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J. Biol. Chem., Vol. 275, Issue 39, 30272-30279, September 29, 2000

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A Novel Pharmacological Approach to Treating Cardiac Ischemia

BINARY CONJUGATES OF A₁ AND A₃ ADENOSINE RECEPTOR AGONISTS^*

Kenneth A. Jacobson^§, Rongyuan Xie, Laura Young^¶, Louis Chang, and Bruce T. Liang^¶

From the  Molecular Recognition Section, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892 and the ^¶ Department of Medicine, Cardiovascular Division and Department of Pharmacology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104

Received for publication, February 23, 2000, and in revised form, July 5, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Adenosine released during cardiac ischemia exerts a potent, protective effect in the heart via activation of A₁ or A₃ receptors. However, the interaction between the two cardioprotective adenosine receptors and the question of which receptor is the more important anti-ischemic receptor remain largely unexplored. The objective of this study was to test the hypothesis that activation of both receptors exerted a cardioprotective effect that was significantly greater than activation of either receptor individually. This was accomplished by using a novel design in which new binary conjugates of adenosine A₁ and A₃ receptor agonists were synthesized and tested in a novel cardiac myocyte model of adenosine-elicited cardioprotection. Binary drugs having mixed selectivity for both A₁ and A₃ receptors were created through the covalent linking of functionalized congeners of adenosine agonists, each being selective for either the A₁ or A₃ receptor subtype. MRS 1740 and MRS 1741, thiourea-linked, regioisomers of a binary conjugate, were highly potent and selective in radioligand binding assays for A₁ and A₃ receptors (K_i values of 0.7-3.5 nM) versus A_2A receptors. The myocyte models utilized cultured chick embryo cells, either ventricular cells expressing native adenosine A₁ and A₃ receptors, or engineered atrial cells, in which either human A₃ receptors alone or both human A₁ and A₃ receptors were expressed. The binary agonist MRS 1741 coactivated A₁ and A₃ receptors simultaneously, with full cardioprotection (EC₅₀ ~0.1 nM) dependent on expression of both receptors. Thus, co-activation of both adenosine A₁ and A₃ receptors by the binary A₁/A₃ agonists represents a novel general cardioprotective approach for the treatment of myocardial ischemia.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Adenosine is released in large amounts during myocardial ischemia and has a potent, protective function in the heart (1). Adenosine also has protective effects when elevated prior to ischemia (2, 3). A brief, ischemic episode can "precondition" the heart, through adenosine receptor-mediated mechanisms, and protect it against injury sustained during a subsequent period of prolonged ischemia. This preconditioning effect can be simulated, in cultures of primary cardiac myocytes and in the isolated heart, by exposure to adenosine agonists selective for either A₁ or A₃ adenosine receptor subtypes, each of which causes a reduction in damage comparable to that induced by prior exposure to ischemia (2-5).

Although both A₁ and A₃ receptors can mediate cardioprotection (5-9), the specific mechanism of protection elicited by each receptor appears to be distinct and associated with activation of either phospholipase C or phospholipase D, respectively (10). Furthermore, the co-activation of A₁ and A₃ receptors may induce a greater protection than activation of each receptor individually. Thus, the protection offered by A₁ and A₃ receptors is not redundant, and in fact the receptors may act together to produce an additive protection (10).

The additivity of protection offered by A₁ and A₃ receptor activation has suggested to us that a single agonist capable of activating both A₁ and A₃, but not A_2A, receptors (6), might represent a new class of highly effective cardioprotective agents. Activation of A_2A receptors has been shown to increase myocyte death in ischemia (6). Because most of the effort in developing potent adenosine agonists has been aimed at pure subtype selectivity (13), it was not immediately obvious which derivatives of adenosine might be utilized to test this concept. A novel means of achieving mixed selectivity for A₁ and A₃ adenosine receptors was to covalently link different functionalized congeners of adenosine (14), each of which has agonist selectivity for one of the desired subtypes. The "binary drug" approach based on chemically functionalized congeners (15) has been utilized in synthesis of combinations of adenosine receptor ligands and biologically active peptides, specifically neurokinin agonists.

Using this novel binary conjugate design for adenosine receptor agonists, as well as a newly developed cardiac myocyte model for adenosine-elicited cardioprotection, our objective was to test the novel concept that simultaneous activation of both A₁ and A₃ receptors exerted a cardioprotective effect that was significantly more potent than the result of activating either receptor individually. The rationale was that it may be possible to achieve tissue selectivity, such that a biological effect would only be observed in cells that have both receptor subtypes.

The models utilized native adenosine A₁ and A₃ receptors in cultured chick embryo ventricular cells, which exhibited the adenosine-elicited cardioprotection characteristic of that found in the intact heart (3, 5-9, 11, 12), or recombinant human receptors expressed in chick atrial myocytes, in which native A₁ receptors were inactivated with a known irreversible antagonist (16). The latter resulted in a recombinant cardiac myocyte model in which the human adenosine receptors were coupled to a well-characterized cardiac cellular response. This study showed that the protective effect mediated by co-activation of both A₁ and A₃ receptors by a single compound was significantly more potent than that produced by activation of either receptor individually. Full protection required stimulation of both receptors and occured within a therapeutic dose window in which only cells expressing both receptors showed this highly potent response.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

8-Cyclopentyl-1,3-dipropylxanthine (DPCPX),¹ N⁶-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), and 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS 1191) were from Research Biochemicals International (Natick, MA). Full-length cDNAs encoding the human adenosine A₁ and A₃ receptors were kindly provided by M. Atkinson, A. Townsend-Nicholson, and P. R. Schofield (Garvan Medical Institute, Sydney, Australia) and were subcloned in the vector pcDNA3 as pcDNA3/hA₁R and pcDNA3/hA₃R. Adenosine was obtained from Sigma Chemical Co. (St. Louis, MO). The pcDNA3 vector was obtained from Invitrogen (Carlsbad, CA). Embryonated chick eggs were from Spafas, Inc. (Storrs, CT).

Synthesis

The adenosine agonist N⁶-[4-(carboxymethyl)phenyl]adenosine (4) was prepared by our previous method (17). All other materials were obtained from commercial sources. Proton nuclear magnetic resonance spectroscopy was performed on a Varian Gemini-300 spectrometer, and all spectra were obtained either in CD₃OD or Me₂SO-D₆. Chemical shifts () relative to tetramethylsilane are given. Chemical-ionization mass spectrometry was performed with a Finnigan 4600 mass spectrometer, and electron-impact mass spectrometry was performed with a VG7070F mass spectrometer at 6 kV.

Synthesis of Amine-functionalized A₃ Adenosine Receptor Agonist N⁶-[3-(Aminomethylethynyl)benzyl]-5'-N-methylcarbamoyladenosine (3a) In a dry reaction vessel flushed with nitrogen gas, IB-MECA (55 mg, 0.107 mmol) was dissolved in a mixture of anhydrous DMF (0.5 ml) and acetonitrile (1 ml). Triethylamine (0.50 ml), triphenylphosphine (2.2 mg, 0.008 mmol), palladium chloride (0.8 mg, 0.004 mmol), and copper(I) iodide (0.2 mg, 0.001 mmol) were added while stirring. Propargylamine (74 mg, 1.08 mmol) was then added, and stirring was continued overnight at 35 °C. The solvent was removed under a stream of nitrogen, and the residue was lyophilized for 30 min. The product was purified using preparative TLC (run twice, Silica Gel 60, 2000 µm, using a mobile phase consisting of chloroform:methanol, 10:1). The product was isolated as an orange solid (22.8 mg, 49% yield). MS (chemical-ionization) m/z 438 (m + 1). The proton NMR spectrum was consistent with the assigned structure, 3a.

This amine was also acetylated, using acetic anhydride, to provide compound 3b (MRS 1525).

Preparation of the Binary Ligand 5 (MRS 1543) by Carboxylate Condensation of Propargylamine Derivative of IB-MECA (3a) with N⁶-[4-(Carboxymethyl)phenyl]adenosine (4) O-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (57 mg, 0.15 mmol) and diisopropylethylamine (27 µl, 0.15 mmol) were added to a solution of 4 (40 mg, 0.1 mmol) in 1 ml of anhydrous DMF. The mixture was stirred at RT for 10 min. Then 3a (44 mg, 0.1 mmol) was added, and the mixture was stirred at RT overnight. TLC separation (eluant: CHCl₃:CH₃OH = 4:1) gave 30 mg of colorless powder of compound 5 (36% yield).

¹H NMR (CD₃OD) : 2.85 (s, 3H), 3.53 (s, 2H), 3.82 (dd, 2H), 4.15 (s, 2H), 4.30 (m, 3H), 4.46 (s, 1H), 4.75 (m, 2H), 4.82 (s, 2H), 5.98 (d, 1H), 6.00 (d, 1H), 7.31 (d, 2H), 7.28-7.40 (m, 4H), 7.76 (d, 2H), 8.23 (s, 1H), 8.29 (s, 1H), 8.34 (s, 1H), 8.35 (s, 1H). MS (FAB): m/z 821 (M⁺ + 1). HR-MS (FAB): Calculated for C₃₉H₄₀CsN₁₂O₉ (M⁺ + Cs): 953.2096, Found: 953.2066. Analysis for C₃₉H₄₀N₁₂O₉: C, 57.07%; H, 4.91%; N, 20.48%. Found: C, 56.89%; H, 4.53%; N, 20.90%.

General Procedure Coupling of Propargylamine Derivative of IB-MECA (3a) with DITC A solution of 1 mmol of DITC (m- or p-isomer) in 1 ml of anhydrous DMF was treated with 0.1 mmol of 3a, and the mixture was allowed to stir at RT for 4-5 h. 100 ml of ether and 50 ml of petroleum ether were added. After standing in a refrigerator for about 10 h, a pale yellow powder precipitated. It was washed several times with ether and petroleum ether to eliminate the excess DITC and unreacted 3a. After drying, product 6 was obtained (yields: m-isomer: 46%; p-isomer: 53%).

6a (m-DITC Derivative)-- ¹H NMR (Me₂SO-d₆) : 2.72 (s, 3H), 4.14 (s, 2H), 4.31 (d, 1H), 4.62 (s, 1H), 4.72 (t, 1H), 4.95 (s, 2H), 5.05 (s, 1H), 5.22 (s, 1H), 5.45 (s, 1H), 5.80 (s, 1H), 5.96 (d, 1H), 7.18-7.86 (m, 8H), 8.42 (s, 1H), 8.52 (s, 1H). MS (FAB): m/z 630 (M⁺ + 1). HR-MS (FAB): Calculated for C₂₉H₂₈N₉O₄S₂ (M⁺ + 1): 630.1706, Found: 630.1747.

6b (p-DITC Derivative)-- ¹H NMR (Me₂SO-d₆) : 2.71 (s, 3H), 4.15 (s, 2H), 4.31 (d, 1H), 4.60 (s, 1H), 4.72 (t, 1H), 4.94 (s, 2H), 5.06 (s, 1H), 5.24 (s, 1H), 5.44 (s, 1H), 5.81 (s, 1H), 5.99 (d, 1H), 7.20 (d, 2H), 7.26-7.36 (m, 4H), 7.80 (d, 2H), 8.39 (s, 1H), 8.50 (s, 1H). MS (FAB): m/z 630 (M⁺ + 1). HR-MS (FAB): Calculated for C₂₉H₂₈N₉O₄S₂ (M⁺ + 1): 630.1706, Found: 630.1713.

Preparation of the Binary Ligands 8a (MRS 1740) and 8b (MRS 1741) by Addition of DITC Derivatives (6a and 6b) to ADAC ADAC, 7 (5.8 mg, 0.01 mmol (16)) was added to a solution of 6a or 6b (0.01 mmol) in 0.2 ml of anhydrous DMF, and the mixture was stirred in a sealed tube under nitrogen at 80 °C for 2 days. Then the reaction mixture was added into 50 ml of ether and refrigerated for about 10 h. The dark brown precipitate from each reaction was collected and recrystallized in methanol. After drying, the pale brown product 8a or 8b was obtained (yields: m: 11%; p: 14%). Purity (>95%) was indicated using TLC.

8a (m-DITC-linked) (MRS 1740)-- ¹H NMR (Me₂SO) : 2.55 (t, 2H), 2.72 (s, 3H), 3.10 (t, 2H), 3.59 (s, 4H), 3.63 (dd, 2H), 3.98 (s, 2H), 4.16 (m, 2H), 4.32 (s, 1H), 4.62 (m, 3H), 4.77 (s, 2H), 5.03 (s, 1H), 5.25 (d, 2H), 5.53 (d, 2H), 5.95 (m, 2H), 7.20 (m, 4H), 7.25-7.40 (m, 6H), 7.55 (d, 2H), 7.83 (m, 4H), 8.18 (s, 1 H), 8.29 (s, 1 H), 8.41 (s, 1H), 8.53 (s, 1H). MS (FAB): m/z 1206 (M⁺ + 1). Analysis for C₅₇H₅₉N₁₇O₁₀S₂·3.2H₂O: C, 54.16%; H, 5.22%; N, 18.84%. Found: C, 54.46%; H, 5.70%; N, 18.41%.

8b (p-DITC-linked) (MRS 1741)-- ¹H NMR (Me₂SO) : 2.57 (t, 2H), 2.70 (s, 3H), 3.11 (t, 2H), 3.59 (s, 4H), 3.62 (dd, 2H), 3.98 (s, 2 H), 4.16 (m, 2 H), 4.32 (s, 1H), 4.62 (m, 3H), 4.79 (s, 2H), 5.03 (s, 1H), 5.25 (d, 2H), 5.53 (d, 2H), 5.95 (m, 2H), 7.20 (m, 4H), 7.25-7.40 (m, 6H), 7.53 (m, 4H), 7.82 (d, 2H), 8.17 (s, 1H), 8.29 (s, 1H), 8.40 (s, 1H), 8.53 (s, 1H). MS (FAB): m/z 1206 (M⁺ + 1). Analysis for C₅₇H₅₉N₁₇O₁₀S₂·3.0H₂O: C, 54.32%; H, 5.20%; N, 18.89%. Found: C, 54.40%; H, 5.66%; N, 18.40%.

Biological Activity

Preparation of Cardiac Myocyte Model of Simulation of Ischemia Atrial and ventricular cells were cultured from chick embryos 14 days in ovo and maintained in culture as described previously (11, 12, 18). All experiments were performed on day 3 in culture, at which time the medium was changed to a HEPES-buffered medium containing (in mM): 139 NaCl, 4.7 KCl, 0.5 MgCl₂, 0.9 CaCl₂, 5 HEPES, and 2% fetal bovine serum, pH 7.4, at 37 °C, before exposing the myocytes to simulated ischemia. Simulated ischemia was induced by exposing the myocytes to 90 min of hypoxia and glucose deprivation in a hypoxic incubator (NuAire), where O₂ was replaced by N₂ as described previously (11, 12). At the end of the 90-min ischemia the extent of myocyte injury was determined, and the myocytes were removed from the hypoxic incubator and re-exposed to room air (normal percentage of O₂). Aliquots of the media were then obtained for creatine kinase activity measurement, which was followed by quantitation of the number of viable cells, as determined by the ability to exclude Trypan blue (6). Measurement of the basal level of cell injury was made after parallel incubation of control cells under normal percentage of O₂. The extent of ischemia-induced injury was quantitatively determined by the percentage of cells killed and by the amount of creatine kinase (CK) released into the media according to a previously described method (11). The amount of CK was measured as enzyme activity (units/mg), and increases in CK activity were determined. The percentage of cells killed was calculated as the number of cells obtained from the control group (representing cells not subjected to any hypoxia or drug treatment) minus the number of cells from the treatment group divided by number of cells in control group multiplied by 100%. The myocyte injury caused by 90 min of ischemia simulated under the present condition appears to be primarily the result of cell lysis due to necrotic death, as evidenced by release of cellular enzymes and proteins (11) and retention of injured cells in the supernatant following low speed centrifugation (6) and not due to apoptosis (as evidenced by lack of DNA laddering, data not shown).

Preparation of Recombinant Cardiac Myocytes Expressing the Human Adenosine Receptor Atrial cardiac myocytes were isolated, maintained in culture for 24 h, and then transfected with either the vector pcDNA3, cDNA encoding the human adenosine A₃ receptor, or cDNAs encoding both adenosine A₁ and A₃ receptors (27). 24 h after the transfection, myocytes were treated for 10 min with the irreversible A₁ antagonist (m-DITC-XAC, 5 µM, synthesized as reported previously (19)) to inactivate the endogenous A₁ receptor as well as any of the exogenous human A₁ receptor. The cells were washed three times with fresh medium and incubated with medium containing 6% fetal calf serum for 24 h. These myocytes were then exposed to simulated ischemia in the presence or absence of agonist. Because the expression of the exogenous receptor is driven by the constitutive cytomegalovirus promoter, any receptor that appears after the m-DITC-XAC treatment is likely the exogenous adenosine receptor. This is supported by the lack of cardioprotective response to the A₁ agonist ADAC in pcDNA3-transfected myocytes after the irreversible A₁ antagonist treatment and the reappearance of an ADAC response in pcDNA3/hA₁R-transfected myocytes (data not shown). These interventions allowed selective expression of only A₁ or A₃ receptors or both A₁ and A₃ receptors in the myocytes.

To directly determine the effect of agonists at the exogenous adenosine receptors, adenosine deaminase was included with the agonists during the 90-min ischemia after the cells were pre-exposed to 5 min of ischemia.

Myocytes can be protected by a 5-min exposure to ischemia before the 90-min ischemia. In this phenomenon, known as "ischemic preconditioning," activation of the adenosine receptor by the endogenous adenosine is required during both the 5-min pre-exposure to ischemia and the 90-min ischemia (6). The protective effect induced by the 5-min exposure to ischemia can be abolished by eliminating the endogenous adenosine with adenosine deaminase (7). The concomitant presence of receptor agonist along with adenosine deaminase during the 90-min simulated ischemia restored the protective effect, allowing a direct determination of the effect mediated by agonist at either the native or the transfected human adenosine receptor.

Radioligand Binding Studies Binding of [³H]R-N⁶-phenylisopropyladenosine ([³H]R-PIA) to A₁ receptors from rat cerebral cortex membranes and of [³H]-2-(4-[(2-carboxyethyl)phenyl]ethylamino)-5'-N-ethylcarbamoyladenosine ([³H]CGS 21680) to A_2A receptors from rat striatal membranes was performed as described previously (20, 21). Adenosine deaminase (3 units/ml) was present during the preparation of the brain membranes, in a preincubation of 30 min at 30 °C, and during the incubation with the radioligands. Binding of [¹²⁵I]N⁶-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyladenosine ([¹²⁵I]AB-MECA) to membranes prepared from Chinese hamster ovary cells stably expressing the human A₃ receptor was performed as described (22). The assay medium consisted of a buffer containing 10 mM Mg²⁺, 50 mM Tris, and 1 mM EDTA, at pH 8.0. The glass incubation tubes contained 100 ml of the membrane suspension (0.3 mg of protein/ml, stored at 80 °C in the same buffer), 50 ml of [¹²⁵I]AB-MECA (final concentration 0.3 nM), and 50 ml of a solution of the proposed antagonist. Nonspecific binding was determined in the presence of 100 µM N⁶-phenylisopropyladenosine (R-PIA).

All non-radioactive compounds were initially dissolved in Me₂SO and diluted with buffer to the final concentration, where the amount of Me₂SO never exceeded 2%.

Incubations were terminated by rapid filtration over Whatman GF/B filters, using a cell harvester (Brandell, Gaithersburg, MD). The tubes were rinsed three times with 3 ml of buffer in each case.

At least five different concentrations of competitor, spanning three orders of magnitude adjusted appropriately for the IC₅₀ of each compound, were used. IC₅₀ values, calculated with the nonlinear regression method implemented in the InPlot program (GraphPad, San Diego, CA), were converted to apparent K_i values using the Cheng-Prusoff equation (23) and K_i values of 1.0 nM ([³H]R-PIA); 14 nM ([³H]CGS 21680); 0.59 nM and 1.46 nM ([¹²⁵I]AB-MECA at human and rat A₃ receptors, respectively).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Novel Drug Design: Creation of New Agonists Activating Both Adenosine A₁ and A₃ Receptors

Based on our previous study demonstrating a synergistic action of A₁ and A₃ adenosine receptors in cardioprotection, we sought to design novel, mixed A₁/A₃ adenosine receptor agonists using a "functionalized congener" approach (14). Novel derivatives were created by the "binary drug" approach, in which two functionalized congeners of adenosine agonists, each of which was selective for either A₁ or A₃ adenosine receptor subtypes, were covalently coupled. This was a means of modulating the selectivity ratio to increase and approximately match affinities at A₁ and A₃ receptors while diminishing affinity at A_2A receptors (6).

The synthesis of an amine-functionalized congener, 3a, derived from the A₃ receptor-selective agonist IB-MECA is shown in Fig. 1. This amine derivative was intended to serve as a common intermediate for two conceptual approaches: binary drugs (15) (Figs. 1 and 2) and amino acid conjugates (14, 24). The amine derivative 3a was adequately reactive toward acylation to form the desired conjugates. The inclusion of a rigid, narrow molecular "rod" in the form of an ethynyl group in 3a as a means of preserving A₃ receptor affinity was predicted by molecular modeling (24). The m-position of the benzyl ring in receptor binding appeared to accommodate long chain extension having minimal steric bulk. This derivative was coupled to an adenosine carboxylic agonist, 4, which was previously shown to form amides that display selectivity for A₁ adenosine receptors (17), to form the binary conjugate 5 (MRS 1543, Fig. 1). Compound 3a was also converted to a simple N-acylated derivative, 3b, which was tested for adenosine receptor affinity. In a separate reaction sequence (Fig. 2) the adenosine amine congener (ADAC, 7) was coupled to 3a, leading to binary conjugates 8a (MRS 1740) and 8b (MRS 1741), containing longer spacers than in 5. These two conjugates were regioisomers that differed only in the substitution pattern of the cross-linking moiety (19) phenylene diisothiocyanate (DITC).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   The synthesis of the binary conjugate MRS 1543. This conjugate is composed of separate adenosine moieties joined covalently and each of which selectively activates either the A1 or A3 receptor. The A1-activating moiety is an N6-phenylcarboxylic congener of adenosine, 4, and the A3 receptor-activating moiety is a phenylpropargylamine derivative, 3a, which is modified at both N6- and 5'-positions. The adenosine receptor affinities are given in Table I. Compound 3b is MRS 1525.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   The synthesis of the binary conjugates MRS 1740 and MRS 1741. These conjugates are composed of separate adenosine moieties, each of which selectively activates either the A1 or A3 receptor. The A1-activating moiety is an amine congener of adenosine and the A3 receptor-activating moiety is a phenylpropargylamine derivative, the synthesis of which is outlined in Fig. 1. The covalent joining of the separate moieties is done through a cross-linking reagent, m- or p-phenylene diisothiocyanate. The adenosine receptor affinities are given in Table I.

Biological Activity

Selectivity at Adenosine A₁ and A₃ Receptors-- As a first indication of selectivity, the receptor binding affinities of the adenosine derivatives were measured in standard binding assays using rat brain A₁ and A_2A receptors and recombinant human A₃ receptors. K_i values for the novel agonists are shown in Table I.

Compound 3b was A₃ receptor-selective, as was the precursor IB-MECA, thus validating the design approach. The amide-linked conjugate MRS 1543, compound 5, was moderately potent and selective for A₃ receptors. The elongated, thiourea-linked, regioisomers MRS 1740 and MRS 1741, 8a and 8b, were highly potent and selective versus A_2A receptors in radioligand binding assays, with K_i values of 0.7-3.5 nM at A₁ and A₃ subtypes.

Biological Activity in the Heart Cell: Novel Cardioprotective Property-- Cultured chick ventricular cells, expressing both native A₁ and A₃ receptors, were used initially as a myocyte model to determine the cardioprotective property of the conjugates. The N-acetylated amine congener 3b (data not shown) and the binary A₁/A₃ receptor agonist 5 (Fig. 3) were able to produce a concentration-dependent preconditioning-like effect, simulating the cardioprotective effect induced by a 5-min exposure to ischemia. The EC₅₀ of 5 was approximately 2 nM. The protective response was partially antagonized by the A₁ antagonist DPCPX and the A₃ antagonist MRS 1191 (Fig. 3A) with a shift of the agonist-mediated dose-response curve to the right. The combination of DPCPX and MRS 1191 caused a complete abolition of the protective response (Fig. 3B). These data suggested that protection mediated by the binary agonist occurs via activation of both adenosine receptors.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   The cardioprotective effect of the binary conjugate MRS 1543 requires the activation of both adenosine A1 and A3 receptors. Cardiac ventricular myocytes were prepared, incubated for 5 min with (A) indicated concentrations of MRS 1543, alone or in the presence of either the A1 receptor antagonist DPCPX (1 µM) or the A3 receptor antagonist MRS 1191 (1 µM), or (B) MRS 1543, alone or in the presence of both DPCPX and MRS 1191. Myocytes were then washed free of the agents and exposed to 90 min of simulated ischemia. The extent of myocyte injury was quantitated as percentage of cardiac cells killed or as CK released (data not shown) at the end of simulated ischemia as described under "Experimental Procedures." Data are the mean and standard error of four experiments. In A, the percentage of cardiac cells killed in the presence of 10 or 30 nM of MRS 1543 plus either antagonist was significantly different from that determined when myocytes were not exposed to any agent. (The asterisk indicates significant difference from data obtained in cells not pre-exposed to MRS 1543 or DPCPX or MRS 1191.) In B, the percentages of cardiac cells killed in the presence of MRS 1543 plus both DPCPX and MRS 1191 were similar to those obtained when myocytes were not exposed to any agent.

To provide further evidence for this concept, a series of experiments were carried out using a novel recombinant cardiac myocyte model expressing only the human adenosine receptor, because endogenous chick A₁ receptors were eliminated by incubation with the irreversible A₁ receptor antagonist, m-DITC-XAC (16, 19). After such pretreatment, these myocytes were unresponsive to the A₁ receptor agonist (data not shown) and remained unresponsive to the agonist following transfection with pcDNA3 (Fig. 4). Because the atrial cardiac myocytes expressed a very low level of native A₃ receptors (25, 26), both before and after treatment with the irreversible A₁ antagonist in myocytes, they were also unresponsive to the A₃ agonist (Fig. 4). Thus, an adenosine receptor-null myocyte was created. Transfection with cDNAs encoding A₁ and A₃ receptors led to the appearance of a cardioprotective response to either agonist, IB-MECA or ADAC. The protection in myocytes expressing the human adenosine receptors was indicated by a reduction in the percentage of cardiac cells killed and creatine kinase (CK) released at the end of the 90-min ischemia. Thus, the specific protective effect of adenosine A₁ or A₃ agonist was due to activation of the human receptors.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Recombinant cardiac myocytes expressing human adenosine A1 and A3 receptors. Atrial cardiac myocytes were prepared, transfected with pcDNA3 or cDNAs encoding the human adenosine A1 and A3 receptors, and subjected to treatment with the irreversible A1 receptor antagonist to create recombinant myocytes whose response to A1 receptor agonist ADAC (10 nM) or the A3 receptor agonist IB-MECA (10 nM) was determined. These myocytes were incubated with ADAC or IB-MECA, washed free of the agonist, and then exposed to 90 min of simulated ischemia. The percentage of cardiac cells killed and amount of CK released were quantitated at the end of the 90-min ischemia. Data are the mean and standard errors of five experiments. The asterisks represent significant difference from myocytes that were transfected with pcDNA3 (one-way ANOVA analysis and post-test comparison, p < 0.01).

Using this novel recombinant cardiac myocyte model, we examined the cardioprotective response to a single binary conjugate compound in myocytes expressing both A₁ and A₃ receptors as compared with myocytes expressing only the A₁ or the A₃ receptor. The cardioprotective responses to the binary conjugates MRS 1741 (Fig. 5A, IC₅₀ ~ 0.1 nM) or MRS 1543 (data not shown) were significantly more potent and efficacious in myocytes expressing both A₁ and A₃ receptors than in myocytes expressing only the A₃ receptor. Thus, transfection of the atrial cardiac myocyte with pcDNA3/hA₃R led to a left shift of the concentration-response curve to MRS 1741, as compared with pcDNA3-transfected myocytes (Fig. 5A). Because pcDNA3/hA3R-transfected atrial myocytes expressed both the A₃ receptor (exogenous) and the A₁ receptor (native), and the pcDNA3-transfected myocytes expressed only the native A₁ receptor, these data were consistent with the concept that the binary conjugate could co-activate both receptors. Similarly, the concentration-response curve in Fig. 5B was significantly left-shifted in myocytes expressing both the exogenous A₁ and A₃ receptors as compared with the curve obtained in myocytes expressing only the exogenous A₃ receptor. These data strongly suggested that a single binary conjugate was capable of activating both adenosine A₁ and A₃ receptors.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   The binary agonist exerts a more pronounced response in myocytes expressing both A1 and A3 receptors than in myocytes expressing either receptor alone. Atrial cardiac myocytes were transfected with (A) pcDNA3 or pcDNA3/hA3R. In B, atrial myocytes were transfected with pcDNA3/hA3R or both pcDNA3/hA1R and pcDNA3/hA3R and then subjected to treatment with m-DITC-XAC. In both A and B, the response to the binary agonist MRS 1741 was then determined as follows. Myocytes were exposed to 5 min of simulated ischemia to precondition the myocytes. Following a 10-min exposure to normal O2, these myocytes were then exposed to 90-min ischemia in the presence of adenosine deaminase alone or with the indicated concentrations of MRS 1741. The percentage of cardiac cells killed and amount of CK released were quantitated at the end of the 90-min ischemia. Data are shown for the percentage of cells killed and are the mean and standard errors of five experiments. Similar data were obtained when CK released was used as the end point (not shown). In A, the response to MRS 1741 in myocytes expressing the native A1 receptor (pcDNA3-transfected) was less than that obtained in myocytes expressing both the native A1 receptor and the exogenous A3 receptor (pcDNA3/hA3R-transfected). (The asterisks represent significant difference from myocytes transfected with pcDNA3 alone, one-way ANOVA analysis and post-test comparison, p < 0.01.) In B, the response to MRS 1741 in myocytes expressing only the exogenous A3 receptor (pcDNA3/hA3R-transfected and treated m-DITC-XAC) was less than that obtained in myocytes expressing both exogenous A1 and A3 receptors (pcDNA3/hA1R- and pcDNA3/h A3R-transfected and treated with m-DITC-XAC) (The asterisks represent significant difference from myocytes transfected with pcDNA3/hA3R alone, one-way ANOVA analysis, and post-test comparison, p < 0.01.)

The cardioprotective effect of a unimolecular, binary agonist that co-activated both receptors was equivalent to that caused by an equimolar mixture of the A₁ agonist ADAC and the A₃ agonist IB-MECA (Fig. 6). The concentrations shown for incubation with both ADAC and IB-MECA refered to the individual concentration of either agonist in the media bathing the myocytes. The response to each concentration of the binary molecule was virtually identical to the response produced by the same concentration of the two constituent agonists present together.

View larger version (10K): [in this window] [in a new window]   Fig. 6.   The response to binary agonist is the same as the response to equivalent concentration of constituent agonists present together. Atrial cardiac myocytes were prepared, transfected with both pcDNA3/hA1R and pcDNA3/hA3R, and subjected to treatment with the irreversible A1 receptor antagonist to create recombinant myocytes, which expressed only the human adenosine A1 and A3 receptors. The responses to MRS 1741 and to the combination of both constituent agonists ADAC and IB-MECA were determined. The concentrations shown for incubation with both ADAC and IB-MECA represent the individual concentration of either agonist, with each agonist present in equal concentrations in the media bathing the myocytes. These myocytes were exposed to 5 min of simulated ischemia to precondition the myocytes. Following a 10-min exposure to normal O2, these myocytes were then exposed to 90-min ischemia in the presence of adenosine deaminase alone or with the indicated concentrations of MRS 1741 or adenosine deaminase plus both ADAC and IB-MECA. The percentage of cardiac cells killed and amount of CK released were quantitated at the end of the 90-min ischemia. Data are shown as the percentage of cells killed and are the mean and standard errors of five experiments. Similar data were obtained when CK released was used as the end point (not shown). The concentration-response curve to MRS 1741 was virtually identical to that obtained in the presence of both ADAC and IB-MECA.

As compared with myocytes transfected with the vector alone, the binary A₁/A₃ receptor agonists caused a significant reduction in both the percentage of cells killed and the amount of CK released in myocytes expressing both human adenosine receptor subtypes. This was demonstrated for binary agonists MRS 1543 and MRS 1740 (Fig. 7, A-D, IC₅₀ ~ 0.1 nM) and for MRS 1741 (data not shown). Thus, the binary agonists appears to be able to co-activate both receptors at the same time.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Binary conjugate of adenosine A1 and A3 agonists can mimic the cardioprotective effect of ischemic preconditioning via the human adenosine A1 and A3 receptors. Atrial cardiac myocytes were prepared, transfected with pcDNA3 or cDNAs encoding the human adenosine A1 and A3 receptor, and subjected to treatment with the irreversible A1 receptor antagonist to create recombinant myocytes whose functional adenosine receptors are predominantly the human A1 and A3 receptors. These myocytes were then exposed to the indicated concentrations of the binary conjugate compounds (A and B) MRS 1543 or (C and D) MRS 1740 for 5 min, washed free of the agent, and then exposed to 90 min of simulated ischemia. The percentage of cells killed (A and C) and the amount of creatine kinase released (B and D) were quantitated at the end of simulated ischemia. Data represent the mean and standard error of four or five experiments. The asterisks represent significant difference from myocytes co-transfected with pcDNA3/hA1R and pcDNA3/h A3R.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Adenosine is a potent cardioprotective agent, capable of exerting a pronounced reduction in the extent of cardiac myocyte injury incurred during myocardial ischemia (1-6, 8, 9). Previous studies have shown that the cardioprotection offered by either receptor was not redundant (6). In fact, activation of both A₁ and A₃ receptors appeared to confer an additive anti-ischemic protective response (10). These data raised the possibility that a single agonist co-activating both receptors might represent a class of new and highly effective cardioprotective agents. Using a combination of novel ligand chemistry, pharmacological, molecular, and cellular approaches, the present study showed that such an agent could concurrently co-activate both receptors. Functionalized congeners, specifically, agonists selective at the adenosine A₁ and A₃ receptors, were combined covalently as binary conjugates. The relatively high molecular weight of the present binary drugs, for compounds 5, 8a, and 8b, would not necessarily be an impediment to bioavailability of the cardioprotective agents, because intravenous administration would be envisioned in the intended application. Pharmacokinetic studies of these and other dual activating adenosine agonists would be appropriate.

The ability of such binary agonists to co-activate both receptors was determined using a newly created recombinant cardiac myocyte model of cardioprotection. The receptor co-activation produced a highly potent anti-ischemic effect within a concentration range in which only cardiac myocytes expressing both receptors responded.

Multiple lines of evidence were obtained to support the concept of greater protection upon receptor co-activation. First, in ventricular myocytes, a binary agonist-mediated response was only partially inhibited by either the A₁ receptor-selective antagonist DPCPX or the A₃ receptor-selective antagonist MRS 1191. In contrast, the combined presence of both antagonists caused a complete abolition of the response to the binary agonist.

Second, in a further series of experiments, chick atrial myocytes were engineered to express only human adenosine receptors. The myocytes were made adenosine receptor-null by irreversible blockade of the endogenous adenosine receptor and were transfected with cDNA for human adenosine receptors. If a binary agonist could co-activate both adenosine A₁ and A₃ receptors, it should cause a much more pronounced response in cardiac myocytes expressing both human adenosine A₁ and A₃ receptors than in myocytes expressing only the human A₃ receptor. The data showed that this was indeed the case. Recombinant myocytes expressing either receptor subtype alone or both receptors were then created to determine the response to the binary agonist or the individual constituent agonists. The concentration-response curve of MRS 1741 was significantly left-shifted in myocytes having both receptors compared with myocytes expressing only the A₃ subtype. Similarly, the binary agonist exerted a more potent and efficacious response in myocytes having both receptors compared with myocytes expressing only the A₁ receptor.

Third, if the binary agonist could co-activate both receptors at the same time, the response produced by one molecule of the binary agonist should be equivalent to that elicited by the combined presence of one molecule of each constituent agonist. The response to each concentration of the binary molecule was virtually identical to the response produced by a combination of each constituent agonist present at the same concentration.

Species differences in the cardioprotective effects of adenosine and also in the A₃ receptor affinities of various ligands, especially antagonists, have been noted (13). In the present study, the critical question of generality across species of the approach of concurrently activating A₁ and A₃ receptors has been partially satisfied (for chick and human).

The binary drug approach has been demonstrated here for a clearly defined target of A₁ and A₃ receptors. The full cardioprotection induced by the conjugates having mixed selectivity occurred at a low concentration, indicating high potency, and was dependent on both receptors being expressed. The possible mechanism that mediates the potent cardioprotection following co-activation of both A₁ and A₃ receptors remains to be determined. The A₁ and A₃ receptors are differentially coupled to phospholipase C and phospholipase D, respectively (10). Although speculative, the diacylglycerol derived from the two phospholipases may confer an additive activation of protein kinase C and K_ATP channels, which in turn causes a greater degree of cardioprotection. Thus, agonists that activate both receptors may be highly protective within a therapeutic dose window in which only cells expressing both receptors respond, thus avoiding or diminishing adenosine-related side effects such as bradycardia and hypotension. Due to the ability of ligands designed using the "functionalized congener" approach (14) to interact potently with these receptors as the intact covalent conjugates, irrespective of the high molecular weight, the possibilities for binary drugs as new therapeutic agents are vast.

	ACKNOWLEDGEMENT

We thank Dr. Neli Melman for carrying out the binding assays.

	FOOTNOTES

^* This work was supported by Grant RO1-HL48225 (NHLBI, National Institutes of Health) and an Established Investigatorship Award (to B. T. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Molecular Recognition Section, Bldg. 8A, Rm. B1A-19, Laboratory of Bioorganic Chemistry, NIDDK, NIH, Bethesda, MD 20892-0810. Tel.: 301-496-9024; Fax: 301-480-8422; E-mail: kajacobs@helix.nih.gov.

Published, JBC Papers in Press, July 7, 2000, DOI 10.1074/jbc.M001520200

	ABBREVIATIONS

The abbreviations used are: DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; AB-MECA, N⁶-(4-amino-3-iodobenzyl)-5'-N-methylcarboxamidoadenosine; ADAC, N⁶-[4-[[[4-[[[(2-Aminoethyl)amino]carbonyl]-methyl]anilino]carbonyl]methyl]phenyl]adenosine; CGS 21680, 2-[4-[(2-carboxyethyl)phenyl]ethylamino]-5'-N-ethylcarbamoyl adenosine; CK, creatine kinase; DITC, phenylene diisothiocyanate; DMF, N,N-dimethylformamide; Me₂SO, dimethyl sulfoxide; IB-MECA, N⁶-(3- iodobenzyl)adenosine-5'-N-methyluronamide; MRS 1191, 3-ethyl 5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate; MRS 1543, conjugate of N⁶-[4-(carboxymethyl)phenyl]adenosine and N⁶-[3-(3-amino-1-propynyl)benzyl]-5'-N-methylcarboxamido adenosine; MRS 1740 and MRS 1741, m- and p-isomers, respectively, of conjugate of ADAC and N⁶-[3-[3-(3-isothiocyanatophenylaminothiocarbonyl)amino-1-propynyl]benzyl]-5'-N-methylcarboxamido adenosine; PIA, N⁶-L-phenylisopropyladenosine; TLC, thin layer chromatography; XAC, 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine; RT, room temperature.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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