, there will generally be 1 to 3 substituents present, preferably 1 substituent. In the fourth embodiment of the invention, the substituents may include those defined in respect of the first embodiment of the invention described above.

According to a fifth embodiment of the invention, the compounds are selected from compounds of formula (la):

wherein X, Ri to R₃ and R₅ to R₁₂ are as defined for formula (I) above;

R4 is selected from hydrogen, alkyl, aryl, halogen, hydroxy, nitro, cyano, alkoxy, aryloxy, COR₇, OCOR₇, C0₂R₇, SR₇, SOR₇, S0₂R₇, S0₂NR₇Rs, CONR₇R₈, CONR₇NR8R₉, CONR₇YNRsR₉, OCO R₇Rg, NR₇R₈NR₇YR , NR₇CORs, NR₇CONRsR₉, NR₇ZCONR₈R₉, NR₇C0₂R8, NR₇ZC0₂R8, N(CORs)COR₉, NR₇S0₂R8, CR₇=NORs, NR₇CONR₈N₉R₁₀,

NR₇CONR₈YNR₉R₁₀, N R₇NR₈COR₉, NR₇YNRsC₂R₉, NR₇NRs CONR₉R₁₀,

NR₇YNR₈CONR₉R₁o, S0₂NR₇NRsR₉, S0₂NR₇YNRsR₉, NR₇S0₂NRsSR₉, NR₇NR₈0₂R₉,

NR₇YNR₈S0₂R₉, NR₇NR8COR₉, NR₇YNRsCOR₉, NR₇N¾R₉, NR₇YNR₈R₉, NRyCSNRgRg, NR₇ YNR$C SNR₉Ri ₀ and NR₇Y R₈CONR₉YNR₁₀R₁1);

Y is a divalent C₂ to C₄ carbon chain; and

Z is a divalent C_\ to C₄ carbon chain,

or a pharmaceutically acceptable salt thereof or prodrug thereof.

In the fifth embodiment of the invention, where any of Ri to R₁₂ is selected from alkyl and alkoxy, in accordance with formula (la) as defined above, then that alkyl group, or the alkyl group of the alkoxy group, may be substituted or unsubstituted. In certain embodiments where any of Ri to R₁₂ is selected from aryl and aryloxy, in accordance with formula (la) as defined above, then the aryl group, or the aryl group of the aryloxy group, is substituted or unsubstituted. Where R_\ and R₂ together form a carbocyclic or heterocyclic ring, or R₅ and Re together form a carbocyclic or heterocyclic ring, in accordance with formula (la) as defined above, then that carbocyclic or heterocyclic ring may be substituted or unsubstituted. Where substituted, there will generally be 1 to 3 substituents present, preferably 1 substituent. In the fifth embodiment of the invention, the substituents may include those defined in respect of the first embodiment of the invention described above.

As used herein, the term "divalent C_\ to C₄ carbon chain" means a chain comprising 1, 2, 3 or carbon atoms, branched or unbranched, and saturated or unsaturated.

As used herein, the term "divalent C₂ to C₄ carbon chain" means a chain comprising 2, 3 or 4 carbon atoms, branched or unbranched, and saturated or unsaturated.

In the compounds of the present invention, preferably X is S. In one embodiment of the invention, Ri and R₂ are independently selected from hydrogen; hydroxy; cyano; alkyl, such as hydroxy-substituted alkyl; and C0₂ R7, wherein R₇ is alkyl. In this embodiment, Ri and R₂ are selected from hydrogen and cyano. In this embodiment, one of R} and R₂ are hydrogen.

In a further embodiment, where Ri is hydroxy, R₂ is not selected from hydroxy, alkoxy and aryloxy. Similarly, in certain embodiments where R₂ is hydroxy, Ri is not selected from hydroxy, alkoxy and aryloxy.

In an alternative further embodiment, where R_\ is selected from hydroxy and SH, R₂ is not selected from hydroxy, alkoxy, aryloxy and SR₇. Similarly, where R₂ is selected from hydroxy and SH, in certain embodiments Ri is not selected from hydroxy, alkoxy, aryloxy and SR₇.

In certain embodiments, Ri and R₂ together form a carbonyl group or an oxime group, in certain embodiments, a carbonyl group. Where R_\ and R₂ together form an oxime group C=N- ORn, in certain embodiments Rn is hydrogen.

In certain embodiments of the invention, Ri and R₂ together form a carbonyl group.

In the compounds of the present invention, in certain embodiments R₃ is aryl, in certain embodiments comprising a five or six membered ring which may be substituted or unsubstituted and which may be carbocyclic or heterocyclic. In certain embodiments t R₃ is monocyclic.

Where R₃ is a five-membered ring, in certain embodiments R₃ is an N, O or S-containing heterocyclic ring, such as a thienyl, furyl pyrrolyl or thiazolyl group, or a thienyl group. Where R₃ is a six-membered ring in certain embodiments R3 is phenyl or an N-containing heterocyclic ring, such as pyridyl.

Where R₃ is substituted, in certain embodiments R₃ is substituted by substituent group(s) selected from halogen, such as fluoro, chloro and bromo, such as chloro; lower alkyl, such as methyl; lower alkoxy, such as methoxy; nitro; and amino, such as dialkylamino, such as dimethylamino.

In certain embodiments, R₃ is selected from thienyl, furyl, pyridyl (such as 2-pyridyl) and phenyl, such as 2-thienyl. Where R₃ is selected from 2-thienyl, the 2-thienyl group in certain embodiments is unsubstituted or substituted by lower alkyl (such as methyl) or halogen (such as chloro or bromo, preferably chloro) or lower alkoxy such as methoxy), and in certain

embodiments is unsubstituted or substituted by lower alkyl (such as methyl), and in certain embodiments is unsubstituted. Where R₃ is selected from furyl, the furyl group in certain embodiments is a 2-furyl group and in certain embodiments is unsubstituted or substituted by lower alkyl (such as methyl). In the compounds of the present invention, in certain embodiments R₅ is selected from hydrogen, alkyl, halogen, hydroxy, nitro, cyano, alkoxy, aryloxy, CO R₇, OCOR , CO2R7, SR₇, SOR7, S0₂R₇, S0₂NR₇R₉, CONR₇R₉, CONR₇NR_gR₉, OCONR₇R8, NR₇Rg, NR₇CORg,

NR₇CONRgR₉, NR₇C0₂R₈, NR₇S0₂R₈, R₇=NORg, NR₇CONR₈NR₉R₁₀, NR₇NRsC0₂R₉, NR₇NR«CONR₉Rio, S0₂NR₇NR₈R₉, NR₇ S0₂NR₉R₉, NR₇NRgS0₂R₉, NR₇NR₈COR₉,

NR₇NRgR₉, NR₇CSNRgR₉, or together with R^ forms a 5, 6 or 7 membered carbocyclic or heterocyclic ring.

In one embodiment of the present invention, R₅ is selected from hydrogen, halogen, alkyl and aryl.

Where R₅ is selected from aryl, in certain embodiments R₅ is an aryl group other than phenyl or an N-containing heteroaromatic group, particularly pyridyl, pyrazinyl pyrimidinyl and pyridazinyl. Where R₅ is an aryl group, in certain embodiments R₅ is an aryl group selected from an O- or S-containing heterocyclic ring, in certain embodiments is an O-containing ring, and in certain embodiments is a 5-membered heterocyclic ring, such as furanyl or thienyl.

In the compounds of the present invention, in certain embodiments R₅ is selected from hydrogen, halogen and alkyl.

In the compounds of the present invention, in certain embodiments R is selected from hydrogen, alkyl, aryl and halogen.

In certain embodiments at least one, and in certain embodiments both of R₅ and R$ are hydrogen. Where R₅ and/or ¾ are selected from alkyl, it is preferred that R₅ and/or ¾ are methyl.

Where any of R_l5 R₂, or R4 to ¾ are independently selected from N R₇ C0₂ Rg, in certain embodiments R₉ is selected from alkyl and aryl.

Where any of R_l5 R₂ or Rj to R6 are independently selected from N R₇ N Rg C0₂ R₉, in certain embodiments R₉ is selected from alkyl and aryl.

Where R4 is selected from N R₇ YN Rg C0₂ R₉, in certain embodiments R₉ is selected from alkyl and aryl.

In the compounds of the present invention where R4 is an N R₇ Rg group, the R₇ and Rg groups may together form a ring to produce a cyclic amino group. The cyclic amino group is a saturated or partially unsaturated cyclic group (i.e. it is non-aromatic), and in certain

embodiments is a saturated cyclic amino group. The cyclic amino group in certain embodiments is a 5-, 6- or 7-membered and in certain embodiments is a 5- or 6-membered cyclic amino group. Where partially unsaturated, in certain embodiments only 1 double bond is present. The cyclic amino group may contain one or more additional heteroatoms, in certain embodiments one or two heteroatoms, wherein the heteroatoms are preferably selected from N, O and S. The cyclic amino groups may be substituted or unsubstituted. Where substituted, there will generally be 1 to 3 substituents present. Substituents may include any of those set out above in respect of the first and second embodiments. In certain embodiments the cyclic amino groups are selected from pyrrolidinyl pyrrolidinonyl, piperidinyl, piperazinyl and morpholinyl groups, , and in certain embodiments selected from pyrrolidinyl groups (such as substituted and in certain embodiments substituted by hydroxy, lower alkyl or hydroxy(lower alkyl)).

In certain embodiments of the present invention, R4 is selected from alkyl (including trifluoromethyl); halogen (preferably chloro); alkoxy (preferably methoxy or ethoxy); SR₇ (preferably alkylthio, preferably methylthio); dialkylamino (preferably dimethylamino); and monoalkylamino, wherein said alkyl groups are substituted or unsubstituted. In certain embodiment, R₄ is unsubstituted alkyl, trifluoromethyl or monoalkylamino (wherein the alkyl groups are substituted or unsubstituted), and in certain embodiments monoalkylamino (in certain embodiments NR₇Rs wherein R₇ is hydrogen, and Rs is substituted or unsubstituted). In this embodiment, where R4 is monoalkylamino or dialkylamino, the alkyl group(s) may be substituted as described above, for instance, by hydroxy, alkoxy, amino or dialkylamino.

Where R4 is alkyl, in certain embodiments R₄ is unsubstituted alkyl (such as saturated alkyl, preferably lower alkyl) or halo-substituted alkyl (such as trifluoromethyl).

In certain embodiments of the invention, R₄ is NR₇Rs. Where R4 is NR₇Rs, in certain embodiments R₇ is lower alkyl or hydrogen. In certain embodiments, R₈ is lower alkyl

(substituted or unsubstituted and, where substituted, in certain embodiments is substituted by hydroxy, alkoxy, a saturated heterocyclic group or aryl (particularly heteroaryl)), cyclic alkyl or aryl.

In a further preferred embodiment, R4 is NH₂.

Where R₄ is NR₇NRsR9, in certain embodiments R₇ is hydrogen. In certain embodiments

R₅ and R₉ are also hydrogen.

In the compounds of formula (I), in certain embodiments R₄ is selected from alkyl (including trifluoromethyl); halogen (such as chloro); alkoxy (such as methoxy or ethoxy, S R₇ (such as methylthio); and a substituted amino group (such as NR₇Rs, NR₇RsCOR9,

NR₇NRgC0₂R₉, NR₇C0₂R₈, ^NRsCONRgRto, NR₇NRsS0₂R9, NR₇R8CSNR₉R₁₀, NR₇NR8R₉ and NR₇COR8, such as NR₇¾). In certain embodiments R₄ is a substituted amino group or alkyl. In certain embodiments y R₄ is a substituted amino group, preferably NR₇Rs wherein R₇ is hydrogen. In the compounds of formula (la), in certain embodiments R₄ is selected from alkyl (including trifluoromethyl); halogen (such as chloro); alkoxy (such as methoxy or ethoxy, S R₇ (such as methylthio); and a substituted amino group (such as NR₇Rs, NR₇YR₈, NR₇YNRsCOR₉, NR₇YNR₈C0₂R₉, NR₇ZC0₂Rs, NR₇YN¾ CONR₉R₁₀, NR₇YNRsS0₂R₉, NR₇YNR CSNR₉R₁₀, NR-₇NR8R9 and N(CORs)COR₉, and in certain embodiments NR₇Rs). In certain embodiments R4 is a substituted amino group or alkyl.

In the compounds of formulae (I) or (la), in certain embodiments where R4 is selected from N R₇ Rs, R₇ is hydrogen or alkyl, and in certain embodiments hydrogen, and Rs is selected from alkyl (such as saturated alkyl), in certain embodiments lower alkyl (such as saturated lower alkyl), substituted or unsubstituted, wherein in certain embodiments the substituent groups on Rs are selected from aryl (such as thienyl, furyl, pyridyl and phenyl); oxygen-containing groups, particularly alcohols (such as hydroxy), ethers (such as alkoxy); acids (such as carboxy); acid derivatives (such as esters such as alkoxycarbonyl), amides (such as alkylcarbonylamino and arylcarbonylamino), carbamates (such as alkoxycarbonylamino and arylalkoxycarbonylamino) and ureas (such as alkylammocarbonylamino, arylaminocarbonylamino and

arylalkylaminocarbonylamino); nitrogen-containing groups, such as amines and thioureas; and saturated heterocyclic groups, such as N- and O-containing groups (such as tetrahydrofuranyl, pyrrolidinyl, pyrrolidinonyl, piperidinyl, piperazinyl and morpholinyl groups).

In the compounds of formulae (I) or (la), in an alternative preferred embodiment where R4 is selected from NR₇Rs, in certain embodiments R₇ is hydrogen or alkyl and such as hydrogen, and R₈ is selected from alkyl (such as saturated alkyl), such as lower alkyl (such as saturated lower alkyl), substituted or unsubstituted and such as substituted, wherein the preferred substituent groups on Rg are selected from aryl (such as phenyl); oxygen-containing groups, alcohols (such as hydroxy), ethers (such as alkoxy) and acid derivatives, particularly esters (such as alkoxycarbonyl) and carbamates (such as alkoxycarbonylamino); nitrogen containing groups, such as amines; and heterocyclic groups, such as saturated N-containing heterocyclic groups (such as pyrrolidinyl, pyrrolidinonyl, piperidinyl, piperazinyl and morpholinyl groups).

In the compounds of formula (la) where R4 is NR₇YN RsCOR₉, in certain embodiments R₇ is hydrogen. In certain embodiments R₈ is also hydrogen. In certain embodiments R₉ is selected from lower alkyl, cyclic alkyl and aryl (such as substituted or unsubstituted phenyl or thienyl).

In the compounds of formula (la) where R₄ is NR₇YNR C0₂R₉ or NR YNR S0₂R₉, in certain embodiments R₇ is hydrogen. In certain embodiments Rs is also hydrogen. In certain embodiments R₉ is selected from lower alkyl (substituted or unsubstituted and, where substituted, may be substituted by halogen (such as chloro) or aryl).

In the compounds of formula (la) where R4 is NR₇YNR CONR₉R₁₀ or

NR₇YNR₈CSNR9R₁₀, in certain embodiments R₇ is hydrogen. In certain embodiments Rg and R₉ are also hydrogen, in certain embodiments Rio is lower alkyl (substituted or unsubstituted), cyclic alkyl or aryl.

In the compounds of formula (la) where R4 is NR₇ZC0₂R8, in certain embodiments R₇ is hydrogen and R is selected from hydrogen and lower alky.

In the compounds of formula (la) where R4 is NR₇YRg, in certain embodiments R₇ is hydrogen. In certain embodiments Rg is aryl, and in certain embodiments is substituted by lower alkyl, lower alkoxy and nitro.

In the compounds of formula (la) where R4 is N(CO Rs)CO R₉, in certain embodiments that R« and R₉ are independently selected from lower alkyl.

In the compounds of formula (la), in certain embodiments Y is a saturated (alkylene) C₂ to C₄ carbon chain and in certain embodiments is unbranched. In certain embodiments, Y is a C₂ or C₃ carbon chain, such as a C₂ carbon chain. In certain embodiments, Y is a divalent C¾CH₂ radical.

In the compounds of formula (la), in certain embodiments Z is a saturated (alkylene) C_\ to C₄ carbon chain and in certain embodiments is unbranched . In certain embodiments, Z is a C_l5C₂ or C₃ carbon chain. In certain embodiments, Z is a divalent CH₂CH₂ radical.

In certain embodiments of the invention, the compounds of the present invention are selected from (2R)-2-(l-Hydroxy-2-propylamino)thieno[3,2-d]pyrimidin-4-yl 2- thienylmethanone, 2-(3 -( 1 H-Imidazol- 1 -yl)propylamino)thieno [3 ,2-d]pyrimidin-4-yl 2- thienylmethanone, (2RS)-2-(l-Hydroxy-2-propylamino)thieno[3,2-d]pyrimidin-4-yl 2- thienylmethanone, 2-(3-Hydroxypropylamino)thieno[3,2-d]pyrimidin-4-yl 2-thienylmethanone, 3-Methyl-N-(2-(4-(2-thienylcarbonyl)thieno[3,2-d]pyrimidin-2-yI)aminoethyl) butanamide, Methyl (2-(4-(2-tWenylcarbonyl)thieno[3,2-d]pyrimidin-2-yl)aminoethyl)carbamate, 2-(2-(lH- Imidazol-4-yl)ethylamino)thieno[3,2-d]pyrimidin-4-yl 2-thienylmethanone, (2RS)-2-(2,3- Dihydroxypropylamino)thieno[3,2-d]pyrimidin-4-yl 2-thienylmethanone, (2R)-2-(2- Hydroxypropylamino)thieno[3,2-d]pyrimidin-4-yl 2-thienylmethanone, 2-(2-

Hydroxyethylamino)thieno[3,2-d]pyrimidin-4-yl 3-methyl-2-thienylmethanone, 2-Chloroethyl (2-(4-(2-thienylcarbonyl)thieno [3 ,2d]pyrimidin-2-yl)aminoethyl)carbamate, (2S)-2-( 1 -Hydroxy- 2-propylamino)thieno[3,2-d]pyrimidin-4-yl 2-thienylmethanone, 2-(3-(lH-Imidazol-l - yl)propylamino)thieno[3,2-d]pyrimidin-4-yl 3-methyl-2-thienylmethanone, N-(2-(4-(2- Thienylcarbonyl)thieno[3,2-d]pyrimidin-2-yl)aminoethyl)cyclohexy lcarboxamide, Ethyl 4-(4- (2-tWenylcarbonyl)t eno[3,2-d]pyrimidin-2-ylamino)butanoate, 2-(2- Pyn^ylmethylamino)thieno[3,2-d]pyrimidin-4-yl 2-thienylmethanone, (2S)-2-(2- Hydroxypropylamino)tWeno[3,2-d]pyrimidin-4-yl 2-thienylmethanone, N-Allyl-N'-(2-(4-(2- tWenylcarbonyl)t eno[3,2-d]pyrimidin-2-yl)aminoethyl) urea, N-(2-(4-(2-

T enylcarbonyl)tWeno[3,2-d]pyrimidin-2-yl)aminoethyl)acetamide , 2-(Tetrahydrofuran-2- ylmethylamino)thieno[3,2-d]pyrimidin-4-yl 2-thienylmethanone, N-Benzyl-N'-2-(4-(2- thienylcarbonyl)thieno[3,2-d]pyrimidin-2-yl)aminoethyl) urea, 2-(2- Hydroxyemylamino)thieno[3,2-d]pyrimidin-4-yl 2-thienylmethanone, Benzyl (2-(4-(2- tWenylcarbonyl)thieno[3,2-d]pyrimidin-2-yl)aminoethyl)carbamate, and 2-Aminothieno[3,2- d]pyrimidin-4-yl 2-thienylmethanone.

In certain embodiments, the compounds of the present invention are selected from: 2- thienyl 2-1rifluoromethyltWeno[3,2-d]pyrirm^in-4-ylmethanone, 2-(2- hydroxyethyl)ammotWeno[3,2-d]pyrimidin-4-yl 2-thienylmethanone, 2-ethylaminothieno[3,2- d]pyrimidin-4-yl 2-thienylmethanone, 2-ethylthieno[3,2-d]pyrimidin-4-yl 2-thienylmethanone, 2-methylaminothieno[3,2-d]pyrimidin-4-yl 2-thienylmethanone, and 2-(2- memoxyemylammo)tWeno[3,2-d]pyrimidin-4-yl 2-thienylmethanone.

Where chiral the compounds of the present invention may be in the form of a racemic mixture of pairs of enantiomers or in enantiomerically pure form.

Formulations and Methods of Administration

The compounds to inactivate A_2AR of the invention may be formulated as

pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms adapted to the chosen route of administration, i.e., orally, intranasally, intradermally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of

microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Examples of useful dermatological compositions which can be used to deliver the compounds of formula I to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949. The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.

The compound is conveniently formulated in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. In one embodiment, the invention provides a composition comprising a compound of the invention formulated in such a unit dosage form.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

Compounds of the invention can also be administered in combination with other therapeutic agents, for example, other agents that are useful for the treatment of atherosclerosis. Accordingly, in one embodiment the invention also provides a composition comprising a compound to inactivate A₂AR, or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, and a pharmaceutically acceptable diluent or carrier. The invention also provides a kit comprising a compound to inactivate A₂AR, or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, packaging material, and instructions for

administering the compound to inactivate A₂AR or the pharmaceutically acceptable salt thereof and the other therapeutic agent or agents to an animal to treat of atherosclerosis.

The following examples are intended to illustrate but not limit the invention.

EXAMPLE 1

Introduction

A_2AR plays a complex role in inflammation and tissue injury. In the context of neurological disease, blocking A₂A appears to be beneficial (Chen JF, Sonsalla PK, Pedata F, Melani A, Domenici MR, Popoli P, Geiger J, Lopes LV, de MA. Adenosine Α2Α receptors and brain injury: broad spectrum of neuroprotection, multifaceted actions and "fine tuning" modulation. Prog Neurobiol 2007; 83(5):310-31). Several A_2AR antagonists are being developed to treat neurological disorders, and some of these are even being assessed in clinical trials (Schwarzschild MA, Agnati L, Fuxe K, Chen JF, Morelli M. Targeting adenosine A2A receptors in Parkinson's disease. Trends Neurosci 2006;29(11):647-54). Notably, many patients with neurodegenerative disease also suffer from vascular disease associated with atherosclerosis. Thus, it is relevant to study whether blocking A_2AR also affects atherosclerosis. To date, however, there have been no reports on the effects of blocking or knocking out A_2AR on atherosclerosis. Therefore, we evaluated whether A_2A deficiency affects atherosclerosis by using mice deficient for both A_2A and Apoe (Apoe^_/ A_2AR ⁷ )·

Materials and Methods

A_2AR ⁷ mice in C57BL/6J background were bred with apoE^~/_ (C57BL/6J background) mice to generate Apoe-7A_2AR^{_ ~} mice and their littermate controls. Chimeric mice with or without A_2AR in their bone marrow-derived cells were produced by bone marrow

transplantation, as described (Huo Y, Zhao L, Hyman MC, Shashkin P, Harry BL, Burcin T, Forlow SB, Stark MA, Smith DF, Clarke S, Srinivasan S, Hedrick CC, Pratico D, Witztum JL, Nadler JL, Funk CD, Ley K. Critical role of macrophage 12/15 -lipoxygenase for atherosclerosis in apolipoprotein E-deficient mice. Circulation 2004; 110(14):2024-31). All animal experiments and care were approved by the University of Minnesota Animal Care and Use Committee, in accordance with AAALAC guidelines.

Results

Atherosclerosis in Apoe^_/ A2AR^_/_ mice

To determine the role of A_2AR in the development of atherosclerotic lesions in vivo, Apoe^-/7A_2AR^{~ -} mice and their littermate Apoe^~_ mice were fed a chow diet or western diet for three months. These mice exhibited no differences in blood pressure, number of circulating leukocytes, differential counts, or blood glucose (Suppl. Table 1, 2, 3). For mice on a western diet, the level of blood alanine aminotransferase (ALT) in Apoe^T ^R^-7- mice was four times higher than that in Apoe^{~ _} mice (Suppl. Table 4). The weight of Apoe^_/7A_2AR^{_ _} mice fed a western diet was 23% greater for males and 12% higher for females compared with sex-matched Apoe^-7- mice fed the same diet. Total blood cholesterol was 45% higher in male Apoe^~/7A_2AR^~/~ mice and 25% higher in females compared with Apoe^{- ~} mice on both chow and western diets; this increase was due solely to increased LDL cholesterol (Table 1 and Suppl. Table 3).

Interestingly, lipid profiles were similar in A_2AR^_/_ and wild type mice on a western diet. In A₂AR^{_ _} and wild type mice on either a chow or western diet, blood IL-6 levels were not detectable. In contrast, blood IL-6 levels were detectable and much higher in Apoe^_/7A₂AR^~/_ mice than in Apoe^{~ _} mice (25 ± 8.2 versus 20 ± 5.8 pg/mL, as shown in Suppl. Table 5). Despite the greater body weight, higher blood cholesterol, and higher proinflammatory cytokine levels, the size of atherosclerotic lesions in the aortas of Apoe^_7A₂AR^{_ _} mice was decreased by 26% in females and 20% in males compared to Apoe^~/_ mice (Fig. la). In addition, the aortic sinuses displayed much smaller lesions in Apoe^_/7A₂AR^~/_ mice than in Apoe^_/~ mice (Fig. lc).

To examine whether A₂AR deficiency could also protect mice from advanced

atherosclerosis, Apoe^_7A₂AR^{~ _} mice and their littermate Apoe^-/~ mice were placed on a Western diet for six months. In accordance with the changes in mice fed a Western diet for three months, Apoe^/A^R^ mice gained more body weight and had a much higher level of blood total cholesterol than Apoe^~/_ mice (Table 1). Aortic atherosclerotic lesions in female Apoe^{_ ~} /A₂AR mice were 51% smaller than those in female Apoe^-~ mice, and the lesions in male

Apoe^_ 7A2AR^_ mice were 55% smaller than those in controls (Fig. lb). These results confirmed the data obtained from mice fed a Western diet for three months, and demonstrated even greater protection against atherosclerosis in Apoe^_/7A₂AR^_/_ mice during a longer period of

atherosclerotic challenge.

The cellular components of atherosclerotic lesions in cross-sections of the aortic sinus were also compared. Macrophages and foam cells were mainly located in the cap and shoulders of lesions in Apoe^"^ and Apoe^_/7A2AR ^~'^~ mice, but the total number of macrophages and foam cells in lesions of Apoe^/A^R^^" mice was greatly diminished (Fig. Id). This was further supported by the lower levels of mRNA encoding the monocyte marker CD68 in lesions of Apoe^-/7A₂AR^-/~ mice than in those of Apoe^_/~mice (Fig. le).

Atherosclerosis in chimeric mice lacking A_2AR and apoE in bone marrow-derived cells (BMDCs)

To determine the influence of leukocyte A₂AR in the formation of atherosclerotic lesions, we studied atherosclerosis in Apoe^{_ ~} chimeric mice fed a Western diet for three months. Apoe^{~ _} mice lacking A₂AR in their BMDCs did not differ from Apoe^_/~ mice in body weight or blood cholesterol level (data not shown). In the aortic sinuses, a 30% reduction in the average size of lesions was observed in chimeric mice lacking A₂AR in their BMDCs compared to that in controls (Fig. If). This finding suggests that protection against atherosclerosis in Apoe^/A^R^^" mice was mainly due to A₂AR deficiency in BMDCs. The presence of macrophages in atherosclerotic lesions of chimeric mice was also assessed; compared with Apoe^_/~ mice, Apoe^_/~ mice lacking A₂A in their BMDCs

demonstrated significantly fewer macrophages in lesions (Fig. lg).

Inflammatory status of atherosclerotic lesions in Apoe^_7A2AR^~/_ mice

Atherosclerosis is a chronic inflammatory disease, and disease progression is usually accompanied by increased inflammation. A₂AR mice and A₂AR-deficient macrophages exhibit increased inflammatory phenotype following inflammatory stimulation. Since Apoe^_/7A₂AR^{_ _} mice developed small atherosclerotic lesions, we speculated that A₂AR-deficient macrophages might react to modified LDL differently from their response to other inflammatory stimuli. To test this possibility, we examined the inflammatory response of A₂AR-deficient macrophages to ox-LDL in an in vivo peritonitis model. On the third day of thioglycollate-induced peritonitis, mice were injected intraperitoneally with ox-LDL. Peritoneal macrophages were collected 30 minutes after the ox-LDL injections. As shown by an electrophoretic mobility shift assay, both wild type and A₂AR-deficient macrophages displayed significant levels of nuclear P65/P50 binding to the NF-κΒ consensus sequence, indicating activation of the NF-κΒ pathway in thioglycollate-elicited macrophages. Compared with wt macrophages, A₂AR-deficient macrophages showed increased NF-κΒ activation before and after ox-LDL treatment (Fig. 2a).

To determine the level of NF-κΒ activation in foam cells present in atherosclerotic lesions of Apoe^-/7A₂AR^{_ ~} mice, sections of atherosclerotic lesions were immunostained with phosphor-p65-specific antibody. Phosphorylation of p65 is an indicator of NF-κΒ activation. Among the macrophages/foam cells present in lesions, many more cells demonstrated positive staining for phospho-p65 in lesions of Apoe^_7A₂AR^{_ ~} mice than in those of Apoe^{- ~} mice (Fig. 2b). In addition to NF-κΒ signaling, we also determined the mRNA levels of proinflammatory cytokines in lesions by real-time RT-PCR. The levels of IL-lb and IL-6 mRNA were much higher in atherosclerotic lesions of Apoe^_ 7A2AR^{- ~} mice than in those of Apoe^{~ _} mice (Fig. 2c). These results indicate that, in an atherosclerotic environment, A₂AR-deficient macrophages exhibited an inflammatory phenotype. Notably, the mRNA level of IL-10, an anti-inflammatory cytokine, was also increased in lesions of Apoe^_ 7A₂AR^~/- mice.

Homing ability of A_2AR-deficient Ly-6C^hl monocytes

Macrophages present in atherosclerotic lesions differentiate from infiltrated Ly-6C^hl monocytes. Therefore, the decreased number of macrophages in aortic lesions of Apoe^_7A₂AR^~

* · hi

mice may be due to a defect in the homing ability of A₂AR-deficient Ly-6C monocytes. To test this possibility, we first measured the expression of homing molecules on circulating Ly- 6C^hl monocytes in Apoe^{~ -} and Apoe^_7A₂AR^{~ _} mice. There were no significant differences in the levels of PSGL-1, L-selectin, LFA-1, VLA-4, or CCR2 between wt and A_2AR-deficient Ly- 6C^hl monocytes (Fig. 3a). Next, we sorted Ly-6C^hl monocytes from splenocytes of wt and A_2AR^{~ _} mice and compared their migration toward MCP-1 and RANTES, the major chemokines that mediate monocyte recruitment to atherosclerotic arteries. A_2AR-deficient Ly-6C^hl monocytes exhibited chemotactic activities similar to those of wt cells, indicating that recruitment of Ly- 6C^hl monocytes to the arterial wall may not be reduced in Apoe-7A_2AR ^~'^~ mice (Fig. 3b). Thus, the decreased number of macrophages observed in atherosclerotic lesions of Apoe^_ A_2AR^-/~ mice was not due to a defect in monocyte recruitment.

Apoptotic foam cells in atherosclerotic lesions of Apoe^TA^R^"7- mice

Apoptosis of macrophages or foam cells during the early stages of atherosclerosis decreases atherosclerosis. To investigate whether this was the mechanism responsible for suppressed atherosclerosis in Apoe^_7A_2AR^_/~ mice, we first performed TUNEL-staining to detect apoptotic cells on cross-sections of atherosclerotic lesions. In lesion areas containing F4/80-positive macrophages, many more cells were positive for TUNEL-staining in lesions of Apoe^_/ A_2AR ^~'^~ mice than in those of Apoe^~/_ mice (Fig. 4a).

Macrophages in the peritoneal cavities of atherosclerotic mice with thioglycollate- induced peritonitis differentiate into foam cells (Li AC, Brown KK, Silvestre MJ, Willson TM, Palinski W, Glass CK. Peroxisome proliferator-activated receptor gamma ligands inhibit development of atherosclerosis in LDL receptor-deficient mice. J Clin Invest 2000;106(4):523- 31). By using this in vivo foam cell formation model, wt and A_2AR-deficient foam cells were generated and assayed by flow cytometry. Among the F4/80-positive foam cells, the percentage of annexin V-positive but Pi-negative cells was 12% for foam cells from Apoe^_/7A₂AR^~/_ mice and 5% for foam cells from Apoe^{~ _} mice (Fig. 4b). Similar results were obtained by TUNEL- staining (Fig. 4c). Caspase-3 is a critical executioner of apoptosis, and the cleaved pi 7 fragment represents its active form. The pi 7 fragment of caspase-3 was detected in foam cells by western blot. The level of pi 7 was much higher in A_2AR-deficient foam cells than in wt cells (Fig. 4d).

Activation of p38 MAPK in A₂AR-deficient macrophages

Activation of A_2AR increases intracellular cAMP, which, in turn, inhibits activation of the intracellular signaling molecule p38 MAPK via the cAMP response element-binding protein-induced dynein light chain. In an in vitro assay using isolated peritoneal macrophages, p38 MAPK activation in response to ox-LDL stimulation was much more robust in A_2AR- deficient macrophages than in wt macrophages (Fig. 5a). Furthermore, the level of ox-LDL- induced active caspase-3 was much higher in A_2AR-deficient macrophages than in wt macrophages (Fig. 5b). To determine whether p38 activation was a possible mechanism for the increased apoptosis of A₂AR-deficient macrophages, A₂AR-deficient macrophages were first pretreated with the p38 inhibitor SB203580, then incubated with ox-LDL for induction of apoptosis. Incubation with ox-LDL elicited apoptosis in 20% of A₂AR-deficient macrophages and 9% of wt macrophages. SB203580 pretreatment decreased ox-LDL-mediated apoptosis in both cases, but this decrease was more pronounced for A₂AR-deficient macrophages than wt cells. The percentage of apoptotic A_2AR-deficient macrophages was reduced almost to the level measured for wt macrophages, indicating that increased p38 activation is the underlying mechanism for apoptosis of A₂AR-deficient macrophages (Fig. 5c).

A₂AR Antagonists suppressed atherosclerosis in Apoe ^{" "} mice

Having demonstrated that A₂AR deficiency caused apoptosis of foam cells and thereafter reduced the size of atherosclerotic lesions, we examined whether A_2AR antagonists exert similar effects. For 6 weeks old male Apoe^{_ "} mice on the western diet, we administered caffeine (through drinking water) or ZM241385 (through a mini-pump planted under the skin) for 4 months. The size of atherosclerotic lesions in the aortas of male Apoe^{~ _} mice was decreased by 41% and 35% with the treatment of caffeine and ZM241385 compared to Apoe^{_ ~} mice treated with the vehicle (Fig. 6a). Consistent with the observation on apoptotic cells in the lesions of Apoe^_/7A₂AR^{_ _} mice, the percentages of apoptotic cells in lesions of Apoe^_/~ treated with caffeine or ZM241385 were much greater than those in lesions of Apoe^-/~ mice treated with the vehicle (Fig. 6b).

Discussion

A₂AR deficiency exacerbates inflammatory reactions and induces severe tissue injury. Our present work demonstrates that, compared to Apoe^_/_mice,

mice have greater body weight, considerably more severe hypercholesterolemia, and increased

proinflammatory cytokines in the blood. These data would predict a severe atherosclerotic phenotype in Apoe^_7A₂AR^{_ "~} mice. Thus, the observed suppression of atherosclerosis in Apoe^~/- /A₂AR^-~ mice was highly unexpected. The initial data showing decreased atherosclerosis in mice fed a Western diet for three months were surprising, and led us to subsequently assess mice fed a Western diet for six months. A₂AR deficiency led to even greater protection against

atherosclerosis when mice were provided a Western diet for a longer period. Results from these two animal studies unambiguously support a protective role for A₂A inactivation in

atherosclerosis.

The protective role of A_2AR deficiency or blockade has mostly been observed in neurological disease models (Chen JF, Sonsalla PK, Pedata F, Melani A, Domenici MR, Popoli P, Geiger J, Lopes LV, de MA. Adenosine A2A receptors and brain injury: broad spectrum of neuroprotection, multifaceted actions and "fine tuning" modulation. Prog Neurobiol

2007;83(5):310-31). Loss or blockade of A₂AR decreases ischemic brain injury and

neurotoxicity in models of Parkinson's disease and Huntington's disease. Blocking A₂AR- mediated glutamate release from the ischemic and nonischemic cortex and striatum has been proposed as the mechanism for these beneficial effects. A recent study found that either global or BMDC-specific A₂AR deficiency in mice attenuated infarct volumes in an ischemic brain injury model (Yu L, Huang Z, Mariani J, Wang Y, Moskowitz M, Chen JF. Selective inactivation or reconstitution of adenosine A2A receptors in bone marrow cells reveals their significant contribution to the development of ischemic brain injury. Nat Med

2004;10(10):1081-7). This protection was associated with a decline in the ischemia-induced expression of several proinflammatory cytokines. Using the same real-time RT-PCR assay, we found that the expression of cytokines in atherosclerotic lesions of Apoe^_7A₂AR ^~'^~ mice was higher than that of Apoe^~_ mice, indicating that the mechanism for protection against atherosclerosis due to A₂AR deficiency differs from that involved in neuroprotection.

A₂AR deficiency has adverse effects in most animal models of peripheral organ diseases.

A₂AR^_/~ mice exhibit extensive liver damage due to prolonged and enhanced expression of proinflammatory cytokines (such as TNF-a, IL-6, and IL-12) in concanavalin A- or endotoxin- induced septic shock and ischemic liver injury models. Additionally, in a renal ischemia reperfusion injury model, plasma creatinine and cytokines are significantly increased in A₂A ^~'^~ mice compared to wt mice. In an adenosine deaminase-deficient model of pulmonary inflammation, A₂AR deficiency causes enhanced pulmonary leukocyte infiltration and mucin production in the bronchial airways, as well as elevated levels of MCP-1 and CXCL1. A₂AR- mediated protection may be achieved via suppression of the generation of reactive oxygen species and proinflammatory cytokines in inflammatory cells. In line with the above studies, we found that proinflammatory cytokines were increased in the circulating blood and atherosclerotic lesions of Apoe^_7A₂AR^_/_ mice.

Macrophage phenotype is modulated through adenosine A_2AR activation. A_2AR agonists synergize toll like receptors to switch macrophages from an Ml (inflammatory) phenotype to an M2 (angiogenic) phenotype (Pinhal-Enfield G, Ramanathan M, Hasko G, Vogel SN, Salzman AL, Boons GJ, Leibovich SJ. An angiogenic switch in macrophages involving synergy between Toll-like receptors 2, 4, 7, and 9 and adenosine A(2A) receptors. Am J Pathol 2003;163(2):711- 21). Thus, due to lack of A₂AR, macrophages in lesions maintain themselves in the Ml phenotype. Indeed, we found that NF-κΒ activation was enhanced in the lesion foam cells of A₂AR-/-/apoE-/- mice. In an in vitro assay, A_2AR deficient macrophages also exhibited increased NF-κΒ activation in response to ox-LDL. It is possible that ox-LDL may stimulate different receptors compared to minimally modified LDL and that the effects of these ligands might discriminate important differences between wild type and A₂AR deficient macrophages (Miller YI, Chang MK, Binder CJ, Shaw PX, Witztum JL. Oxidized low density lipoprotein and innate immune receptors. Curr Opin Lipidol 2003;14(5):437-45). Nevertheless, results from both in vivo and in vitro setups confirm the inflammatory phenotype of A₂AR-deficient macrophages and foam cells under atherosclerotic conditions. Contrary to the general concept that suppression of macrophage inflammatory reactions reduces atherosclerosis, inhibition of NF-κΒ activity by deletion of IKK2 decreases macrophage inflammatory phenotype, but enlarges atherosclerotic lesions (Kanters E, Pasparakis M, Gijbels MJ, Vergouwe MN, Partouns-Hendriks I, Fijneman RJ, Clausen BE, Forster I, Kockx MM, Rajewsky K, Kraal G, Hofker MH, de Winther MP. Inhibition of NF-kappaB activation in macrophages increases atherosclerosis in LDL receptor- deficient mice. J Clin Invest 2003;112(8): 1 176-85). Therefore, enhanced macrophage inflammatory phenotype may not directly lead to an increase in atherosclerotic lesion size.

The size of atherosclerotic lesions is directly related to the number of foam cells within the lesions, which is balanced by monocyte recruitment, macrophage apoptosis, and macrophage emigration from lesions. We found no significant difference in monocyte homing ability between wt and A_2AR-deficient monocytes. However, the number of macrophages in

atherosclerotic lesions of Apoe^_7A₂AR^{~ _} mice was less than that of Apoe^{_ _} mice. This led us to examine whether A₂AR deficiency induces macrophage apoptosis in atherosclerotic lesions.

Macrophage or foam cell apoptosis occurs during all stages of atherosclerosis and plays a different role in atherosclerosis depending on the stage at which it occurs. During late stages of atherosclerosis, apoptosis contributes to the formation of necrotic cores and to lesion vulnerability. In contrast, during the early stages of atherosclerosis, apoptosis decreases the number of foam cells and the size of atherosclerotic lesions. In lesions of Apoe^-/7A₂AR ^~/_ mice, most apoptoic cells were localized in the subendothelial space, indicating early apoptosis of foam cells. In response to oxLDL treatment, A₂AR-deficient macrophages exhibited increased p38 MAPK activation. This may result from a change in signaling associated with intracellular cAMP. Elevation of cAMP following A₂AR occupancy inhibits activation of p38 via the cAMP response element-binding protein-induced dynein light chain, and p38 activation has been linked to apoptosis. A recent study showed that p38 mediates caspase-3 activation and apoptosis in macrophages stimulated with ATP and H₂0₂. A₂AR-deficient macrophages challenged with modified LDL may utilize similar pathways, because the p38 inhibitor can inhibit caspase-3 activation and apoptosis. We have attempted to elucidate molecular mechanisms underlying the apoptosis of A₂AR-deficient macrophages, but we have yet to find a difference in the levels of Bcl-2, Bax, and Bcl-XL between wt and A₂AR-deficient macrophages.

Activation of A₂AR using agonists dramatically inhibits inflammation and protects against tissue injury. A₂AR activation protects against ischemia in the myocardium, kidney, liver, spinal cord, and brain. Additionally, administration of A₂AR agonists improves survival in mouse models of endotoxemia and sepsis, and attenuates inflammation and injury in

lipopolysaccharide-induced lung injury, diabetic nephropathy, and inflammatory bowel disease. A₂AR agonists inhibit foam cell formation and vascular remodeling after injury.

In summary, our data provide evidence that A₂A inactivation protects against atherosclerosis. A₂A deficiency increases p38 activation in macrophages and foam cells, and this modulation in signaling induces activation of caspase-3. The latter drives foam cells toward apoptosis, thus reducing the size of atherosclerotic lesions. This study suggests that A₂AR inactivation represents a new direction for anti-atherosclerotic therapies.

Although the foregoing specification and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto.

All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising,"

"having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is

incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein.

Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED IS:

1. A substance to inactivate A₂A receptors (A₂AR) on cells for the treatment or to inhibit the formation of atherosclerotic lesions.

2. The substance of claim 1, wherein the cells are bone-marrow-derived cells (BMDC).

3. A substance to inactivate A₂A on cells to induce apoptosis of foam cells.

4. The substance of any one of claims 1-3, wherein the substance is caffeine, ZM241385, or istradefylline (KW-6002).

5. The substance of any one of claims 1-3, wherein the compound is caffeine or

ZM241385.

6. The substance of claim 1 , wherein the substance is a compound of formula (I):

wherein: X is O or S;

Ri and R₂ are independently selected from hydrogen, alkyl, aryl, hydroxy, alkoxy, aryloxy, cyano, nitro, C0₂R₇, COR₇, OCOR₇, CONR₇R_fj, CONR₇NR₈R₉,

OCONR₇R«, NR₇R₉, NR₇CORs, NR₇CONRsR₉, NR₇C0₂R„, NR₇S0₂R₈,

NR₇CONRsNR₉R₁₀, NR₇NR,jC0₂R₉, NR₇NR8CONR₉Ri₀, NR₇S0₂ R« R₉, S0₂R₇, SOR₇, SR₇ and S0₂NR₇Rs, or R] and R₂ together form a carbonyl group (C=0), an oxime group (C=NORn), an imine group (C^NRn) or a hydrazone group

(C= NRnR₁₂), or R_\ and R₂ together form a 5, 6 or 7 membered carbocyclic or heterocyclic ring;

R₃ is alkyl or aryl;

R4, R₅ and R are independently selected from hydrogen, alkyl, aryl, halogen, hydroxy, nitro, cyano, alkoxy, aryloxy, COR₇, OCOR₇, C0₂R₇, SR₇, SOR₇, S0₂R₇, S0₂NR₇R8, CONR₇R8, CONR₇NR«R9, OCONR^, NR₇R_¾, NR₇CORg, NR₇CONRgR9, NR₇C0₂Rs, NR₇S0₂R8, CR₇=NOR₈, NR₇CONR NR₉R₁₀, NR₇NRsC0₂R₉,

NR₇NR8CONR₉R₁₀, S0₂NR₇NR8R₉, NR₇S0₂N¾R₉, NR₇NR₈S0₂R₉, NR₇NR₈COR₉, NR₇NRg R₉ and NR₇CSNR₈R₉, or R₅ and R^ together form a 5, 6 or 7 membered carbocyclic or heterocyclic ring; and

R₇, Rg, R₉, R₁₀, Rn and R₁₂ are independently selected from hydrogen, alkyl and aryl,

or a pharmaceutically acceptable salt thereof or prodrug thereof.

7. The substance of any one of claims 1-6, further comprising one or more additional anti- atherosclerotic compounds.

8. The substance of claim 7, wherein the one or more additional anti-atherosclerotic

compounds are caffeine, ZM241385, or istradefylline (KW-6002).

9. A composition comprising a compound to inactivate A_2AR for use in the treatment of atherosclerosis, wherein the compound to inactivate A_2AR is to be administered to a patient that has atherosclerotic lesions or is at risk for developing atherosclerotic lesions.

10. The composition of claim 9, wherein the compound to inactivate A_2A is caffeine,

ZM241385, or istradefylline (KW-6002).

11. The composition of claim 9, wherein the compound to inactivate A_2A is caffeine or ZM241385.

12. The composition of claim 9, wherein the compound to inactivate A_2AR is the compound of Formula (I) or a pharmaceutically acceptable salt thereof or prodrug thereof.

13. A method of inhibiting formation of atherosclerotic lesions by administering an effective dose of a compound to inactivate A_2AR in cells in a patient in need thereof.

14. The method of claim 13, wherein the cells are bone-marrow-derived cells (BMDC).

15. A method of inducing apoptosis of foam cells by administering an effective dose of a compound to inactivate A_2AR on cells in a patient in need thereof.

16. The method of any one of claims 13-15, wherein the compound is caffeine, ZM241385, or istradefylline (KW-6002).

17. The method of any one of claims 13-15, wherein the compound is the compound

Formula (I) or a pharmaceutically acceptable salt thereof or prodrug thereof.

18. The method of any one of claims 13-17, further comprising administering one or more additional anti-atherosclerotic compounds.

19. The method of claim 18, wherein the one or more additional anti-atherosclerotic

compounds are caffeine, ZM241385, or istradefylline (KW-6002).