Cl-IB-MECA [2-Chloro-N6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide] Reduces Ischemia/Reperfusion Injury in Mice by Activating the A Adenosine Receptor

Department of Pharmacology and Toxicology , Medical College of Wisconsin, Milwaukee, Wisconsin, United States
Journal of Pharmacology and Experimental Therapeutics (Impact Factor: 3.97). 01/2007; 319(3):1200-10. DOI: 10.1124/jpet.106.111351
Source: PubMed


We used pharmacological agents and genetic methods to determine whether the potent A(3) adenosine receptor (AR) agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (Cl-IB-MECA) protects against myocardial ischemia/reperfusion injury in mice via the A(3)AR or via interactions with other AR subtypes. Pretreating wild-type (WT) mice with Cl-IB-MECA reduced myocardial infarct size induced by 30 min of coronary occlusion and 24 h of reperfusion at doses (30 and 100 mug/kg) that concomitantly reduced blood pressure and stimulated systemic histamine release. The A(3)AR-selective antagonist MRS 1523 [3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate], but not the A(2A)AR antagonist ZM 241385 [4-{2-7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol], blocked the reduction in infarct size provided by Cl-IB-MECA, suggesting a mechanism involving the A(3)AR. To further examine the selectivity of Cl-IB-MECA, we assessed its cardioprotective effectiveness in A(3)AR gene "knock-out" (A(3)KO) mice. Cl-IB-MECA did not reduce myocardial infarct size in A(3)KO mice in vivo and did not protect isolated perfused hearts obtained from A(3)KO mice from injury induced by global ischemia and reperfusion. Additional studies using WT mice treated with compound 48/80 [condensation product of p-methoxyphenethyl methylamine with formaldehyde] to deplete mast cell contents excluded the possibility that Cl-IB-MECA was cardioprotective by releasing mediators from mast cells. These data demonstrate that Cl-IB-MECA protects against myocardial ischemia/reperfusion injury in mice principally by activating the A(3)AR.

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Available from: Zhi-Dong Ge
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    • "We have previously described Langendorff perfusion of mouse hearts.[22], [26]–[28] Briefly, once excised, the hearts were mounted on a Langendorff apparatus and perfused retrogradely through the aorta at a constant pressure of 80 mmHg with Krebs-Henseleit buffer containing (in mM) NaCl 118, NaHCO3 25, KCl 4.7, MgCl2 1.2, CaCl2 2.5, KH2PO4 1.2, EDTA 0.5, and glucose 11. The buffer was continuously bubbled with a mixture of 95% oxygen/5% carbon dioxide via an in-line filter (5 µm pore size). "
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    ABSTRACT: Endothelial-myocardial interactions may be critically important for ischemia/reperfusion injury. Tetrahydrobiopterin (BH4) is a required cofactor for nitric oxide (NO) production by endothelial NO synthase (eNOS). Hyperglycemia (HG) leads to significant increases in oxidative stress, oxidizing BH4 to enzymatically incompetent dihydrobiopterin. How alterations in endothelial BH4 content impact myocardial ischemia/reperfusion injury remains elusive. The aim of this study was to examine the effect of endothelial-myocardial interaction on ischemia/reperfusion injury, with an emphasis on the role of endothelial BH4 content. Langendorff-perfused mouse hearts were treated by triton X-100 to produce endothelial dysfunction and subsequently subjected to 30 min of ischemia followed by 2 h of reperfusion. The recovery of left ventricular systolic and diastolic function during reperfusion was impaired in triton X-100 treated hearts compared with vehicle-treated hearts. Cardiomyocytes (CMs) were co-cultured with endothelial cells (ECs) and subsequently subjected to 2 h of hypoxia followed by 2 h of reoxygenation. Addition of ECs to CMs at a ratio of 1∶3 significantly increased NO production and decreased lactate dehydrogenase activity compared with CMs alone. This EC-derived protection was abolished by HG. The addition of 100 µM sepiapterin (a BH4 precursor) or overexpression of GTP cyclohydrolase 1 (the rate-limiting enzyme for BH4 biosynthesis) in ECs by gene trasfer enhanced endothelial BH4 levels, the ratio of eNOS dimer/monomer, eNOS phosphorylation, and NO production and decreased lactate dehydrogenase activity in the presence of HG. These results demonstrate that increased BH4 content in ECs by either pharmacological or genetic approaches reduces myocardial damage during hypoxia/reoxygenation in the presence of HG. Maintaining sufficient endothelial BH4 is crucial for cardioprotection against hypoxia/reoxygenation injury.
    Full-text · Article · Jul 2013 · PLoS ONE
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    • "The large size of the dog also precluded extensive studies to investigate potential molecular mechanisms by which LUF6096 provides protection from ischemia/reperfusion injury. Earlier studies suggest that A 3 AR activation may promote cell survival by facilitating opening of the K ATP channel in cardiac myocytes and suppressing the robust inflammatory response that occurs during reperfusion (Auchampach et al., 1997b; Black et al., 2002; Ge et al., 2006, 2010; Wan et al., 2008). Although we found that LUF6096 functioned effectively as a PAM of the dog A 3 AR, its pharmacological profile versus the dog receptor was slightly different from that observed previously with the human A 3 AR (Heitman et al., 2009; Göblyös and IJzerman, 2011). "
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    ABSTRACT: Adenosine is increased in ischemic tissues where it serves a protective role by activating adenosine receptors (ARs), including the A₃ AR subtype. We investigated the effect of N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarboxamide (LUF6096), a positive allosteric modulator of the A₃ 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 (EC₅₀ 114.3 ± 15.9 nM) with the canine A₃ AR in a guanosine 5'-[γ-[³⁵S]thio]triphosphate binding assay. LUF6096 increased the maximal efficacy of the partial A₃ AR agonist 2-chloro-N⁶-(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 A₃ AR allosteric enhancer in an in vivo model of infarction.
    Full-text · Article · Jan 2012 · Journal of Pharmacology and Experimental Therapeutics
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    • "Of the four subtypes, A1 and A3ARs present in the heart are well known to mediate the cardioprotective effects of adenosine [19-22]. However, unlike the A1AR, when the A3AR is activated by selective agonists, such as Cl-IB-MECA 1 (Figure 1), it does not induce detrimental hemodynamic side effects, such as negative chronotropy, inotropy and dromotropy [20,23-25]. "
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    ABSTRACT: When stimulated by small molecular agonists, the A3 adenosine receptor (AR) mediates cardioprotective effects without inducing detrimental hemodynamic side effects. We have examined pharmacologically the protective properties of a multivalent dendrimeric conjugate of a nucleoside as a selective multivalent agonist for the mouse A3AR. A PAMAM dendrimer fully substituted by click chemistry on its peripheral groups with 64 moieties of a nucleoside agonist was shown to be potent and selective in binding to the mouse A3AR and effective in cardioprotection in an isolated mouse heart model of ischemia/reperfusion (I/R) injury. This conjugate MRS5246 and a structurally related model compound MRS5233 displayed binding Ki values of 0.04 and 3.94 nM, respectively, and were potent in in vitro functional assays to inhibit cAMP production. A methanocarba (bicyclo[3.1.0]hexane) ring system in place of ribose maintained a North conformation that is preferred at the A3AR. These analogues also contained a triazole linker along with 5'-N-methyl-carboxamido and 2-alkynyl substitution, previously shown to be associated with species-independent A3AR selectivity. Both MRS5233 and MRS5246 (1 and 10 nM) were effective at increasing functional recovery of isolated mouse hearts after 20 min ischemia followed by 45 min reperfusion. A statistically significant greater improvement in the left ventricular developed pressure (LVDP) by MRS5246 compared to MRS5233 occurred when the hearts were observed throughout reperfusion. Unliganded PAMAM dendrimer alone did not have any effect on functional recovery of isolated perfused mouse hearts. 10 nM MRS5246 did not improve functional recovery after I/R in hearts from A3AR gene "knock-out" (A3KO) mice compared to control, indicating the effects of MRS5246 were A3AR-specific. Covalent conjugation to a versatile drug carrier enhanced the functional potency and selectivity at the mouse A3AR and maintained the cardioprotective properties. Thus, this large molecular weight conjugate is not prevented from extravasation through the coronary microvasculature.
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