Description of the Related Art: The heart possesses intrinsic protective mechanisms to provide tolerance to injurious stimuli such as ischemia-reperfusion. Recent research has focused on understanding and harnessing such endogenous cardioprotective mechanisms. The autacoid adenosine has been proposed to function as an endogenous cardioprotectant (Ely and Beme (1992) Circulation 85. 893-904). Exogenous or endogenous activation of A adenosine receptors (A,ARs) protects the heart from injury during global ischemia (Lasley et al. (1990) Circulation 85. 893-904), and improves bioenergetic and mechanical recovery in reperfused myocardium (Angello et al.

(1990) Am. J. Physiol. 260. H193-H200; Lasley et al, (1990); Zhao et al. (1993) Circulation 88: 709-719: Finegan et al. (1996) J. Pharmacol. 118:355-363). A brief period of ischemia or brief exposure to adenosine. protects the heart from damaging effects of subsequent ischemic episodes: a phenomenon known as "preconditioning" (Parratt (1994) TIPS. 15: 19-25). Adenosine appears to be of central importance as a mediator of ischemic preconditioning in a range of species (Liu et al. (1991) Circulation 84: 350-356; Lawson and Downey (1993) Cardiovasc. Res. 27: 542-550; Parratt. 1994: Headrick (1996a) J. alJol. Cell. Cardiol. 28, 1227-1240). There is evidence that more than one of the four adenosine receptor subtypes may contribute to myocardial protection. but the A,AR appears to be primarily responsible for ischemic preconditioning (Mizimura et al. (1996) Circ. Res. 79: 415-423.).

Additionally, A1AR activation has been shown to provide protection from ischemic injury in other tissues (von Lubitz et al. (1988) Eur. J Pharmacol. 302, 43-48; Heurteauz et al. (1995) Proc. Natl. Acad Sci. USA 92: 4666-4670) providing evidence that ALARM activation may have broad protective effects in other organ systems.

Despite evidence implicating endogenous adenosine as a mediator of cardioprotection during ischemic episodes. controversy remains regarding the ability of adenosinergic therapy to reduce ischemic or post-ischemic injury (e.g., Vander Heide and Reimer (1996) Cardiovasc. Res. 31: 711-718).

In view of the aforementioned deficiencies anendant with the prior art methods of reducing cardiac injury during ischemia it is clear that there exists a need in the art for such a method.

SUMMARY OF THE INVENTION Accordingly, one object of this invention is to provide a method for protecting a mammalian heart from damage due to ischemia and/or hypoxia by providing to the heart an amount of exogenous adenosine receptors effective to prevent ischemia- and/or hypoxia-induced damage to the heart.

Another object of the invention is to provide a transgenic mammal having a greater number of adenosine receptors expressed on the surface of its cardiac cells than a non-transgenic control mammal.

Still another object of the invention is to provide a cell culture system of mammalian cardiac cells stably transformed by an expression vector containing a DNA sequence encoding an adenosine receptor.

Yet a further object of the invention is to provide a method for testing the ability of a drug to modulate cardiac response to ischemia by administering the drug to the transgenic mammal. measuring the coronary resistance and/or contractile function of the transgenic mammal to which the drug was administered. and correlating improved coronary vascular function and/or increased coronan contractile function to the ability of the drug to modulate cardiac response to ischemia.

Still another object of the invention is to provide a method for protecting or treating a mammalian heart by providing to the heart an amount of a substance effective to upregulate the expression of adenosine receptors on cells of the heart.

With the foregoing and other objects advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the preferred embodiments of the invention and to the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings. wherein: Figure 1 illustrates the demonstration of the A,AR transgene by Southern and Northern blotting. Figure 1A is a Southern blot demonstrating presence of transgene in lines 1 and 5. Figure 1B is a Northern analysis demonstrating presence of transgene in lines 1 and 5.

Figure 2 shows the characterization of [3H]CPX binding to membranes prepared from the hearts of transgenic mice over-expressing A,AR. The inset shows Scatchard plots of the same data. Values are means=s.e.m. of triplicate determination of a single experiment.

Figure 3 demonstrates the time to ischemic contracture (TIC) in globally ischemic hearts from control and transgenic animals. Control and transgenic hearts were either untreated (n=ll and 13, respectively) or pre-treated with 50 uM 8-(p- sulphophenyl) theophylline (8-SPT. n=15 and 6. respectively). Additionally. control hearts were also treated with N6-Cyclopentxladenosine (CPA n=6). Values shown are means+s.e.m.* P<0.05 transgenic vs. control hearts: t P<0.05 treated vs. untreated control hearts.

Figure 4 shows postischemic recoveries. Figure 4A illustrates diastolic tension. Figure 4B illustrates developed tension. Figure 4C illustrates coronary flow during 30 min global ischemia and 30 min reperfusion in hearts from control (n=ll) and transgenic mice (n=13). Values shown are meansis.e.m. * P<0.05 transgenic vs. control hearts.

DESCRIPTION OF THE PREFERRED EMBODINtENTS Given that ischemia elicits a large increase in interstitial adenosine (Van Wylen et al. (1992) Am. JPhysiol. 262: H1934-H1938; Headrick. 1 996a), one possibility which has received little attention is that it may prove difficult to pharmacologically enhance an intrinsic response or mechanism that is normally maximally active during ischemia-reperfusion. .4n alternative approach taken by the present inventors which proves more effective is to increase the number of functional receptors present.

There is evidence suggesting that "up-regulation" of myocardial A,AR can reduce ischemic injury (Rudolphi et al. (1989) Veurosci. Lett. 103. 275-280).

Transgenic manipulation provides the oppormnity for substantial modification of receptor function. Transgenic models of G-protein coupled receptors have been employed in assessment of cardiac function and provide unique opportunities for study of receptor signaling (Koch et al. (1996) Circ. Res. 78 511-516.). Recently, over-expression of heat shock protein has been shown to improve myocardial responses to ischemia-reperfusion (Plumier et al. (1995) J. Clin. Invest. 95: 1854- 1860, Marberetal. (1995) J. Clin. Invest. 95:1446-1456).

The present invention is directed to the delivery of adenosine receptors to heart tissue. either directly, via a recombinant expression system (including methods referred to as "gene therapy" as disclosed in U.S. Patent No. 5,399,346, which is incorporated herein by reference in its entirety), or via transgenic technology.

In a preferred embodiment of the present invention. a recombinant expression system is provided to produce biologically active animal or human adenosine receptors. In one embodiment. the adenosine receptors are co-expressed with heat shock protein (Plumier et al. (1995), which is incorporated herein by reference in its entirety) or other molecules which upregulate adenosine receptors in the heart.

In another embodiment, the adenosine receptors are over-expressed and a pharmaceutical composition comprising adenosine. adenosine agonists. adenosine analogs, and/or compounds which increase the efficacy of adenosine binding to the adenosine receptor are also administered to the patient. In some cases, molecules which bind to adenosine and/or its receptor (including antibodies thereto) may increase the efficacv of adenosine binding. The pharmaceutical may also include, either alone or in combination with the above-identified factors, compounds which act to up-regulate adenosine receptor activity and/or expression. Such compounds include, for example. theophylline (Wu et al. (1989) Circulation Research 65:1066-1077; Lee et al. (1993) Am. J. Physiol.264:H1634-H1643, which are incorporated herein by reference in their entireties).

In yet another embodiment, a substance such as a drug, a protein or an antibody is administered to a mammal, in an amount effective to increase the expression or upregulate the expression of the endogenous adenosine receptor gene. In particular. the substance may interact with the endogenous adenosine receptor promoter to effect such upregulation. The endogenous adenosine receptor would be considered "upregulated" if the number of adenosine receptors on the surface of cardiac cells were increased in comparison with a control mammal. in which no such administration had occurred. Preferably, the number of adenosine receptors would be increased by 5-20%, more preferably 20-100%, and most preferably more than 100%.

The use of overexpression of adenosine receptors in the heart is based in part on the fact that adenosine has been shown to be useful in myocardial protection (U.S. Patent No. 4.888,783 and Ely and Berne (1992). both of which are incorporated herein by reference in their entireties).

The nucleotide sequence of the rat A1 adenosine receptor is known (Reppert et al. (1991) Mol. Endo. 5:1037-1048; Mahan et al. (1991) Mol. Pharmacol. 40:1- 7), which are incorporated herein by reference in their entireties). Likewise, the dog (Schiffman et al. (1990) Brain Res.519:333-337), bovine (Olah et al. (1992) J.

Biol. Chem. 267:10764-10770; Tucker et al. (1992) FEBS Lett. 297:107-111), human (Libert et al. (1992) Biochem. Biophys. Res. Commun. 187:919-926; Townsend-Nicholson et al. (1992) Mol. Brain Res. 16:365-370; Ren et al. (1994) J. Biol. Chem. 269:3103-3110), rabbit (Bhatacharya et al. (1993) Gene 128:285- 288) and guinea pig (Meng et al. (1994) Mol. Brain Res. 26:143-155) A, adenosine receptors have also been cloned (all of which are incorporated herein by reference in their entireties.) The nucleotide sequence of the A3 adenosine receptor is also known: Zhou et al, Proc. Natl. Acad. Sci. USA 89:7432-7436 (1992); Linden et al, Mol.

Pharmacol. 44:524-532 (1993) [sheep]; Sajjadi et al. Biochim. BiophNs. Acta 1179:105-107 (1993) [human]; Salvatore et al. Proc. NatI. Acad. Sci. USA 90:10365-10369 (1993) [human]: .9rmstrong et al, CardiovascularRes. 28:1049- 1056 (1994); Linden, TiPS 15:298-306 (1994); and Strickler et al, J. Clin. invest.

98:1773-1779 (1996), all of which are incorporated herein by reference in their entireties.

The corresponding sequences from any other mammal, may be identified using these sequences (or fragment thereof) as probes. or by synthesizing primers for amplification of the desired sequence using polvmerase chain reaction (PCR) (U.S. Patent Nos. 4,683,202 and 4.683,195) based on the known sequences. In particular, the probe or primer is chosen from a region exhibiting a high degree of homology among the known sequences. The sequence encoding the adenosine receptor may also be modified to enhance its binding to its ligand and/or to increase the myocardial protective activity deriving therefrom. It is the specificity of the adenosine receptor for adenosine (and its analogs and/or agonists) and the direct involvement of the adenosine receptor in the chain of events leading to mycocardial protection from ischemia, that offers the promise of a broad spectrum of diagnostic and therapeutic utilities. It is known that adenosine exerts its modulatory effects through the adenosine receptor which is coupled to guanine nucleotide binding proteins (G proteins) and action on adenylyl cyclase (Reppert et al. (1991)).

The present invention naturally contemplates several means for preparation and introduction of the adenosine receptor. including as illustrated herein known recombinant techniques. and the invention is accordingly intended to cover such synthetic preparations within its scope. The knowledge of the cDNA and amino acid sequences for the adenosine receptor facilitates its reproduction by such recombinant techniques, and accordingly, the invention extends to expression vectors prepared from the known DNA sequence, fragments or derivatives thereof. for expression in host systems by recombinant DNA techniques. and to the resulting transformed hosts and cells.

By "fragments and derivatives thereof is meant any fragment of the nucleotide sequence encoding the adenosine receptor which retains the ability to bind to adenosine, its agonists and/or analogs, and to effect myocardial protective activity. Such activity can be assayed for in vivo by measuring binding using a labeled adenosine receptor agonist or antagonist. The label for use in vivo may be any label known in the art which is detectable via a particular imaging technique. such as computerized tomography (CT), magnetic resonance imaging (MRI) or the like. The activity can be assaying for in vitro using a labeled adenosine agonist or antagonist in a binding assay, an immunofluorescence assay, enzyme-linked immunosorbent assay (ELISA), Western blot, and the like, using labeled ligand or antibody, either directly or in a sandwich-type assay. The label may be radioactive, may be an enzyme detectable by a color reaction, may be fluorescent (such as fluorescein, rhodamine. Lucifer Yellow. AMCA blue, Texas Red or the like) or may be a compound such as biotin or deoxygenin which is detectable using labeled avidin or antibody thereto. respectively. Other labels are well known in the an which are suitable for detection using known assays.

Other in vivo tests which can be used to confirm the activity of a fragment or derivative. or the presence of the introduced adenosine receptor, fragment or derivative thereof are physiological assays such as coronary resistance, contractile function and heart rate.

In addition, assays may be performed both in VlVo and in vitro for receptor binding activity by analyzing G protein/adenylate cyclase activity. or any other activity downstream from adenylate cyclase which leads to the cardioprotective effects.

The presence of the adenosine receptor coding sequence in the transformed or transgenic host may be confumed using known assays, such as Southern blot or PCR. The expression of the adenosine receptor can be confirmed using Northern blot and RNA-PCR to detect mRNA and Western blot and/or ELISA to detect protein.

As noted above, the nucleic acid encoding the adenosine receptor may be introduced using a suitable expression vector or via transgenic technology.

Plasmids suitable for transgenic expression of proteins are well known in the art (Plumier et al. (1995); Adolph et al. (1993) J. Biol. Chem. 268:5349-5342, both of which are incorporated herein by reference in their entireties). Suitable vectors for introduction of the adenosine receptor coding sequence via an expression vector include. but are not limited to retroviral and adenoviral vectors, as well as defective adenoviruses (e.g. adeno-associated virus). Such vectors are described in the following patents and publications. which are incorporated herein by reference in their entireties: WO 9639830, WO 9633698, WO 9630535, WO 9627021, WO 9626742, WO 9626281, WO 9618372, WO 9617072, WO 9616087, WO 9611946, WO 9600112, WO 9600038. WO 9533840, WO 9616163, WO 9520975. WO 9411506, WO 9402605. WO 9306223. and WO 9220698.

The invention includes an assay system for screening of potential drugs effective to modulate cardiac response to ischemia by potentiating the action of adenosine at the adenosine receptor. In one instance, the test drug could be administered to a cellular sample with adenosine, which activates the adenosine receptor and associated adenylate cyclase system. or an extract containing the adenosine receptor, to determine its effect upon the binding activity of adenosine to its receptor, by comparison with a control.

The assay system could more importantly be adapted to identify drugs or other entities that are capable of binding to the adenosine receptor, thereby inhibiting or potentiating the activity thereof. Such assay would be useful in the development of drugs that would aid in the protection of the heart from the deleterious effects of ischemia.

In a further embodiment. the present invention relates to certain therapeutic methods which would be based upon the activity of the adenosine receptor, its subunits. or active fragments thereof. or upon agents or other drugs determined to possess the same activity. A first therapeutic method is associated with the prevention of the manifestations of conditions causally related to or following from decreased blood flow to the heart. and comprises administering via gene therapy or transgenic technology the adenosine Al receptor or active fragments or subunits thereof. either individually or in mixture with each other in an amount effective to prevent the development of those conditions in the host. In addition. drugs or other binding partners to the adenosine receptor may also be administered to potentiate the activity of the adenosine receptor.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature.

See, e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-m [Ausubel, R. M. ed.

(1994)]; "Cell Biology: A Laboratory Handbook" Volumes I-m [J. E. Celis, ed.

(1994))]; "Current Protocols in Immunology" Volumes I-E [Coligan, J. E., ed.

(1994)]; "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames & S.J. Higgins eds. (1985)]; "Transcription And Translation" [B.D. Hames & S.J. Higgins, eds. (1984)]; "Animal Cell Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

Therefore if appearing herein. the following terms shall have the definitions set out below.

The terms "adenosine receptor." "adenosine A, receptor," "A1 receptor," "A,AR," "adenosine A3 receptor," "A3 receptor," "A3AR" and any variants not specifically listed. may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins and extends to those proteins having the amino acid sequence data described in the literature. and the profile of activities set forth herein and in the claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate. for example, such as modifications obtained through site-directed mutagenesis. or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms "adenosine receptor." "adenosine A, receptor," and "A, receptor," "A,AR," "adenosine A3 receptor," "A3 receptor." "AAR" are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.

"Exogenous" means that the material provided, whether it is a nucleic acid sequence, a protein. a drug, etc., is either not present in the cell or animal to which it is administered prior to administration, or is not present at the same level as it is following administration. "Exogenous" may mean "heterologous," i.e., a gene derived from a species other than the species to which it is administered, or "exogenous" may mean that the gene is derived from the recipient species, but is naturally present in only a single. or limited number of copies per cell. compared to the treated animal or cell. "Exogenous" may also mean protein which is derived from the recipient species or a heterologous species which is administered to provide a level of the protein in the recipient animal or cell which is higher than that which naturally occurs in the recipient animal or cell.

A "replicon" is any genetic element (e.g., plasmid. chromosome. virus) that functions as an autonomous unit of DNA replication in vivo: i.e.. capable of replication under its own control.

A "vector" is a replicon. such as plasmid. phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form. or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule. and does not limit it to any particular tertiary forms.

Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e.. the strand having a sequence homologous to the mRNA).

An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.

A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences. cDNA from eukaryotic mRNA. genomic DNA sequences from eukaryotic (e.g., mammalian) DNA. and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators. and the like, that provide for the expression of a coding sequence in a host cell.

A "promoter sequence' is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.

Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

Eukaryotic promoters will often. but not always. contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA. which is then translated into the protein encoded by the coding sequence.

A "signal sequence" can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide. that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

The term "oligonucleotide," as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.

The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature. source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands.

Therefore, the primer sequence need not reflect the exact sequence of the template.

For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.

As used herein, the terms " restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes. each of which cut double-stranded DNA at or near a specific nucleotide sequence.

A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example. the transforming DNA may be maintained on an episomal element such as a plasmid.

With respect to eukaryotic cells. a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

Two DNA sequences are "substantially homologous" when at least about 75 % (preferably at least about 80%, and most preferably at least about 90 or 95 %) of the nucleotides match over the defined length of the DNA sequences.

Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under. for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See. e.g.. Maniatis et al., supra; DNA Cloning, Vols. I & II, supra: Nucleic Acid Hybridization, supra.

Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.

A "heterologous'' region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene).

Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4.816,397 and 4,816,567.

An "antibody combining site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.

The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope. including those portions known in the art as Fab, Fab', F(ab') and F(v), which portions are preferred for use in the therapeutic methods described herein.

Fab and F(ab ' )2 portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example. U.S. Patent No. 4,342,566 to Theofilopolous et al. Fab' antibody molecule portions are also well-known and are produced from F(ab'S portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. An antibody containing intact antibody molecules is preferred herein.

The phrase "monoclonal antibody" in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites. each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction. such as gastric upset. dizziness and the like, when administered to a human.

The phrase "therapeutically effective amount is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent. most preferably by at least 90 percent, a clinically significant change in the S phase activity of a target cellular mass, or other feature of pathology such as for example, conditions associated with ischemia.

A DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term "operatively linked" includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal. such a start signal can be inserted in front of the gene.

The term "standard hybridization conditions ' refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65"C for both hybridization and wash. However, one skilled in the art will appreciate that such "standard hybridization conditions" are dependent on particular conditions including the concentration of sodium and magnesium in the buffer. nucleotide sequence length and concentration. percent mismatch. percent formamide. and the like. Also important in the determination of "standard hybridization conditions" is whether the two sequences hybridizing are RNA-RNA. DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20"C below the predicted or determined Tm with washes of higher stringency, if desired By the phrase "excessive energy demands" is meant any occurrence wherein myocardial energy needs exceed supply. An example of an "excessive energy demand" is what occurs during exercise.

As discussed earlier. in addition to providing the adenosine receptor, binding partners or other ligands or agents exhibiting mimicry to the adenosine receptor or control over its production, may be prepared in pharmaceutical compositions, with a suitable carrier and at a strength effective for administration by various means to a patient experiencing an adverse medical condition associated with ischemia for the treatment thereof. A variety of administrative techniques may be utilized, among them parenteral techniques such as subcutaneous. intravenous and intraperitoneal injections, catheterizations and the like. Average quantities of the agent may vary and in particular should be based upon the recommendations and prescription of a qualified physician or veterinarian.

Also, antibodies including both polyclonal and monoclonal antibodies. and drugs that modulate the production or activity of the adenosine receptor and/or its subunits may possess certain diagnostic applications and may for example. be utilized for the purpose of detecting and/or measunng the presence or functionality of the adenosine receptor. For example. the adenosine receptor or its subunits may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example fused mouse spleen lymphocytes and myeloma cells.

Likewise. small molecules that mimic or antagonize the activity(ies) of the adenosine receptor may be discovered or synthesized. and may be used in diagnostic and/or therapeutic protocols.

The present invention further contemplates therapeutic compositions useful in practicing the therapeutic methods of this invention. In addition to providing the adenosine receptor via transgenic or gene therapy protocols, the adenosine receptor may also be administered directly, via various "drug" delivery devices, including but not limited to liposomes, immunostimulating complexes (ISCOMs) and the like. A subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of an adenosine receptor. polypeptide analog thereof or fragment thereof. as described herein as an active ingredient. In a preferred embodiment. the composition comprises a ligand or agent antigen capable of modulating the specific binding of adenosine (or other suitable adenosine receptor agonist) with the adenosine receptor on the surface of a target cell. In addition, where the adenosine receptor is provided via genetic means, ligands for the receptor or agents which potentiate the activity of the adenosine receptor may be prepared and administered by such means.

The preparation of therapeutic compositions which contain polypeptides, analogs or active fragments as active ingredients is well understood in the art.

Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.

Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired. the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.

A polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.

Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric. mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium. calcium. or ferric hydroxides. and such organic bases as isopropylamine. trimethylamine. 9-ethylamino ethanol, histidine. procaine. and the like.

The term 'unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent: i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of adenosine receptor binding capacity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual.

However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10. and more preferably one to several. milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated.

The therapeutic compositions may further include an effective amount of the adenosine receptor, ligand thereof or agent potentiating the activity of the adneosine receptor, and one or more of the following active ingredients: an antibiotic, a steroid.

Another feature of this invention is the expression of the DNA sequences disclosed herein. As is well known in the art, DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate host.

Such operative linking of a DNA sequence of this invention to an expression control sequence, of course. includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include, but are not limited to derivatives of SV40. cytomegalovirus. retroviruses, adenoviruses and adeno- associated viruses.

Any of a wide variety of expression control sequences -- sequences that control the expression of a DNA sequence operatively linked to it -- may be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include. for example. the early or late promoters of SV40, CMV. vaccinia. polyoma or adenovirus. and other sequences known to control the expression of genes of eukaryotic cells or their viruses, and various combinations thereof.

It will be understood that not all vectors. expression control sequences and hosts will function equally well to express the DNA sequences of this invention.

Neither will all hosts function equally well with the same expression system.

However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this invention. For example. in selecting a vector, the host must be considered because the vector must function in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors will normally be considered. These include. for example, the relative strength of the system. its controllability. and its compatibility with the particular DNA sequence or gene to be expressed. particularly as regards potential secondary structures.

Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture.

It is further intended that adenosine receptor analogs may be prepared from nucleotide sequences of the protein complexisubunit. Analogs, such as fragments. may be produced, for example, by pepsin digestion. Other analogs, such as muteins, can be produced by standard site-directed mutagenesis of adenosine receptor coding sequences. Analogs exhibiting "adenosine receptor activity" such as small molecules, whether functioning as promoters or inhibitors, may be identified by known in vivo and/or in vitro assays.

As mentioned above. a DNA sequence encoding an adenosine receptor can be prepared synthetically rather than cloned. The DNA sequence can be designed with the appropriate codons for the adenosine receptor amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al.. J. Biol. Chem., 259:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes which will express adenosine receptor analogs or "muteins" Alternatively. DNA encoding muteins can be made by site-directed mutagenesis of native adenosine receptor genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.

A general method for site-specific incorporation of unnatural amino acids into proteins is described in Christopher J. Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, Peter G. Schultz. Science. 244: 182-188 (April 1989). This method may be used to create analogs with unnatural amino acids.

The presence of adenosine receptors on cells can be ascertained by the usual immunological procedures applicable to such determinations. A number of useful procedures are known. Three such procedures which are especially useful utilize either the adenosine receptor labeled with a detectable label, antibody Ab, labeled with a detectable label, or antibody Ah, labeled with a detectable label. The procedures may be summarized by the following equations wherein the asterisk indicates that the particle is labeled, and "AR" stands for the adenosine receptor: A. AR* + Ab, = AR*Ab, B. AR + Ab* = ARAb,* C. AR + Ab, + Ab2* = ARAb,Ab2* The procedures and their application are all familiar to those skilled in the art and accordingly may be utilized within the scope of the present invention. The "competitive" procedure, Procedure A. is described in U.S. Patent Nos. 3,654,090 and 3.850.752. Procedure C. the "sandwich" procedure. is described in U.S.

Patent Nos. RE 31.006 and 4.016.043. Still other procedures are known such as the "double antibody, ' or "DASP" procedure.

In each instance, the AR forms complexes with one or more antibody(ies) or binding partners and one member of the complex is labeled with a detectable label. The fact that a complex has formed and. if desired. the amount thereof. can be determined by known methods applicable to the detection of labels.

It will be seen from the above. that a characteristic property of Abn is that it will react with Ab,. This is because Ab! raised in one mammalian species has been used in another species as an antigen to raise the antibody Am. For example.

Ab2 may be raised in goats using rabbit antibodies as antigens. Ah, therefore would be anti-rabbit antibody raised in goats. For purposes of this description and claims, Abl will be referred to as a primary or anti-AR antibody, and Ab, will be referred to as a secondary or anti-Ab, antibody.

The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.

A number of fluorescent materials are known and can be utilized as labels.

These include, for example, fluorescein, rhodamine. auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.

The AR or its binding partner(s) can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope may be selected from 3H, 14C 32pal 355, 36Cl, 51cur. 57Coo 58Co, 59Fe, 9'Y, l25I, 131I, and 86rye.

Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric. spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides. diisocyanates, glutaraldehvde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, fS-glucuronidase, B-D-glucosidase, B-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. U.S. Patent Nos. 3,654,090: 3,850,752; and 4.016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.

In accordance with the above. an assay system for screening potential drugs effective to modulate the activity of the adenosine receptor may be prepared. The adenosine receptor may be introduced into a test system. and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the cells' response to ischemia.

The present inventors have found that a specific increase in myocardial adenosine receptor density enhances tolerance to ischernia. In a specific embodiment. the cardiac specific a-myosin heavy chain promoter (Subramaniam et al. (1991) J. Biol. Chem. 266, 24613-24620) was used to over-express A,AR cDNA in a murine model. The inventors have also characterized nvo lines expressing the transgene and documented the response of hearts from these animals to global normothermic ischemia and reperfusion. The results indicate that over-expression of myocardial adenosine receptors protects the heart from ischemic damage.

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.

EXAMPLES MATERIALS AND METHODS Transgenic Construct. The Eco RI-ho I fragment of the rat Al cDNA (Reppert et al., 1991) was sub-cloned into the Sal I site of a construct containing the alpha myosin heavy chain promoter with a MEF-2 mutation (Adolph. et al.. 1993) and the human growth hormone polyadenylation signal. This promoter results in high level expression in the heart of mature animals along with some aortic expression (Adolph et al. 1993). The A,AR cDNA promoter construct was digested with Not I and purified for injection into the pronuclei of single cell fertilized mouse embryos (Hogan et al. (1986) (Cold Spring Harbor Lab Press. Plainview. NY)).

Transgene Detection. Mice were screened for the presence of the transgene by Southern analysis. Mouse genomic DNA was digested with Eco RI and probed with an a-NIHC promoter fragment. Eco RI digestion of the native a-MHC promoter resulted in a 2.6 kb fragment and digestion of the transgenic a-MHC A! construct resulted in a 1.6 kb fragment. Thus. transgenic mice demonstrated a 1.6 kb fragment from the transgenic promoter plus the 2.6 kb fragment from native promoter. Since there is one native a-MHC copy per genome, relative transgenic copy number was determined by comparing the density of the transgenic a-MHC band to the native band.

Message Determination. Total RNA was isolated using the method of Chomczynski and Sacchi [(1987) Anal. Biochem. 162:156-159]. Northern analysis of RNA was performed by electrophoresis under denaturing conditions in a MOPS/ formaldehyde 1.2% agarose gel and transfer to a charged nylon membrane (Zetaprobe, Biorad) by capillary action in high salt (20 X SSPE). Membranes were then UV cross-linked. A1AR cDNA was labeled by random priming with hybridization and washes at 65 C according to the method of Church and Gilbert [(1974) Proc. Natl. Acad. Sci., USA 81:1991-1995].

Membrane Preparation. Hearts from transgenic positives and negatives were homogenized in 10 vol of ice-cold buffer (10 mM EDTA. 10 mM HEPES, 0.1 mM berizainidine, pH 7.4). Homogenate was centrifuged at 48,000 g for 10 min. The pellet was resuspended in 30 ml of buffer with EDTA reduced to 1 mM, re- centrifuged and washed twice more by re-suspension/centrifugation. The final pellet was re-suspended in 1 vol of the appropriate buffer for assays. Membrane suspensions were stored at -80°C. Protein was determined by the Lowry method using bovine serum albumin for standards.

Ligand Binding. A,AR density and equilibrium dissociation constants in heart membranes from control and transgene positive animals were determined by quantitation of specific binding of an adenosine A1 receptor antagonist, 8- cyclopentyl,l-1.3-[3H]-dipropylxanthine ([3H]CPX, 0.1 to 7 nM) using standard techniques (Cothran et al. (1995) Biol. Neonate 68:111-118)). Briefly, 100 uL aliquots of membrane (0.2-0.7 mg protein in negative animals and 10 -15 ag protein in transgene positive animals) were incubated with adenosine deaminase (5 U/ml) in membrane buffer (50 mM Tris-HCl. 5 mM MgCl" pH 7.4) with radio-ligand. After a 2 hr incubation at 21 "C, 3 ml of ice-cold rinse buffer ( 10 mM Tris HCl, 5 mM MgCl2. pH 7.4) was added to each sample. Membranes were collected onto Whatman GF/C glass fiber filters which were washed three times or 10 sec with ice- cold buffer (10 mM Tris MCI, 5 mM MgCl2. pH 7.4). Radioactivity trapped on filters was counted. Non-specific binding was determined by adding 10 cm N6- phenylisopropyladenosine (R-PIA) to displace specific binding of [3H]CPX.

The number of high affinity agonist binding sites. a measure of G-protein coupled receptors. was determined using the agonist radioligand l25lABA (Linden et al. (1985) Circ. Res. 56:279-284). Specific binding was fit to a single site binding model using non-linear least squares curve fitting of the un-transformed data to calculate receptor density and dissociation constants. To compare receptor density between transgenic and control hearts, Bm; was reported as fmol receptor per mg protein. Coupling was defined as the ratio of specific '25IABA/ [3H]CPX binding.

The A.AR structure activity profile was determined by calculating K sK's K sK's of competing drugs. To calculate the Ka,, Ka,, [3H]CPX or -5IABA was added to tubes at - 50% of the Kd for the radiolabelled ligand. and competing ligand was added over a range of concentrations. IC50 values were calculated using a three parameter logistic equation: B = B0 - (Bo - Ns)[I]/(IC50 + [I], K, values were precisely calculated from IC50, B,,, the concentration of radioactive ligand and the Kd (Linden. 1982).

Langendorff Perfused Heart Model. Male and female mice (7-9 weeks.

21.8+0.421.80.4 21.8+0.421.80.4 g body wot.) wot.) were anesthetized with 50 mg.kg-' sodium pentobarbital. a thoracotomy performed and hearts excised into ice-cold perfusion fluid. The aorta was cannulated and hearts retrogradely perfused at a pressure of 80 mmHg with modified Krebs buffer containing (in mM): NaCI, 120: NaHCOa,NalICO3, NaHCOa,NalICO3, 25: KCl 4.7: 1.9; CaCl2,1.25: Mg2SOd 1.2; glucose. 15; and EDTA, 0.05. Buffer was equilibrated with 95% 02, 5% CO2 at 37"C, giving a pH of 7.4. Hearts were bathed in perfusate in a water jacketed bath maintained at 37"C. The left ventricle was vented with a polyethylene apical drain. Coronary perfusion was monitored via an ultrasonic flow- probe in the aortic perfusion line. To assess contractile function a stainless steel hook was attached to the ventricular apex and connected to a Grass FT03C strain gauge. Transducer position was adjusted to yield a diastolic tension of 1.0 g.

Apicobasal displacement was continuously measured via a Could RS3400 physiograph and the signal electronically processed to yield heart rate and +dP / dt.

After 20 min of perfusion at intrinsic heart rate hearts were switched to electrical pacing at 6 Hz (12 ms square wave. voltage 207O in excess of threshold) and allowed to stabilize for a further 10 min before experimentation.

Ischemia-Reperfusion Studies. Ischemia was produced by clamping the aortic cannula and simultaneously bubbling the bathing perfusate with 95% N2 / 5% CO2 to reduce PO. Pacing was stopped during ischemia and resumed on reperfusion.

Two studies were performed. In the initial study time to ischemic contracture (TIC) was measured in control and transgenic hearts in the absence and presence of 50 uM 8-(p-sulphophenyl)theophylline (8-SPT). A group of control hearts was also pre-treated with 20 nM N6-cyclopentyladenosine (CPA). The hearts were subjected to 10 min of global normothermic ischemia. TIC was defined as time between cessation of coronary flow and the point at which diastolic tension increased by 0.2 g (20% above basal tension). In the second study, control and transgenic hearts were subjected to 20 min of global normothermic ischemia followed by 30 min of reperfusion. Recovery of developed tension, diastolic tension and coronary flow was assessed during reperfusion.

Data Analysis and Statistical Comparisons. Baseline function in control and transgenic hearts was assessed via a t-test, and TIC values were compared using a one-way analysis of variance with Newman-Keuls post-hoc test for individual comparisons. Functional changes during ischemia-reperfusion were statistically analyzed by two-way analysis of variance for repeated measures with Newrnan-Keuls post-hoc test. In all tests significance was accepted for P<0.05.

RESULTS Transgenic Animals. Two positive transgenic lines were established and bred for analysis (Lines 1 and 5). Fig. Ia is a Southern blot demonstrating the presence of the transgene in lines 1 and 5. Northern analysis revealed abundant message in hearts from lines 1 and 5 (Fig. Ib). Consistent with previous findings (Matherne et al.. 1996), A,AR message is too low to be detected by standard northern analysis in a control heart.

Receptor Binding. Ligand binding data revealed that transgenic hearts express approximately 1000-fold higher AiAR than control hearts (Table 1). A,AR ligand binding was specific and saturable in transgenic tissue (Fig. 2), and the calculated Kd for CPX was comparable in control and transgenic hearts (Table 1).

Receptor coupling was estimated by comparison of the number of high affinity agonist ('25IABA) binding sites with total binding sites for the antagonist ([3H]CPX). Line 1 displayed a higher number of coupled receptors than line 5 despite lower total receptor number (Table 1), and thus possessed a higher percentage of functionally coupled receptors. Calculated K,Y K,Y values for different ligands (agonists and antagonists) are shown in Table 1. The calculated K,K K,K values for theophylline, CPX, CGS-21680CGS -21680 CGS-21680CGS -21680 and CPA are similar to published values (Bruns et al., (1987) Naunvn Schmiedebergs Arch. Pharmacol. 335:59-63; Bruns and Pugsley, (1987) In: Topics and PerspectivesandPerspecrives and PerspectivesandPerspecrives in Adenosine Research. eds. Gerlach and Becker.

Springer-Verlag. Berlin. pp. 59-73: Luthin et al.. (1995) AXlol.Io[ AXlol.Io[ Pharmacol. 47:307- 313). There were no detectable differences in K, values between the two transgenic lines.

Table 1. Ligand binding to membrane preparations from control and transgenic hearts Line Line I Hearts Line S Hearts | | Control Hearts Antagonist and Agonist Binding Properties [3HlCPXBmaxI3HICPXB,, [3HlCPXBmaxI3HICPXB,, 657=965*657965t 657=965*657965t | | 1069=102*t 8i18il 8i18il (fir'ojmg!) (fir'ojmg!) (n=lS) (n=80 (n=l l) [3H]CPXKd 0.92=0.09 1.12=0.09 0.73+0.17 (nM) (n=15) (n=8) (n=ll)(n=l l) (n=ll)(n=l l) '25lABA Bmax 47512547525 47512547525 244i8t (fmol.mg')(fmol.mg~') (fmol.mg')(fmol.mg~') (n=6)(n-) (n=6)(n-) (n=6) %Coupling 7.7i 1.0%7.7rut1.0% 7.7i 1.0%7.7rut1.0% 2.4t0.3%t Ligand KiK; KiK; Values (n=6 in all cases) Theo suM)(M) suM)(M) 17.0i1.817.0l.8 17.0i1.817.0l.8 10.0+2.2 CPX (nM) 0.4=0.3 2.0i0.6 CGS 01MCGS M) O.4tO.2 CGS 01MCGS M) O.4tO.2 |0.4io.2 |0.2iO.I |0.4io.2 |0.2iO.I | lO.2O. I | lO.2O. I CPA (nM) 0.3it.2 0.3it.2 0.3rut0.1 0.3rut0.1 Total and coupled receptor number was assessed from the B max for antagonist (CPX) and agonist (ABA), respectively. Agonist and antagonist K,Y K,Y values were determined as described in the Methods. Theo, theophylline; CPX, 1,3-dipropyl-8- cyclopentylxanthine; COGS, COGS, CGS21680; CPA, N6-Cyclopentyladenosine. All binding studies were performed in triplicate. * P<0.05 transgenic vs. control; t P<0.05 line 5 vs. line 1. Coupling is defined as Bma, agonist / B zB may B zB may antagonist.

Baseline Function in Perfused Hearts. Baseline functional parameters for hearts from control and transgenic animals are shown in Table 2. There were no statistical differences between baseline functional parameters in hearts from lines 1 and 5. Thus. throughout the analysis, data for lines 1 and 5 are combined.

Table 2. Baseline Functional Parameters in Control and Transgenic Hearts Developed Heart Rate +dT/dt (g.s-')(g.s') (g.s-')(g.s') Coronary Tension (g) (beats.min-')(beats.min') (beats.min-')(beats.min') Resistance (mmHg.ml '.g~') Intrinsic Heart Rate I Control(n=9) 1.59+0.18 318+14 | | 30.0+1.4 5.92+0.65 Transgenic 1.18+0.10* 248+ lOt248i l Ot 248+ lOt248i l Ot 28.6+1.6 6.41+0.43 (n=10) Electrically Paced at 6 Hz Control(n-9) 1.61+0.11 360 32.1+1.5 6.3211.13 Transgenic 1.56+0.10 360 32.8+1.7 7.591.457.59s 1.45 7.591.457.59s 1.45 (n=l1) Functional parameters were measured after 30 min of normal aerobic perfusion at a perfusion pressure of 80 mmHg. Diastolic tension was adjusted to 1.0 g in both groups. All values are means i s.e.m. * P<0.05 transgenic vs. control hearts.

There was no difference in coronary resistance, a slight but significant reduction in contractile function (indexed by developed tension). and a significant reduction in heart rate in hearts from transgene positive animals. When rate was normalized between groups by pacing, no differences were detected in contractile function, indicating that the apparent difference in contractility is due to rate- dependent change in contractile function (e.g. a manifestation of the positive staircase phenomenon).

Functional Effects of Ischemia-Reperfusion in Control and Transgenic Hearts. Ischemic injury was assessed by TIC in control hearts and transgenic hearts.

As shown in Fig. 3, TIC was 10 min in control hearts. This was reduced to 7 min by treatment with 8-SPT but was unaltered by treatment with the A agonist CPA.

Alternatively, TIC was significantly prolonged to 14 min in transgenic hearts. The improvement in transgenic hearts was reduced by competitive antagonism with 8- SPT (Fig. 3).

Functional parameters for control and transgenic hearts following 20 min of global normothermic ischemia are shown in Fig. 4. Global normothermic ischemia rapidly abolished contractile function and caused a gradual rise in diastolic tone.

Diastolic tension rose to a maximum of 1.93+0.12 gin control hearts and 1.85+0.10 g in transgenic hearts. In the first 2 min of reperfusion there was a rapid initial recovery of contractile function. At 2 min of reperfusion developed tension recovered to 60% of the pre-ischemic value in transgenic hearts but to only 15% in control hearts (P<0.05). This may reflect enhanced myocardial viability following the ischemic insult in transgenic hearts. At the end of reperfusion, developed tension recovered to only 30% of the pre-ischemic value in control hearts while it recovered to 45% in transgenic hearts (P<0.05). Diastolic tension was initially 50% above baseline in control hearts and remained elevated at 25% above the pre-ischemic value in control hearts. In contrast, diastolic tension was initially 25% above pre- ischemic levels in transgenic hearts and ultimately recovered to pre-ischemic levels after 30 min reperfusion(P<0.05). Coronary flow did not differ between the two groups, displaying an initial hyperemia at the onset of reperfusion followed by gradual decline to a final flow of ~120% of baseline in both control and transgenic hearts (Fig. 4).

DISCUSSION The primary goal of this study was to study the impact of over-expressinP AlAR in transgenic mouse heart on cardiovascular responses to ischemia-reperfusion injury. There is considerable disagreement about the impact of adenosinergic therapy on functional and metabolic responses to ischemia-reperfusion. and conflicting results have been obtained regarding the ability of adenosinergic therapy to limit infarct size (e.g. Toombs petal, (1992) Circulation 86:986-994; Zhao et al., 1994: Vander Heide and Reimer. 1996). Endogenous mechanisms may normally produce maximal or near maximal cardioprotection. Indeed. given the evidence that competitive antagonism of adenosine receptors does worsen the response to ischemia-reperfusion (Angello et al.. 1990: Lasley et al.. 1990: Zhao et al.. 1993; Finegan et al.. 1996), and the well documented elevations in interstitial adenosine to high levels (>1 ,aM) during ischemia (Van Wylen et al., 1992: Headrick, 1996a), it seems highly likely that the endogenous adenosine response is normally active and near maximal in ischemic tissue. This being the case. it may be ineffective to attempt to harness adenosinergic cardioprotection via classical pharmacological strategies alone (e.g.. infusion of agonists. allosteric enhancers. or inhibitors of uptake and breakdown) during severe ischemic episodes. The advent of genetic engineering and the ability to transgenicallv engineer tissues with modified receptor number and/or function provides an opportunity to circumvent this problem. If a response is potentially cardioprotective but normally near maximal it is possible to enhance protection by increasing the number of functional receptors. Here a model is described in which the present inventors over-expressed the cardiac A!AR. and present evidence that this provides enhanced tolerance to ischemia. AAR over- expression improves myocardial recovery of function on reperfusion.

Characteristics of Myocardial A,ARs on Control and Transgenic Mice. The affinity of CPX binding is singular in control and transgenic hearts. Agonist and antagonist K, values for transgenic hearts are similar to values for rat AlAR (Bruns et al.. 1987; Bruns and Pugsley. 1987). Hence. over-expressed A,ARs in transgenic lines appear to bind ligands normally. Interestingly. receptor coupling was significantly higher in line 1 than line 5 despite lower total receptor number. This may reflect an upper limit for G-protein coupling in the myocyte. There may be competition between receptors for G-proteins at high levels of expression.

Functional Effects of Over-expression of Myocardial A,ARS. One effect caused by over-expressing A1ARs was a reduction in heart rate. Under these conditions the heart is not subject to neurohumoral influences and the heart rate difference reflects a change in intrinsic rate (ie.. intrinsic firing of SA nodal cells).

Since endogenous adenosine reduces heart rate in isolated hearts from rats. rabbits and guinea pigs under various conditions (Belardinelh et awl . (1989) Prog.

Cardiovasc. Dis.32:73-97) ; Headrick and Willis. (1988) PfieuegersArch. 412:618- 623), and this is an A1AR response. it is not surprising that over-expression of A,ARs leads to reduced heart rate. In the absence of a change in A,AR affinity (Table 1). this indicates that endogenous adenosine levels are sufficient to activate over-expressed AIRS. This result is consistent with modest changes in resting heart rate with adenosine antagonism in other species (Headrick and Willis. 1988: Headrick. Ç 1996b) Am J. Phvsioi. 270:H897-H906.

Contractile function and coronary resistance were not altered by A1AR over- expression when heart rate was normalized between groups (Table 2). These data suggest that the A,AR does not play a significant role in modifying baseline contractile function. Comparable coronary resistance in control and transgenic hearts suggests a lack of effect of transgenic manipulation on the function of A,ARs that mediate coronary dilation.

Ischemic and Post-Ischemic Function in Transgenic versus Control Hearts.

Following abolition of contractile activity during ischemia the myocardium undergoes contracture - (increased resting ventricular tension). The mechanism of contracture is not fully understood but may involve rigor bond formation as a result of impaired glycolytic ATP production (Kingsley er al., (1991) Am J. Physio.

261:H469-H478.) Time to onset of contracture is an indicator of the severity of the ischemic insult. In control hearts a reduction in TIC in response to 8-SPT was observed, implicating prolongation of TIC by endogenous adenosine. This is consistent with the observations of Lasley and colleagues in rat heart (1990). TIC was not increased by pre-treatment of control hearts with the Ai agonist CPA at a dose (20 nM) which was found to reduce heart rate by more than 50% (data not shown). Taken together, these observations indicate that endogenous adenosine prolongs TIC in mouse heart and that the endogenous response is maximal. being resistant to pharmacologic augmentation. Since very high levels of extracellular adenosine are predicted under these conditions (Van W N len ef al.. 1992; Headrick.

1996a) it is not surprising that CPA was ineffective in prolonging TIC in ischemic mouse heart. Consequently, while it is possible to inhibit effects of endogenous adenosine with high levels of a competitive antagonist, protection was not enhanced with CPA. This observation, together with the fact that over-expression of AlAR significantly prolonged the TIC. indicates that receptor number can limit the degree of cardioprotection afforded by endogenous adenosine in the ischemic mouse heart.

Provision of additional receptors provides further protection when pharmacological manipulation is ineffective. This is a potentially important point in terms of development of cardioprotective therapies for ischemic myocardium - lack of response to an exogenous receptor agonist may reflect maximal endogenous activity.

Under these conditions targeting receptor expression is a more appropriate strategy.

This observation is relevant to studies in which there is conflicting data regarding the ability of exogenous adenosinergic therapy to reduce injury from ischemia- reperfusion (e.g. Vander Heide and Reimer. 1996).

During reperfusion following 20 min of global ischemia there were two principle differences in contractile recovery between control and transgenic hearts.

Initial contractile recovery (e.g.. at 2 min reperfusion) was markedly higher in transgenic versus control hearts. This pronounced "hyper-contractile" state may reflect the viability of ischemic myocardial cells immediately following ischemic insult and prior to the onset of reperfusion injury (Marber et al., 1995: Plumier et al..

1995). This would be consistent with contracture data - over-expression of A,ARs improves cellular function viability during the ischemic insult itself. The effect of A,AR over-expression in reperfused tissue was predominantly the result of reduced diastolic tone, which recovered to pre-ischemic levels in transgenic hearts but remained significantly elevated in control hearts (25% above baseline), consistent with observations in post-ischemic myocardium from other species (Meissner and Morgan, (1995) Am. J. Physio. 268:H100-H1 II: Headrick, 1996a). As a result. developed tension was greater in transgenic versus control hearts. Since coronary flow recovered to similar values in both groups, the differences in contractile recovery do not appear to result from differences in "no-reflow". It is therefore concluded that activation of A,ARs by endogenous adenosine improves contractile recovery predominantly via a reduction in post-ischemic diastolic dysfunction.

While not intending to be bound by theory, post-ischemic elevation in diastolic tone may reflect altered Ca2 handling with enhanced diastolic -Ca'~ (Meissner and Morgan. 1995). Endogenous adenosine acting via A,ARs may modify Ca2 handling at the level of the sarcolemma and/or at the sarcoplasmic reticulum. Indeed. A receptors are functionally coupled to K=-ATP channeis which can limit Cw entry when activated (Kirsch et al. (1990) Am. J Physiol 259:H820- 6). K+-ATP channel openers have been shown to be cardioprotective (Grover (1994) Cardiovasc. Res. 28:778-782) and effects of adenosine can be attenuated by K+-ATP channel blockers (Toombs et al.. (1993) Cardiovasc. Res. 27:623-629).

Alternatively, adenosine may modify post-ischemic energy metabolism such that the SR-ATPase can more effectively sequester Ca2 Receptor-mediated improvement of post-ischemic bioenergetic state by endogenous adenosine has been demonstrated (Angello et al., 1991; Headrick, 1996a). It is also possible that A,AR activation may directly modify SR channel function.

The present invention shows that A,AR activation by endogenous adenosine is beneficial in ischemic-reperfused myocardium, and that A,AR over-expression provides increased ischemic tolerance in the mouse heart. During ischemia the endogenous adenosine response appears to be near maximal precluding additional benefit from exogenously applied adenosine agonists. However, transgenic over- expression of A,ARs provides additional cardioprotection. A,AR mediated cardioprotection during ischemia is therefore limited by receptor number and/or coupling. During reperfusion, A,AR over-expression significantly improves contractile recovery, predominantly as a result of reduced diastolic dysfunction. The data demonstrate for the first time that genetic manipulation of the A,ARs is an effective method of improving outcome from ischemia-reperfusion when conventional pharmacological approaches may be less effective.

Having now fully described the invention. it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.