Protection from myocardial ischemia/reperfusion injury by a positive allosteric modulator of the A ₃ adenosine receptor

Adenosine is increased in ischemic tissues where it serves a protective role by activating adenosine receptors (ARs), including the A ₃ AR subtype. We investigated the effect of N-{2-[(3,4- dichlorophenyl)amino] quinolin-4-yl}cyclohexanecarboxamide (LUF6096), a positive allosteric modulator of the A ₃ AR, on infarct size in a barbital-anesthetized dog model of myocardial ischemia/reperfusion injury. Dogs were subjected to 60 min of coronary artery occlusion and 3 h of reperfusion. Infarct size was assessed by macrohistochemical staining. Three experimental groups were included in the study. Groups I and II received two doses of vehicle or LUF6096 (0.5 mg/kg i.v. bolus), one administered before ischemia and the other immediately before reperfusion. Group III received a single dose of LUF6096 (1 mg/kg i.v. bolus) immediately before reperfusion. In preliminary in vitro studies, LUF6096 was found to exert potent enhancing activity (EC ₅₀ 114.3 ± 15.9 nM) with the canine A ₃ AR in a guanosine 5′-[γ-[ ³⁵S]thio]triphosphate binding assay. LUF6096 increased the maximal efficacy of the partial A ₃ AR agonist 2-chloro-N ⁶-(3- iodobenzyl)adenosine-5′-N-methylcarboxamide and the native agonist adenosine more than 2-fold while producing a slight decrease in potency. In the dog studies, administration of LUF6096 had no effect on any hemodynamic parameter measured. Pretreatment with LUF6096 before coronary occlusion and during reperfusion in group II dogs produced a marked reduction in infarct size (∼50% reduction) compared with group I vehicle-treated dogs. An equivalent reduction in infarct size was observed when LUF6096 was administered immediately before reperfusion in group III dogs. This is the first study to demonstrate efficacy of an A ₃ AR allosteric enhancer in an in vivo model of infarction.