WO1999043326A1

WO1999043326A1 - Use of a cardiac purinoceptor to effect cellular glucose uptake

Info

Publication number: WO1999043326A1
Application number: PCT/US1999/003881
Authority: WO
Inventors: Bruce T. Liang; Morris Birnbaum; Kendall Harden
Original assignee: The Trustees Of The University Of Pennsylvania
Priority date: 1998-02-24
Filing date: 1999-02-23
Publication date: 1999-09-02
Also published as: AU2781899A

Abstract

The invention provides a method of determining whether a compound is a glucose uptake modulator, including use of a cell comprising an isolated nucleic acid encoding a glucose uptake-enhancing myocardial purinoceptor. The invention also includes a method of enhancing glucose uptake into a cell such as a mammalian cardiac myocyte, a method of affecting the activity of an intracellular enzyme, and pharmaceutical compositions for use in these methods. The method of enhancing glucose uptake into a cell may be used to minimize ischemic cardiac damage, such as that attributable to angina pectoris, chronic stable angina, unstable angina, post-myocardial infarction angina, myocardial infarction, cardiac arrhythmia, coronary artery disease, diabetes mellitus, and cardiac ischemia attributable to schock, stress, or exertion.

Description

USE OF A CARDIAC PURINOCEPTOR TO EFFECT CELLULAR GLUCOSE UPTAKE

FIELD OF THE INVENTION The field of the invention is cellular glucose uptake and physiological processes and disorders associated therewith, such as diabetes and ischemia.

BACKGROUND OF THE INVENTION Cardiovascular diseases associated with diabetes account for a large proportion of the mortality and morbidity experienced by diabetic patients. For example, the association of diabetes mellitus with atherosclerosis is well known. Cardiomyopathy in diabetic patients often leads to contractile failure independent of coronary artery disease. Studies of diabetic animals indicate that most alterations in cardiac proteins and cardiac structures result from metabolic changes that occur independent of diseases of blood vessels, and that these alterations precede development of coronary artery disease. Diabetic patients exhibit abnormal systolic and diastolic contractile function (Litwin et al., 1990, J. Clin. Invest. 86:481-488; Davidoff et al., 1990, Hypertension Dallas 15:643-651; Fein et al., 1980, Circ. Res. 47:911-921; Zarich et al., 1989, Curriculum Cardiol. 118:1000-1012; Galderisi et al., 1991, Am. J. Cardiol. 68:85-89). The basis for this cardiac dysfunction is not understood. The incidence of heart failure is higher in both patients afflicted with insulin-dependent diabetes mellitus and patients afflicted with the non-insulin dependent form of the disease than among the population in general. Heart failure occurs more frequently in diabetic patients having coronary artery disease than in non- diabetic patients having the same degree of coronary artery disease (Kannel et al., 1974, Am. J. Cardiol. 34:29-34).

- 1 - Epidemiological studies indicate that diabetic patients experience greater mortality following myocardial infarction than non-diabetic patients having a similar extent of coronary artery disease (Stone et al., 1989, J. Am. Coll. Cardiol. 14:49-57; Smith et al., 1984, Am. J. Cardiol. 54:718-721). Together, these studies suggest that differences exist between myocardium of diabetic patients and myocardium of normal patients, and that these differences contribute to cardiovascular mortality and morbidity in diabetic patients.

Even among non-diabetic humans, cardiac ischemic disorders are a significant cause of mortality and morbidity. For example, acute myocardial infarction (a cardiac ischemic disorder) causes 35% of deaths of men between ages 35 and 50 in the United States.

Although cardiac myocytes derive most of their energy from catabolism of fatty acids, ATP derived from glucose utilization is a particularly important energy source for myocytes during periods of cardiac ischemia induced by stress or physical exertion (Weiss et al, 1989, J. Gen. Physiol. 94:911-935; Sebbag et al., 1996, Circ.

(Suppl.) 94:2140; Moret, 1980, In: Hearts and Heart-like Organs. Vol. 2, Bourne, Ed., Academic Press, New York, pp. 333-388)). Cardiac myocytes obtain glucose either by transporting glucose across the sarcolemma or by depolymerization of glycogen stored within individual myocytes. Glycolytically-derived ATP delays myocyte death during periods of ischemia (Sebbag et al., 1996, Circ. (Suppl.) 94:2140). Clinical and experimental studies have demonstrated that increased glucose uptake and metabolism by cardiac myocytes during periods of cardiac ischemia are associated with preserved myocardial function and improved recovery during reperfusion.

Cardiac myocytes express both the insulin-responsive glucose transporter GLUT4 and the GLUT1 glucose transporter, which is less responsive to insulin (Bell et al., 1990, Diabetes Care 13:198-208; Slot et al., 1991, Proc. Natl. Acad. Sci. USA 88:7815-7819). Because glucose transport across the sarcolemma is the rate- limiting step of basal glucose utilization in cardiac myocytes, enhanced glucose transport enables enhanced glucose utilization. For example, providing insulin to a

2 - cardiac myocyte enhances glucose uptake effected by GLUT4, enabling enhanced glucose utilization (Manchester et al., 1994, Am. J. Physiol. 266:E326-333).

Mice that have been engineered such that they do not express functional GLUT4 have grossly underdeveloped hearts (Katz et al., 1995, Nature 377:151-155). It was hypothesized that GLUT4 deficiency in these engineered mice causes low energy supply in cardiomyocytes, leading to myocardial hypertrophy and dysfunction. Rats which exhibit diabetes induced by administration of streptozotocin to the rats (hereinafter, "streptozotocin-induced diabetic rats" or "SID rats") are a commonly used model of insulin-dependent diabetes in humans. SID rats exhibit systolic and diastolic contractile dysfunction similar to that exhibited by diabetic humans. In both SID rats and diabetic humans, this dysfunction is manifested as decreased peak twitch contractile amplitude and increased contraction and relaxation time (Ren et al., 1997, Am. J. Physiol. H148-158). Lower GLUT4 activity is observed in hearts obtained from SID rats than in hearts obtained from normal rats (Hall et al., 1996, Am. J. Physiol. 271 :H2320-2329; Gerrits et al., 1993, J. Biol. Chem. 268:640-644). Hearts obtained from Yucatan micropigs which exhibit diabetes induced by administration of streptozotocin (hereinafter, "SID micropigs") also exhibit lower GLUT4 activity than hearts obtained from normal micropigs (Hall et al., 1996, Am. J. Physiol. 271 :H2320- 2329; Gerrits et al., 1993, J. Biol. Chem. 268:640-644). These observations suggest that dysfunctional glucose transport in the heart can contribute to development of cardiomyopathy.

SID rats have been used as a model for insulin-dependent diabetes mellitus (Ren et al., 1997, Am. J. Physiol. H148-158; Hall et al., 1996, Am. J. Physiol. 27 H2320-2329; Gerrits et al., 1993, J. Biol. Chem. 268:640-644). The Zucker Diabetic Fatty (ZDF) rat can be used as a model for diabetes, and is believed to exhibit a phenotype which closely resembles the onset, course, and symptoms of non-insulin- dependent diabetes mellitus in humans (Peterson et al., 1990, Inst. Lab. Anim. Res.

News. 32:16-19; Friedman et al., 1991, Am. J. Physiol. (Endocrinol. ):E782-

788).

- 3 - In cardiac myocytes, skeletal muscle cells, and adipocytes, insulin stimulates cellular uptake and storage of glucose by promoting translocation of GLUT4 from intracellular sites of sequestration to the plasma membrane (Pressin et al., 1992, Annu. Rev. Physiol. 54:911-930). Mediators other than insulin also augment glucose uptake by cardiac myocytes by enhancing the activity of protein kinase C (PKC) or by stimulating one or more of the G_j- and G -coupled receptors (Kish et al., 1996, J. Biol. Chem. 271:26561-26568; Todaka et al., 1996, Biochem. J. 315:875-882). The receptor(s), if any, at which these non-insulin mediators exert their effect on glucose uptake have not been characterized. Furthermore, the ability of these non-insulin mediators to stimulate glucose transport in vivo in cardiac myocytes has not been described. It is known that not all G -coupled receptors are able to stimulate glucose uptake. For example, stimulation of the muscarinic cholinergic receptor is not coupled with enhanced glucose uptake. The physiological significance of these non-insulin mediators is unknown. Recently, another protein has been identified which resides in the same intracellular compartment as GLUT4 in muscle and adipocytes. This protein is alternately designated NP165 or insulin-responsive aminopeptidase (IRAP), and translocates in response to insulin (Kandror et al., 1994, Proc. Νatl. Acad. Sci. USA 91:8017-8021; Mastick et al., 1994, J. Biol. Chem. 269:6089-6092). IRAP co-localizes with GLUT4 in tubulovesicular structures in the heart (Martin et al., 1997, J. Cell Sci.

110:2281-2291). Unlike GLUT4, surface IRAP is easily biotinylated in intact tissue culture cells, allowing quantitation of the abundance of cell surface IRAP (Ross et al., 1997, Biochem. Biophys. Res. Commun. 239:247-251).

Adenosine 5'-triphosphate (ATP) is a ubiquitous regulatory agent that is released as a signaling molecule into the extracellular space surrounding cardiac myocytes under both physiologic and pathophysiologic conditions (Swain, 1986, In: The Heart and Cardiovascular System. Fozzard et al., ed., Raven Press.; Borst et al., 1991, Circ. Res. 68:797-806). The sources of extracellular cardiac ATP appear to be multiple. Extracellular cardiac ATP is released from sympathetic nerve endings in the myocardium and from platelets, endothelium, and possibly from hypoxic myocardium

- 4 - (Pearson et al., 1979, Nature 281:384-386; Clemens et al., 1980, J. Physiol. (London) 312:143-158; Forrester et al., 1977, J. Physiol. (London) 268:371-390; Fredholm et al, 1982, Acta Physiol. Scand. 116:285- 295; Vial et al., 1987, J. Mol. Cell Cardiol. 19:187-197).

Extracellular ATP, acting at one or more of the purinergic P2 receptors, stimulates hydrolysis of phosphatidylinositol-(4,5)bisphosphate and increases cardiomyocyte contractility (Danziger et al., 1988, Cell Calcium 9:193-199; Scamps et al., 1990, Circ. Res. 67:1007-1016; DeYoung et al., 1989, Am. J. Physiol. 257:C750- C758). The nucleotide-activated P2 receptors are distinct from purinergic Pi receptors of myocytes, which are activated by adenosine. The P2 receptors have been classified into the P2X and P2Y subtypes (Bumstock et al., 1996, Drug Develop. Res. 38:67-71). The following table outlines the order of potency of the various agonists for each subtype.

Table 1. Efficacy and Potency of P2 Purinoceptor Agonists.

15 Purinoceptor Relative Efficacy and Potency of Agonists

P2X! 2-MeSATP² > ATP; α,β-meATP³ is active

P2X₂ 2-MeSATP > ATP; α,β-meATP is inactive

P2X₃ 2-MeSATP > ATP ;α,β-meATP is active

P2X₄ ATP > 2-MeSATP; α,β-meATP is inactive

20 P2X₅ ATP > 2-MeSATP > ADP⁴; α,β-meATP is inactive

P2X₆ ATP > 2-MeSATP > ADP; ,β-meATP is inactive

P2X₇ BzATP⁵ > ATP > 2-MeSATP >ADP » UTP

Table 1 (continued)

P2Y_j 2-MeSADP > ADP > 2-MeSATP; UTP and α,β-meATP are inactive

P2Y₂ ATP = UTP = UTPγS⁷ > Ap₄A⁸; ADP and UDP are inactive

P2Y₆ UDPβS⁹ > UDP » (UTP, ATP, and ADP)

P2Y₄ UTP » ATP; UDP is inactive

P2Y_n ATP> 2-MeSATP » ADP;

UTP and UDP are inactive

Notes: Relative Efficacy and Potency of Agonists refers to the maximum response effected upon administration of the agonist and the concentration of agonist that effects half-maximal effect, respectively.

² 2-MeSATP is 2-methylthio ATP.

³ α,β-meATP is α,β-methylene ATP. ADP is Adenosine 5'-diphosphate.

⁵ BzATP is 2',3*-(O)-(4,benzoyl benzoate) ATP.

⁶ 2-C1-ATP is 2'-chloro ATP.

⁷ UTPγS is gamma-thio UTP.

⁸ A_D A ^S diadenosine tetraphosphate

⁹ UDPβS is beta-thio UDP.

Several of the P2X and P2Y receptors which have been cloned can be blocked using antagonists such as suramin, reactive blue-2 (RB-2), and pyridoxal- phosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium (PPADS; Bumstock et al., 1996, Drug Dev. Res. 38:67-71; Harden et al., 1995, Annu. Rev. Pharmacol. Toxicol. 35:541-579). However, none of these molecules are effective antagonists of P2X₄ or P2X₆ receptors (Bumstock et al., 1996, Drug Dev. Res. 38:67-71; CoUo et al., 1996, J. Neurosci. 16:2495-2507). For example, suramin and PPADS inhibit the rat P2X₄ receptor with an IC^Q value of 178 micromolar (Garcia-Guzman et al., 1997, Mol. Pharmacol. 51:109-118). The resistance of the P2X₄ and P2Xg receptors to suramin, RB-2, and PPADS distinguishes these two receptors from other P2X receptors and

- 6 - from P2Y receptors. Adenosine 3',5'-diphosphate (A3P5P) and adenosine 3',5'- diphosphosulfate (A3P5PS) are selective antagonists of the P2Y_j receptor, and exhibit little or no antagonist activity at any other P2Y receptor (Boyer et al., 1996, Mol. Pharmacol. 50:1323-1329). There has heretofore been insufficient understanding of the myocardial differences between diabetic and non-diabetic patients and the role of abnormal glucose transport in development of diabetic cardiomyopathy. As a result, design of therapeutic agents for treatment of diabetic heart disease has been hindered. The teachings of the present invention permit this hindrance to be overcome, both by providing methods to identify such therapeutic agents and by providing therapeutic agents and methods of using them in both diabetic and non-diabetic mammals.

BRIEF SUMMARY OF THE INVENTION The invention relates to a method of enhancing glucose uptake into a mammalian myocyte. This method comprises contacting the myocyte and an agonist of a glucose uptake-enhancing myocardial purinoceptor. Glucose uptake into the myocyte is thereby enhanced. The agonist and the myocyte may, for example, be contacted by providing (e.g. intravenously) a pharmaceutical composition comprising the agonist to the mammal. The agonist may alternatively be provided together with another active ingredient such as one or more of glucose, potassium, and insulin. The mammal to which the pharmaceutical composition is administered may, for example, be one experiencing cardiac ischemia (e.g. a human afflicted with a disorder selected from the group consisting of angina pectoris, chronic stable angina, unstable angina, post- myocardial infarction angina, myocardial infarction, cardiac arrhythmia, coronary artery disease, diabetes mellitus, and cardiac ischemia attributable to shock, stress, or exertion) or a mammal at risk for experiencing cardiac ischemia (e.g. a human who is imminently to undergo surgery or one who is afflicted with one of the above-mentioned disorders).

The myocyte with which the agonist is contacted is preferably a cardiac myocyte such as a P2 Y₂ purinoceptor or a P2 Y₄ purinoceptor. The agonist may, for

- 7 - example, be selected from the group consisting of ATP, UTP, ATPγS, UTPγS, diadenosine tetra-phosphate, a diuridine polyphosphate, a base-substituted UTP analog, a ribose-substituted UTP analog, and a phosphate-substituted UTP analog. Preferably, the analog is UTP or diadenosine tetra-phosphate. The invention also relates to a method of enhancing glucose uptake into a mammalian cell. This method comprises providing a glucose uptake-enhancing myocardial purinoceptor to the cell, whereby glucose uptake into the cell is enhanced. The purinoceptor may be provided to the cell, for example, by providing an expression vector to the cell, wherein the expression vector comprises a nucleic acid encoding the purinoceptor. Of course, an agonist of the purinoceptor may also be provided to the cell after providing the expression vector to the cell.

The invention further relates to a method of minimizing ischemic cardiac damage in a mammal afflicted with a cardiac ischemic disorder. This method comprises administering to the mammal a pharmaceutical composition comprising an agonist of a glucose uptake-enhancing myocardial purinoceptor in an amount sufficient to enhance glucose uptake into cardiac myocytes of the mammal. Cardiac damage is thereby minimized.

The invention further relates to a method of minimizing ischemic cardiac damage in a mammal at risk for experiencing cardiac ischemia. This method comprises administering to the mammal a pharmaceutical composition comprising an agonist of a glucose uptake-enhancing myocardial purinoceptor in an amount sufficient to enhance glucose uptake into cardiac myocytes of the mammal. Cardiac damage is πiinimized in the event of cardiac ischemia.

In another aspect, the invention relates to a method of maintaining the viability of a cardiac cell extracted from a mammal. This method comprises contacting the cell with a composition comprising glucose and an agonist of a glucose uptake- enhancing myocardial purinoceptor after extracting the cell from the mammal. The viability of the cell is thereby maintained.

The invention also relates to a method of increasing the level of activity of an enzyme in a mammalian cell. This method comprises contacting the cell with an

- 8 - agonist of a glucose uptake-enhancing myocardial purinoceptor. The level of activity of the enzyme in the cell is thereby increased. The enzyme may, for example, be selected from the group consisting of phospholipase C, protein kinase C, and phosphatidylinositol 3-kinase. The invention further relates to a method of effecting transmembrane translocation of a protein in a mammalian cell. This method comprises contacting the cell with an agonist of a glucose uptake-enhancing myocardial purinoceptor. Transmembrane translocation of the protein is thereby effected. The protein may, for example, be selected from the group GLUT1, GLUT4, and IRAP, whereby In still another aspect, the invention relates to a method of determining whether a test compound is a modulator of a glucose uptake-enhancing myocardial purinoceptor. This method comprises incubating a cell comprising the purinoceptor in the presence of a glucose analog and in the presence or absence of the test compound; and assessing uptake of the analog into the cell. A difference between uptake of the analog into the cell in the presence of the test compound and uptake of the analog into the cell in the absence of the test compound is an indication that the test compound is a modulator of the purinoceptor. The purinoceptor may, for example, be selected from the group consisting of a P2 Y purinoceptor and a P2Y₄ purinoceptor, and the glucose analog may, for example, be selected from the group consisting of glucose and 2- deoxyglucose. The cell may be substantially any cell, but is preferably one selected from the group consisting of a mammalian myocyte, a rat cardiac ventricular myocyte, a cultured human myocyte, a myocyte obtained from a Zucker Diabetic Fatty rat, a myocyte contained within a Zucker Diabetic Fatty rat, a myocyte obtained from a streptozotocin-induced diabetic rat, a myocyte contained within a streptozotocin- induced diabetic rat, a myocyte obtained from a streptozotocin-induced diabetic micropig, a myocyte contained within a streptozotocin-induced diabetic micropig, and a transformed mammalian cell comprising an isolated nucleic acid encoding the purinoceptor.

9 - The invention also relates to a transformed mammalian cell comprising an isolated nucleic acid encoding a mammalian glucose uptake-enhancing myocardial purinoceptor such as a P2Y₂ purinoceptor or a P2Y₄ purinoceptor. The cell may, for example, be selected from the group consisting of a cardiac myocyte, a cardiac ventricular myocyte, a rat cardiac ventricular myocyte, a human myocyte, a human skeletal myocyte, a human cardiac myocyte, and a human cardiac ventricular myocyte.

The invention further relates to a pharmaceutical composition comprising an agonist of a mammalian glucose uptake-enhancing myocardial purinoceptor and at least one additional ingredient, such as one selected from the group consisting of insulin, potassium, and glucose.

The invention still further relates to the use of an agonist of a mammalian glucose uptake-enhancing myocardial purinoceptor for preparation of a pharmaceutical composition for minimizing cardiac damage attributable to cardiac ischemia.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a bar graph which depicts uptake of 2-deoxyglucose into isolated rat cardiac ventricular myocytes. The data corresponding to bar "2" relate to experiments in which basal glucose uptake from medium which did not comprise insulin was assessed. The data corresponding to bar "3" relate to experiments in which glucose uptake from medium which comprised 300 nanomolar insulin was assessed. The data corresponding to bar " 1 " relate to experiments in which glucose uptake from medium which comprised 10 micromolar cytochalasin B was assessed.

Figure 2 is a bar graph which depicts the amount of inositol phosphate detected in isolated rat cardiac ventricular myocytes incubated in various media. The media used were: for the experiments corresponding to bar "1 ," non-supplemented medium; for the experiments corresponding to bar "2," medium supplemented with 10 micromolar ATP; for the experiments corresponding to bar "3," medium supplemented with 10 micromolar UTP; for the experiments corresponding to bar "4," medium supplemented with 10 micromolar 2-methylthio adenosine 5'-triphosphate (2-

- 10 - MeSATP); and for the experiments corresponding to bar "5," medium supplemented with 10 micromolar α,β-methylene-ATP.

Figure 3 depicts the magnitude of contractions exhibited by isolated cardiac myocytes before and after addition of 10 micromolar ATP to the assay mixture. Figure 4 depicts the magnitude of contractions exhibited by isolated cardiac myocytes before and after addition of 1 micromolar 2-MeSATP and before and after addition of 10 micromolar 2-MeSATP to the assay mixture.

Figure 5 is a bar graph which depicts uptake of 2-deoxyglucose into isolated rat cardiac ventricular myocytes. The data corresponding to bar "I" relate to experiments in which 2-deoxyglucose uptake from medium which did not comprise insulin or 2-MeSATP was assessed. The data corresponding to bars "2" and "3" relate to experiments in which 2-deoxyglucose uptake from medium which comprised 1 micromolar and 10 micromolar, respectively, 2-MeSATP was assessed. The data corresponding to bar "4" relate to experiments in which 2-deoxyglucose uptake from medium which comprised 100 nanomolar insulin was assessed. The data corresponding to bar "5" relate to experiments in which 2-deoxy glucose uptake from medium which comprised 100 nanomolar insulin and 10 micromolar 2-MeSATP was assessed.

Figure 6 is a bar graph which depicts uptake of 2-deoxyglucose into isolated rat cardiac ventricular myocytes. The data corresponding to bar " 1 " relate to experiments in which basal 2-deoxyglucose uptake from medium which did not comprise UTP was assessed. The data corresponding to bars "2," "3," and "4" relate to experiments in which 2-deoxyglucose uptake from medium which comprised 1 micromolar, 10 micromolar, and 100 micromolar, respectively, UTP was assessed. Figure 7 is a bar graph which depicts uptake of 2-deoxyglucose into isolated rat cardiac ventricular myocytes. The data corresponding to bar "V relate to experiments in which basal 2-deoxyglucose uptake from medium which did not comprise UTP or insulin was assessed. The data corresponding to bar "2" relate to experiments in which basal 2-deoxyglucose uptake from medium which comprised 100 nanomolar insulin was assessed. The data corresponding to bar "2" relate to

- 11 - experiments in which basal 2-deoxyglucose uptake from medium which comprised 100 nanomolar insulin and 10 micromolar UTP was assessed.

Figure 8 is a bar graph which depicts uptake of 2-deoxyglucose uptake by isolated rat cardiac ventricular myocytes in the presence and absence of diadenosine tetraphosphate. The concentration of diadenosine tetraphosphate was 1 micromolar in the experiments corresponding to the bar labeled "2" and 10 micromolar in the experiments corresponding to the bar labeled "3." The experiments corresponding to the bar labeled " 1 " were performed in the absence of diadenosine tetraphosphate. These results are typical of three sets of experiments; error bars represent the standard error.

Figure 9 is a graph which depicts results of an experiment described herein which establishes the pre-ischemic conditioning ability of UTP on cultured chick ventricular myocytes. The data presented in this graph represent the percentage of myocytes killed by simulated ischemia following pre-treatment with the indicated concentration of UTP. The data are representative of four sets of experiments; error bars represent the standard error.

Figure 10 is a bar graph which depicts the results of an experiment described herein which establishes the cardioprotective effect of UTP even after the onset of ischemia. Cultured chick ventricular myocytes were incubated under simulated ischemic conditions in the presence (bar "2") or absence (bar " 1 ") of 10 micromolar UTP, and the percentage of myocytes killed by 90 minutes incubation under these conditions was assessed. The data are representative of three sets of experiments; error bars represent the standard error.

Figure 11 is a bar graph which depicts the results of an experiment described herein which established that pre-treatment of isolated adult rat ventricular myocytes with 10 micromolar UTP prior to simulated ischemia reduced the amount of creatine kinase ("CK") released by the myocytes during ischemia (represented by the data corresponding to bar "2"), relative to CK release by myocytes not pre-treated with UTP prior to simulated ischemia (represented by the data corresponding to bar "1").

- 12 - The data are representative of three sets of experiments; error bars represent the standard error.

DETAILED DESCRIPTION The invention relates to the discovery that certain P2Y purinoceptors, namely the P2 Y₂ receptor and the P2Y₄ receptor, are involved in regulating cardiac glucose transport in cardiac myocytes. These purinoceptors are herein designated glucose uptake-enhancing myocardial purinoceptors (GUEMPs).

This invention also relates to the discovery that agonists of GUEMPs can minimize or prevent damage to, or death of, myocytes which express GUEMPs. The invention thus includes methods and pharmaceutical compositions which are useful for minimizing or preventing ischemic cardiac damage, such as that attributable to angina pectoris, chronic stable angina, unstable angina, post-myocardial infarction angina, myocardial infarction, coronary artery disease, or diabetes mellitus. Definitions As used herein, the following terms have the meanings defined herein.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

A "glucose uptake-enhancing myocardial purinoceptor" ("GUEMP") is a purinoceptor which is normally expressed by a cardiac myocyte of a mammal and which, in the presence of an agonist of the GUEMP, enhances glucose uptake into the myocyte.

A protein is "normally expressed" by a cell if a naturally occurring form of the cell expresses the protein. As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property by which it is characterized. A functional enzyme, for example, is one which exhibits the characteristic catalytic activity by which the enzyme is characterized.

13 A GUEMP "enhances" glucose uptake into a cell if, in the presence of a GUEMP agonist, either the rate or the amount of glucose uptake into the cell is greater when the cell comprises the GUEMP than when the cell does not comprise the GUEMP. A "GUEMP agonist" is a compound which, when contacted with a cell which comprises a GUEMP, enhances glucose uptake into the cell.

A cell comprises an "isolated nucleic acid" if the cell comprises a nucleic acid, such as a DNA, an RNA, or a fragment of one of these, which has been separated from the sequences which flank it in a naturally occurring state. By way of example, an isolated nucleic acid may be a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, such as the sequences adjacent to the fragment in a genome in which it naturally occurs. "Isolated nucleic acid" also includes a nucleic acid which has been substantially purified from other components which naturally accompany the nucleic acid. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or vims; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule such as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion, independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequences.

A nucleic acid is "normally present" in a cell if a naturally occurring form of the cell comprises the nucleic acid.

A cell is "transformed" with an isolated nucleic acid if, after the isolated nucleic acid is provided to the interior of the cell (e.g. using a electroporation method or a gene vector such as a plasmid or a virus vector), the cell expresses a gene product

(i.e. an RNA or a protein) encoded by the isolated nucleic acid.

The "viability" of a cardiac myocyte obtained from a mammal refers to either or both of the ability of the myocyte to remain alive after it is extracted from the mammal and its ability to resume its contractile function after implantation into a second mammal.

- 14 - A "glucose analog" means glucose or any compound which can be recognized by a glucose uptake protein of a cell and transported into the cell by that protein. By way of example, glucose and 2-deoxyglucose are glucose analogs.

The term "pharmaceutically acceptable carrier" means a chemical composition with which the active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject.

The term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition and which is not deleterious to the subject to which the composition is to be administered.

A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

A "cardiac ischemic disorder" is a disease or disorder in a mammal which causes, results from, or has as a symptom, a deficiency between the amount of oxygen supplied to one or more cardiac myocytes of the mammal and the metabolic need of the myocyte(s) for oxygen. Examples of cardiac ischemic disorders include, but are not limited to, angina pectoris, chronic stable angina, unstable angina, post- myocardial infarction angina, myocardial infarction, coronary artery disease, or diabetes mellitus.

A mammal is "at risk for experiencing cardiac ischemia" if it is foreseeable that the mammal will experience cardiac ischemia. Examples of such mammals include, but are not limited to, mammals afflicted with a cardiac ischemic disorder, mammals which are imminently to undergo surgery involving stopping contractions of the mammal's heart or occluding blood flow to all or part of the myocardium, and mammals which are experiencing cardiac ischemia.

15 A mammal is "imminently to undergo surgery" if the mammal is scheduled to undergo a surgical operation within the ensuing 24 hours and if cardiac ischemia is a known or foreseeable complication of the surgery. The surgical operation may be one which involves incising and opening the skin of the mammal or one which is performed using minimally invasive devices (e.g. intravenous or intraarterial catheters). By way of example, a human patient scheduled to undergo coronary bypass surgery or open heart surgery within the ensuing 24 hours and a human patient being prepared for emergency coronary angioplasty are mammals imminently to undergo surgery. Description

The invention includes a method of enhancing glucose uptake into a mammalian myocyte. The method comprises contacting the myocyte with an agonist of a GUEMP. By providing the agonist to the myocyte, glucose uptake into the myocyte is enhanced. Enhancing glucose uptake by mammalian myocytes is useful as a therapeutic modality for diseases and disorders related to undesirable myocyte ischemia. Such diseases and disorders include, but are not limited to, ischemic cardiac damage, such as that attributable to angina pectoris, chronic stable angina, unstable angina, post-myocardial infarction angina, myocardial infarction, coronary artery disease, or diabetes mellitus. Application of the method to a mammalian cardiac myocyte is contemplated, as is application of the method to a skeletal myocyte.

The agonist may be provided to the myocyte to enhance basal glucose uptake or to enhance insulin-dependent glucose uptake. The agonist may be an agonist of any GUEMP, although an agonist of either the P2Y₄ purinoceptor or, preferably, the P2Y₂ purinoceptor is preferred. By way of example, the agonist may be ATP, UTP, diadenosine tetraphosphate (AP4A), adenosine 5'-(γ-thio)triphosphate (ATPγS), uridine 5'-(γ-thio)triphosphate (UTPγS), or potential synthetic molecules such as diuridine polyphosphates or various base-, ribose-, or phosphate- substituted analogs of UTP. Of these, AP4A and UTP are preferred. The mammal may be any mammal, but is preferably a human for therapeutic uses or a research mammal such as a rat or a micropig for screening or other testing purposes (e.g. pre-clinical testing). When the

- 16 - mammal is a human, it is preferably a human afflicted with a cardiac ischemic disorder (e.g. coronary artery disease or angina pectoris), a human afflicted with diabetes mellitus, a human who is experiencing or has experienced an acute cardiac ischemic event (e.g. a myocardial infarction), or a human imminently to undergo surgery (e.g. a human scheduled to undergo cardiac bypass surgery within the next 10 minutes to 24 hours). When the agonist is administered to a human patient afflicted with a cardiac ischemic disorder, it may be administered either while the patient is experiencing the disorder or to a patient at risk for experiencing the disorder (e.g. prior to a period of strenuous exercise to be undertaken by a patient afflicted with chronic stable angina). For example, the invention includes a method of minimizing ischemic cardiac damage associated with diabetes in a mammal afflicted with diabetes. The method comprises administering to the mammal a composition comprising an agonist of a GUEMP. Administration of the GUEMP enhances glucose uptake into cardiac myocytes of the mammal, thereby minimizing ischemic cardiac damage. The invention also includes a method of minimizing ischemic cardiac damage associated with an acute ischemic cardiac event in a mammal. The method comprises administering to the mammal a composition comprising an agonist of a GUEMP. Administration of the GUEMP enhances glucose uptake into cardiac myocytes of the mammal, thereby minimizing ischemic cardiac damage. Acute ischemic cardiac events include any physiological conditions under which a cardiac tissue of a mammal receives an insufficient supply of oxygen. Such events include, but are not limited to, myocardial infarction, ischemia associated with cardiac arrhythmia, angina pectoris, temporary cardiac ischemia, and ischemia attributable to shock, stress, or exertion. GUEMP agonists may also be administered (e.g. intravenously) to enhance normal (i.e. non-diseased) human cardiovascular performance for a brief or extended period (e.g. during an athletic competition or a military operation).

The method of enhancing glucose uptake into a mammalian cell by providing a GUEMP agonist to the cell may be performed by administering a pharmaceutical composition to the mammal. The pharmaceutical composition may be administered to the mammal by substantially any route which will result in contact of

- 17 - the cell and the agonist. By way of example, providing the agonist intravenously permits the agonist to reach a cardiac myocyte by way of the blood stream. The composition may also be administered topically to the myocardium (e.g. during open- chest surgery or using an endoaortic catheter). The route by which the agonist is administered to the cell is not critical, although it is understood that certain routes of administration are less efficient than others. Selection of a route of administration is within the skill of the ordinary artisan, once the particular agonist and the anatomical location of the cell to which the agonist is desired to be provided are identified.

The invention encompasses the preparation and use of medicaments and pharmaceutical compositions comprising a GUEMP agonist as an active ingredient.

Such a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. Administration of one of these pharmaceutical compositions to a subject is useful for enhancing glucose uptake into a cell (e.g. a cardiac myocyte) in the subject, as described elsewhere in the present disclosure. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to mammals of all sorts.

- 18 - Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various mammals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, and mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. Other contemplated formulations include projected nanoparticles and liposomal preparations.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.

As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and

100% (w/w) active ingredient.

In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents. Particularly contemplated additional agents include glucose, potassium, and insulin.

19 Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion. As used herein, an "oily" liquid is one which comprises a carbon- containing liquid molecule and which exhibits a less polar character than water.

A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surface active agents include, but are not limited to, sodium lauryl sulphate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, com starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known

20 lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U.S. Patents numbers 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use. Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid

21 paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol. Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

- 22 Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in- water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents. A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.

Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e. about 20 °C) and which is liquid at the rectal temperature of the subject (i.e. about 37°C in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations may further

23 comprise various additional ingredients including, but not limited to, antioxidants and preservatives.

Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for vaginal administration. Such a composition may be in the form of, for example, a suppository, an impregnated or coated vaginally-insertable material such as a tampon, a douche preparation, or a solution for vaginal irrigation.

Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e. such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, douche preparations may be administered using, and may be packaged within, a delivery device adapted to the vaginal anatomy of the subject. Douche preparations may further comprise various additional ingredients including, but not limited to, antioxidants, antibiotics, antifungal agents, and preservatives.

As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through

24 the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular, or intrasternal injection and intravenous, intraarterial, or kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous admimstration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules, in multi-dose containers containing a preservative, or in single-use devices for auto-injection or injection by a medical practitioner. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3 -butane diol, for example. Other acceptable diluents and

25 solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally- administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low- boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter

- 26 - greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0J to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration preferably have an average diameter in the range from about 0J to about 200 nanometers.

The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the invention. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered in the manner in which snuff is taken i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

27 - Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may further comprise one or more of the additional ingredients described herein. A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle or droplet size in the range from about 0J to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a OJ-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops may further comprise buffering agents, salts, or one or more other of the additional ingredients described herein. Other ophthalmalmically- administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents,

- 28 - demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences.

Mack Publishing Co., Easton, PA, which is incorporated herein by reference.

A pharmaceutical composition of the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. Alternatively, the dosage may be expressed in terms of an amount sufficient to effect a concentration of at least about 0.01 micromolar, and preferably at least about 0J micromolar, in a cardiac tissue of the subject (e.g. left or right ventricle muscle tissue). The dosage is preferably one which is sufficient to effect a concentration of the active agent of from about 0J micromolar to about 10 micromolar in a cardiac tissue of the subject throughout the period during which cardiac ischemia is experienced or anticipated. It is understood that the ordinarily skilled physician or veterinarian will readily determine and prescribe an effective amount of the compound to enhance glucose uptake into a cell in the subject. In so proceeding, the physician or veterinarian may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. It is further understood, however, that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the severity of the ischemia or glucose deficit being n inimized or prevented. Another aspect of the invention relates to a kit comprising a pharmaceutical composition of the invention and an instructional material. As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which is used to communicate the usefulness of the pharmaceutical composition of the invention for enhancing glucose uptake into a cell in

29 a subject or for administering such a composition via an intravenous route. The instructional material may also, for example, describe an appropriate dose of the pharmaceutical composition of the invention. The instructional material of the kit of the invention may, for example, be affixed to a container which contains a pharmaceutical composition of the invention or be shipped together with a container which contains the pharmaceutical composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the pharmaceutical composition be used cooperatively by the recipient. The invention also includes a kit comprising a pharmaceutical composition of the invention and a delivery device for delivering the composition to a subject. By way of example, the delivery device may be a squeezable spray bottle, a metered-dose spray bottle, an aerosol spray device, an atomizer, a dry powder delivery device, a self-propelling solvent/powder-dispensing device, a syringe, a needle, a tampon, or a dosage measuring container. The kit may further comprise an instructional material as described herein.

The invention also includes a method of enhancing glucose uptake into a mammalian cell, the method comprising providing a GUEMP to the cell. By providing the GUEMP to a cell, the cell is better able to obtain glucose from the medium in which it is located. Enhancement of cellular glucose uptake mediated by the GUEMP may be effected by a physiological agonist of the GUEMP or by an agonist identified using the screening method of the invention, for example. The GUEMP may be provided to the cell in the form of a protein, for example, embedded in a liposome or another membrane-like structure, or in the form of a nucleic acid, such as an expression vector encoding the GUEMP. The cell may be substantially any mammalian cell, but is preferably a myocyte, more preferably a cardiac myocyte of a human afflicted with a cardiac ischemic disorder.

The invention also includes a screening method of determining whether a test compound is a glucose uptake modulator. This screening method comprises incubating a cell in the presence of a glucose analog and further in the presence or

- 30 - absence of the test compound, wherein the cell comprises a glucose uptake-enhancing myocardial purinoceptor (GUEMP). Uptake of the glucose analog into the cell is subsequently assessed. A difference between uptake of the analog into the cell in the presence of the test compound and uptake of the analog in the absence of the test compound is an indication that the test compound is a glucose uptake modulator. Thus, if glucose uptake is greater in the presence of the test compound than in the absence of the test compound, then the test compound enhances glucose uptake into the cell.

The glucose analog used in the screening method of the invention may be glucose, 2-deoxyglucose, a radioactively labeled analog, or any other compound which can be recognized by a glucose uptake protein of a cell. If the cell is incubated in the presence of both a glucose analog and a known glucose uptake modulator such as, for example, insulin, then the screening method may be used to determine whether the test compound potentiates the effects of the known glucose uptake modulator on cellular glucose uptake. By way of example, if the known glucose uptake modulator is insulin, and if cellular glucose uptake is greater in the presence of the test compound and insulin, compared with cellular glucose uptake in the presence of insulin alone, then the test compound potentiates the glucose uptake effect of insulin.

The GUEMP used in the screening method of the invention may be any purinoceptor capable of modulating glucose uptake in cardiac myocytes. By way of example, the GUEMP may be a P2Y₂ purinoceptor or a P2 Y₄ purinoceptor. Preferably, the GUEMP is a P2Y purinoceptor.

The cell used in the screening method of the invention may be any cell which either naturally expresses the GUEMP or has been engineered to express the GUEMP. Cells which naturally express GUEMPs include, but are not limited to, mammalian myocytes, rat cardiac ventricular myocytes, and human cardiac ventricular myocytes. The cell used in the screening method of the invention can be a cell which is obtained from or which is contained within a research animal such as, for example, a ZDF rat, an SID rat, or an SID micropig, as described herein.

The invention also includes a cell comprising an isolated nucleic acid encoding a GUEMP. Such a cell is useful in the screening method of the invention.

- 31 - Methods of providing an isolated nucleic acid to a cell and expressing that nucleic acid within the cell are known in the art and are not described herein in detail. Nucleic acids encoding the each of the P2Y₂ receptor and the P2Y₄ receptor have been described, and the deduced amino acid sequence of each of these receptors has also been described (Chen et al., 1996, Endocrinology 137:1833-1840; Parr et al, 1994, Proc.

Natl. Acad. Sci. U.S.A. 91:3275-3279; Lustig et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5113-5117; Communi et al., 1995, J. Biol. Chem. 270:30849-30852; Nguyen et al., 1995, J. Biol. Chem. 270:30845-30848). Methods of making and manipulating a nucleic acid and methods of providing a nucleic acid to a cell are well known in the art, and are not described herein in detail (see, e.g. Sambrook et al., 1989,

Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York; Ausubel et al., 1993, Current Protocols in Molecular Biology. Green & Wiley, New York).

The cell to which the isolated nucleic acid encoding the GUEMP is provided may be any cell. However, it is believed that delivery of the isolated nucleic acid to a myocyte in vitro or ex vivo using, for example, a gene therapy approach is useful to enable prevention of damage to the myocyte associated with insufficient glucose uptake. Similarly, delivery of the isolated nucleic acid to a myocyte in vivo using a viral or non- viral vector is useful to enable prevention of damage to the myocyte associated with insufficient glucose uptake. Conditions which are known to mediate myocyte damage and which are associated with insufficient glucose uptake into the myocyte include ischemia and diabetes. Thus, it is preferred that the cell to which the isolated nucleic acid encoding a GUEMP is provided is a myocyte of the cardiovascular system of a mammal. Such a cell may be, for example, a cardiac myocyte, a cardiac ventricular myocyte, a rat cardiac ventricular myocyte, a human myocyte, a human skeletal myocyte, a human cardiac myocyte, and a human cardiac ventricular myocyte.

This method may further comprise providing an agonist of the purinoceptor to the cell after providing the purinoceptor to the cell. By way of example, cells in a cardiac muscle tissue of a human patient may be transformed by

- 32 - administering to those cells an expression vector encoding a P2Y₂ purinoceptor (e.g. a purinoceptor having the same amino acid sequence as the patient's own purinoceptor or a purinoceptor having a consensus human amino acid sequence). Thereafter (e.g. hours, days, weeks, months or more later), an agonist of the P2Y purinoceptor such as UTP or AP4A may be administered to the patient intravenously in order to enhance glucose uptake into the cells transformed with the expression vector.

The invention further includes a method of maintaining the viability of a cardiac cell extracted from a mammal. This method comprising contacting the cell, tissue, or heart with a composition comprising glucose and an agonist of a GUEMP. This treatment enhances glucose uptake into the myocytes obtained from the mammal, and increases both the survival of the myocytes during the extra-corporeal period and the ability of the myocytes to resume their contractile function upon transplantation into another mammal. The cardiac cell may be a single cardiac cell obtained, for example, from an animal tissue, a portion of a cardiac tissue sample obtained from the mammal, or all or a substantial portion of a heart of the mammal. Preferably, the mammal is a human or animal from which the heart may be extracted for transplantation into a human. This treatment is especially advantageous for storage of a human heart during the period from when it is extracted from the cadaver of a donor, optionally transported to a different geographical location, until it is prepared for implantation into a recipient in need of a heart transplant.

The invention also includes a method of affecting the activity of an enzyme in a mammalian cell. This method comprises contacting the cell with an agonist of a GUEMP, whereby the activity of the enzyme in the cell is either enhanced or depressed. It is believed that the ability of a GUEMP agonist to affect enzyme activities is attributable to interaction of one or more GUEMPs with at least one G- protein. It is further believed that this G protein, which may be the G protein G , is able to interact either with the enzymes directly or with proteins which control expression of the enzymes. GUEMP agonists may be used to affect the activity of numerous enzymes including, but not limited to, phospholipase C, protein kinase C, and phosphatidylinositol 3-kinase. GUEMP agonists may also be used to increase

- 33 - transmembrane translocation, and thus activity, of proteins such as GLUT1, GLUT4, and IRAP.

The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention should in no way be constmed as being limited to these Examples, but rather should be constmed to encompass any and all variations which become evident as a result of the teaching provided herein.

Example 1 The experiments presented in this Example establish that the P2Y purinoceptor is involved in enhancing glucose uptake in cardiac myocytes.

The materials and methods used in the experiments presented in this

Example are now described. Isolation of Cardiac Myocytes and Determination of 3 H-2-Deoxyglucose Uptake

Rat ventricular myocytes were isolated following collagenase-mediated dispersion as described (Xu et al., 1996, Am. J. Physiol. 270:H1655-H1661). Several preparations of collagenase were tested for the ability to consistently produce viable rod-shaped ventricular myocytes that exhibit insulin responsiveness, and the lot that yielded myocytes which exhibited the greatest response to insulin was used. Isolated myocytes were transferred to laminin-coated six- well plates and maintained in DMEM containing 6% (v/v) fetal bovine serum for twenty-four hours. The myocytes were washed with KRP medium, which comprised 136 millimolar NaCl, 4.7 millimolar KCl, 10 millimolar NaPO₄, 0.9 millimolar MgSO 0.9 millimolar CaCl₂, and 0.2% (w/v) bovine serum albumin (BSA). The pH of KRP medium was 7.4. A preparation of bovine serum albumin exhibiting no insulin-like activity was used. Myocytes were then incubated in 1 milliliter of Leibovitz's L-15 media containing 0.2% (w/v) BSA for two hours. L-15 medium has been described (Garcia et al., 1989, J. Biol. Chem. 264:19994-19999). The myocytes were then washed twice more with KRP medium, and were incubated for an additional thirty minutes in KRP medium.

To assess the effects of insulin or a potential GUEMP agonist on H-2- deoxyglucose uptake into myocytes, each agent was added to the culture medium, and

- 34 - the myocytes were incubated for twenty minutes prior to addition of H-2- deoxy glucose and unlabeled 2-deoxy glucose to the cells. Briefly, myocytes were incubated under the tissue culture conditions described herein for either about three or about five hours in the presence or in the absence of insulin, and the extent of H-2- deoxyglucose uptake was then quantitated. Non-specific ^JH-2-deoxyglucose uptake was assessed using 10 micromolar cytochalasin B in place of insulin and the agonist.

Measurement of the Phosphoinositide Response and Measurement of PKC Activity in

Intact Cardiac Myocytes

Intracellular concentrations of inositol phosphates were assessed according to a described method (Berridge et al., 1983, Biochem. J. 212:473-482), modified as described (Podrasky et al., 1997, Am. J. Physiol. 273:H2380-2387). LiCl was added to the assay mixture to inhibit inositol monophosphate phosphatase. A known assay for PKC activity in intact myocytes was used (Toullec et al, 1991, C. J.

Biol. Chem. 266:15771-15781; Blackshear et al., 1993, J. Biol. Chem. 268:1501-1504). The results of the experiments presented in this Example are now described.

Insulin Causes a Marked Enhancement of Glucose Uptake Into Adult Rat Cardiac

Ventricular Myocytes

H-2 -deoxyglucose uptake into adult rat cardiac myocytes was about 5- to 8-fold greater in the presence of insulin than in the absence of insulin following three hours of incubation, as shown in Figure 1. After five hours incubation, the difference

-3 between the extent of ^JH-2-deoxyglucose uptake in the presence of insulin and the extent in the absence of insulin was similar to that observed after three hours incubation. Non-specific H-2-deoxyglucose uptake was less than 20% of the total uptake, as assessed in the presence of 10 micromolar cytochalasin B.

These observations indicate that insulin causes a marked enhancement of glucose uptake into adult rat cardiac ventricular myocytes. It was therefore concluded that these myocytes are a useful model for investigating mammalian cardiac glucose transport.

35 - Presence of a G -Coupled P2 Purinoceptor in Cardiac Myocytes

Experiments were performed to determine cellular physiological or biochemical responses caused by incubating adult rat cardiac ventricular myocytes with

ATP. As indicated in Figure 2, each of ATP and UTP was capable of effecting more than a three-fold enhancement of intracellular inositol phosphate production.

This observation is consistent with the presence on the cells of a P2 purinergic receptor, the receptor being capable of stimulating of phospholipase C (PLC) activity in cardiac myocytes. Treatment of myocytes with pertussis toxin had no effect on the ability of ATP to enhance inositol phosphate production, suggesting that a G protein is involved

VI in ATP-mediated enhancement of PLC activity. It was observed that ATP is capable of increasing the contractile amplitude of the cardiac myocytes, as indicated by the results presented in Figure 3.

As shown in Figure 4, treating cardiac myocytes with 2-MeSATP, a P2 purinoceptor agonist, caused a increase in contractile amplitude similar to that caused by treating myocytes with ATP. However, as shown in Figure 2, 2-MeSATP is not capable of enhancing intracellular PLC activity in myocytes. Furthermore, the increase in contractile amplitude of myocytes mediated by 2-MeSATP is not prevented by treating the myoctes with U-73122, an inhibitor of PLC. Therefore, the P2 receptor which is capable of enhancing intracellular PLC activity appears to be different from the receptor(s) which is capable of enhancing cardiac myocyte contractile amplitude. UTP Stimulates a Pronounced Increase in Basal and Insulin-Dependent Glucose Uptake into Cardiac Ventricular Myocytes

The first step used to identify the P2 purinoceptor that mediates enhancement of cardiac glucose uptake was to examine the effect of UTP and 2-

MeSATP on glucose uptake. The results shown in Figure 5 indicate that 2-MeSATP did not affect either basal or insulin-dependent glucose uptake into cardiac myocytes. The results shown in Figures 6 and 7 indicate that UTP is capable of enhancing both basal and insulin-dependent glucose uptake into cardiac myocytes. ATP is equally as efficacious as UTP in stimulating glucose uptake into cardiac myocytes. These

- 36 - observations are consistent with mediation of glucose uptake enhancement by either the P2Y₂ receptor or the P2Y₄ receptor in cardiac myocytes. The results of these experiments demonstrate that adult rat ventricular myocytes represent a useful model for identifying nucleotide agonist modulators of glucose uptake. Availability of SID and ZDF Rats

Tail vein injection of 50 milligrams streptozotocin induced diabetes in the rat three weeks later. Blood glucose level in streptozotocin-treated rats was 606 ± 55 milligrams per deciliter (mean ± standard error; n=5); the corresponding level was 203 ± 18 milligrams per deciliter (n=4) in normal age-matched rats which were not treated with streptozotocin. Furthermore, the average body weight of streptozotocin- treated rats (206 ± 50 grams, n=5) was lower than the average body weight of normal age-matched rats which were not treated with streptozotocin (415 ± 20 grams, n=4). Isolated cardiac myocytes have been prepared from both SID rats and ZDF rats.

Example 2 Determining The Role of P2X and P2Y Receptors in Mediating Glucose Uptake

Because P2X receptors are ligand-gated channels, rather than G protein- coupled receptors, it is not likely that a P2X receptor mediates enhancement of glucose uptake by ATP. Nevertheless, the role of various P2X receptors may be investigated by assessing the effect of various P2 receptor antagonists on ATP-mediated enhancement of glucose uptake. Because 2-MeSATP does not enhance glucose uptake, it is unlikely that any of the P2X₄, P2X₅, and P2X₆ receptors are involved. P2X₄ and P2X₆ receptors are insensitive to antagonism mediated by suramin, reactive blue-2, or PPADS. Therefore, the ability of these antagonists to antagonize ATP- or UTP- mediated enhancement of glucose uptake rules out the involvement of either of these P2X receptors in such enhancement. Neither the P2X receptor nor the P2Xγ receptor is expressed in the heart. Therefore, these two receptors are not involved in ATP- mediated enhancement of glucose uptake. P2Xι receptors and P2Xo receptors can be activated by ,β-meATP and may be involved in ATP-mediated enhancement of glucose uptake. The potential involvement of the P2X_j receptor or the P2X3 receptor may be tested by investigating whether α,β-meATP enhances glucose uptake into the

- 37 - cardiac myocyte. If α,β-meATP does not enhance glucose uptake, then neither the P2X_j receptor nor the P2Xτ receptor is involved.

Because UTP induces a considerable enhancement of glucose uptake, it is likely that either the P2 Y receptor or the P2Y₄ receptor is the receptor that mediates enhancement of glucose uptake. The potency and the efficacy of various P2Y receptor agonists, such as those listed in Table 1, may be used to identify the P2Y receptor which is involved.

Because 2-MeSATP is the most potent and efficacious agonist of the P2Y_j receptor and because 2-MeSATP does not stimulate glucose uptake, it is unlikely that the P2Y_| receptor is involved in enhancing glucose uptake in cardiac myocytes.

A3P5P and A3P5PS, which are antagonists of the P2Yι receptor but are not antagonists of the other three P2Y receptors, may be used to rule out involvement of the P2Y_j receptor in enhancing glucose uptake. If P2Y_j receptor is involved, then these two antagonists will prevent enhancement of glucose uptake by ATP. mRNA encoding P2Yg receptor is not expressed in the heart; therefore

P2Yg receptor is likely not involved in enhancing glucose uptake in cardiac myocytes. The involvement of the P2Yg receptor may be tested by investigating whether UDP enhances glucose uptake and whether the potency and efficacy with which UDP enhances uptake are greater than those of UTP, ATP or ADP. To differentiate involvement of the P2Y₂ receptor from involvement of the P2Y₄ receptor in enhancing glucose uptake in cardiac myocytes, the potency and efficacy with which each of UTP and ATP enhances glucose uptake may be compared. If the potency and efficacy of ATP is similar to that of UTP, then the P2Y₂ receptor is involved. Data described in Example 1 indicated that ATP and UTP are equally efficacious for enhancing glucose uptake, suggesting the involvement of P2Y₂ receptor. To further test whether the P2 Y₂ receptor is involved, the ability of diadenosine tetra-phosphate (AP4A) compounds to enhance glucose uptake is investigated. AP4A is an agonist of the P2Y₂ receptor, but is not an agonist of the P2 Y₄ receptor. If it is determined that UTP is much more potent and efficacious than ATP, or if it is determined that UDP and AP4A are not capable enhancing glucose

- 38 - uptake, then the P2 Y₄ receptor is involved in enhancing glucose uptake in cardiac myocytes. The ATP analog ATPγS also is much more potent at the P2Y₂ than at the P2Y₄ receptor.

Example 3 Pre-Ischemic Conditioning of Cardiac Myocytes

The experiments presented in this example demonstrate that P2Y₂ receptor agonists have a ischemia-protective effect upon myocytes if the myocytes are contacted with the agonist before or during ischemia.

In a first set of experiments, adult rat cardiac ventricular myocytes were incubated, as described herein, in the presence and absence of the P2Y₂ receptor agonist, diadenosine tetraphosphate (AP4A). As indicated in Figure 8, the results of these experiments confirm that AP4A is an agonist of glucose uptake mediated by the P2Y receptor.

In another set of experiments, the ischemia-protective effect of P2Y₂ receptor agonists was demonstrated. In these experiments, the materials and methods used were the same as those described in Liang (1996, Am. J. Physiol. 271 (Heart Circ. Physiol.40):H1769-H1777; "the Liang reference"), except as described herein. Rather than pre-treating myocytes using a brief period of ischemia, as in the Liang reference, myocytes were pre-treated by exposing them to 0.1 to 100 micromolar UTP for five minutes, exposing the myocytes to UTP-free medium for 10 minutes, and then exposing the myocytes to 90 minutes of simulated ischemia. As indicated in Figure 9, cell mortality was reduced by pre-treatment with UTP, a P2Y receptor agonist. These data demonstrate that treating cells which comprise a functional P2Y₂ receptor (e.g. cardiac myocytes) with an agonist of that purinoceptor prior to the onset of ischemia reduces cell mortality during ensuing ischemia.

As indicated in Figure 10, the ischemia-protective effect of P2Y receptor agonists also reduces cell mortality attributable to ischemia when the myocyte is exposed to the agonist (i.e. UTP in Figure 10) during, but not before, simulated ischemia. In the experiments corresponding to Figure 10, isolated chick ventricle myocytes were incubated as described in the Liang reference. During a 90 minute

- 39 - period of simulated ischemia, the myocytes were incubated in medium comprising 10 micromolar UTP. As indicated in Figure 10, the survival rate of the myocytes incubated in the presence of UTP was much greater than the survival rate of myocytes incubated in the absence of UTP. The ischemia-protective effect of P2Y receptor agonists may be achieved by contacting cardiac myocytes with a P2Y₂ receptor agonist for a brief (e.g. 10 minute) period, as described above, or by contacting the myocytes and the agonist for a longer period, as demonstrated in the following set of experiments. Isolated rat ventricular myocytes were incubated for 24 hours in Dulbecco's modified Eagle's medium containing 6% (v/v) fetal bovine serum in the presence or absence of 10 micromolar UTP. The myocytes were washed free of UTP (if present), and were then incubated under simulated ischemic conditions for 45 minutes. The amount of creatine kinase (CK) released into the medium was determined using standard methods in order to quantitate the extent of myocyte injury (Oliver, 1955, Biochem. J. 61:116; Rosalki, 1967, J. Lab. Clin. Med. 69:696). As indicated in Figure 11, myocytes which had been incubated in the presence of UTP prior to the onset of ischemia released less CK, and thus sustained less ischemic injury, than myocytes which were not pre- incubated in UTP-containing medium. These results confirm that the ischemia- protective effect of UTP may be achieved by contacting cardiac myocytes with UTP for either a brief (e.g. 10 minute) or extended (24 hour) period prior to the onset of ischemia.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be constmed to include all such embodiments and equivalent variations.

40 -

Claims

What is claimed is:

1. A method of enhancing glucose uptake into a mammalian myocyte, said method comprising contacting the myocyte and an agonist of a glucose uptake- enhancing myocardial purinoceptor, whereby glucose uptake into the myocyte is enhanced.

2. The method of claim 1, wherein said agonist and the myocyte are contacted by providing a pharmaceutical composition comprising said agonist to the mammal.

3. The method of claim 2, wherein said pharmaceutical composition is provided intravenously to the mammal.

4. The method of claim 3, wherein said pharmaceutical composition further comprises an active ingredient selected from the group consisting of glucose, potassium, and insulin.

5. The method of claim 2, wherein said pharmaceutical composition is administered to a mammal experiencing cardiac ischemia.

6. The method of claim 5, wherein the mammal is afflicted with a disorder selected from the group consisting of angina pectoris, chronic stable angina, unstable angina, post-myocardial infarction angina, myocardial infarction, cardiac arrhythmia, coronary artery disease, diabetes mellitus, and cardiac ischemia attributable to shock, stress, or exertion.

7. The method of claim 2, wherein said pharmaceutical composition is administered to a mammal at risk for experiencing cardiac ischemia.

41

8. The method of claim 7, wherein said pharmaceutical composition is administered to a mammal which is imminently to undergo surgery.

9. The method of claim 7, wherein the mammal is afflicted with a disorder selected from the group consisting of angina pectoris, chronic stable angina, unstable angina, post-myocardial infarction angina, myocardial infarction, cardiac arrhythmia, coronary artery disease, diabetes mellitus, and cardiac ischemia attributable to shock, stress, or exertion.

10. The method of claim 1 , wherein the myocyte is a cardiac myocyte.

11. The method of claim 1 , wherein the purinoceptor is selected from the group consisting of a P2 Y₂ purinoceptor and a P2Y₄ purinoceptor.

12. The method of claim 11 , wherein the purinoceptor is a P2Y₂ purinoceptor.

13. The method of claim 1, wherein said agonist is selected from the group consisting of ATP, UTP, ATP╬│S, UTP╬│S, diadenosine tetra-phosphate, a diuridine polyphosphate, a base-substituted UTP analog, a ribose-substituted UTP analog, and a phosphate-substituted UTP analog.

14. The method of claim 13, wherein said agonist is selected from the group consisting of UTP and diadenosine tetra-phosphate.

15. The method of claim 1, wherein the mammal is a human.

16 . A method of enhancing glucose uptake into a mammalian cell, said method comprising providing a glucose uptake-enhancing myocardial purinoceptor to the cell, whereby glucose uptake into the cell is enhanced.

- 42 -

17. The method of claim 16, wherein said purinoceptor is provided to the cell by providing an expression vector to the cell, wherein said expression vector comprises a nucleic acid encoding said purinoceptor.

18. The method of claim 17, further comprising providing an agonist of said purinoceptor to the cell after providing said expression vector to the cell.

19. A method of minimizing ischemic cardiac damage in a mammal afflicted with a cardiac ischemic disorder, said method comprising administering to the mammal a pharmaceutical composition comprising an agonist of a glucose uptake- enhancing myocardial purinoceptor in an amount sufficient to enhance glucose uptake into cardiac myocytes of the mammal, whereby cardiac damage is minimized.

20. A method of minimizing ischemic cardiac damage in a mammal at risk for experiencing cardiac ischemia, said method comprising administering to the mammal a pharmaceutical composition comprising an agonist of a glucose uptake- enhancing myocardial purinoceptor in an amount sufficient to enhance glucose uptake into cardiac myocytes of the mammal, whereby cardiac damage is minimized in the event of cardiac ischemia.

21. A method of maintaining the viability of a cardiac cell extracted from a mammal, said method comprising contacting the cell with a composition comprising glucose and an agonist of a glucose uptake-enhancing myocardial purinoceptor after extracting the cell from the mammal, whereby the viability of the cell is maintained.

22. A method of increasing the level of activity of an enzyme in a mammalian cell, said method comprising contacting the cell with an agonist of a glucose uptake-enhancing myocardial purinoceptor, wherein the enzyme is selected from the group consisting of phospholipase C, protein kinase C, and

- 43 phosphatidylinositol 3 -kinase, whereby the level of activity of the enzyme in the cell is increased.

23. A method of effecting transmembrane translocation of a protein in a mammalian cell, said method comprising contacting the cell with an agonist of a glucose uptake-enhancing myocardial purinoceptor, wherein the protein is selected from the group GLUT1, GLUT4, and IRAP, whereby transmembrane translocation of the protein is effected.

24. A method of determining whether a test compound is a modulator of a glucose uptake-enhancing myocardial purinoceptor, said method comprising incubating a cell comprising said purinoceptor in the presence of a glucose analog and in the presence or absence of the test compound; and assessing uptake of said analog into said cell, whereby a difference between uptake of said analog into said cell in the presence of the test compound and uptake of said analog into said cell in the absence of the test compound is an indication that the test compound is a modulator of said purinoceptor.

25. The method of claim 24, wherein said purinoceptor is selected from the group consisting of a P2 Y₂ purinoceptor and a P2Y₄ purinoceptor.

26. The method of claim 24, wherein said glucose analog is selected from the group consisting of glucose and 2-deoxyglucose.

27. The method of claim 24, wherein said cell is selected from the group consisting of a mammalian myocyte, a rat cardiac ventricular myocyte, a cultured human myocyte, a myocyte obtained from a Zucker Diabetic Fatty rat, a myocyte contained within a Zucker Diabetic Fatty rat, a myocyte obtained from a streptozotocin- induced diabetic rat, a myocyte contained within a streptozotocin-induced diabetic rat, a myocyte obtained from a streptozotocin-induced diabetic micropig, a myocyte

- 44 - contained within a streptozotocin-induced diabetic micropig, and a transformed mammalian cell comprising an isolated nucleic acid encoding said purinoceptor.

28. A transformed mammalian cell comprising an isolated nucleic acid encoding a mammalian glucose uptake-enhancing myocardial purinoceptor.

29. The cell of claim 28, wherein said purinoceptor is selected from the group consisting of a P2 Y purinoceptor and a P2Y purinoceptor.

30. The cell of claim 28, wherein said mammalian cell is selected from the group consisting of a cardiac myocyte, a cardiac ventricular myocyte, a rat cardiac ventricular myocyte, a human myocyte, a human skeletal myocyte, a human cardiac myocyte, and a human cardiac ventricular myocyte.

31. A pharmaceutical composition comprising an agonist of a mammalian glucose uptake-enhancing myocardial purinoceptor and at least one additional ingredient selected from the group consisting of insulin, potassium, and glucose.

32. Use of an agonist of a mammalian glucose uptake-enhancing myocardial purinoceptor for preparation of a pharmaceutical composition for minimizing cardiac damage attributable to cardiac ischemia.

- 45 -