The broad objective of this program is to perform preclinical experimentation on animal models of myocardial ischemia to elucidate the mechanisms of cellular death in the myocardium and development of the subsequent CHF and to evaluate the potential of different therapeutic modalities. The ultimate goal is to limit the extent of myocardial damage and to prevent or attenuate the development of CHF. Erythropoietin (EPO), a cytokine known to stimulate erythropoiesis and widely used to treat the anemia of different etiology, had been recently reported to suppress apoptosis and to reduce the extent of damage in brain tissue following experimental ischemia or trauma. We had reported that, similar to brain tissue, a single systemic injection of recombinant human EPO following coronary ligation reduce apoptosis in the myocardial area at risk, attenuated the early left ventricular remodeling, and, eight weeks later, resulted in the reduction of the infarct size and the extent of structural and functional deterioration of the heart. We had also reported the results of studies that defined therapeutic doses and the therapeutic window of rhEPO in the rat model of MI. However, repeated dosing of rhEPO obviously causes a marked elevation of hematocrit. Moreover, it has been reported that even a single injection of rhEPO resulted in a significant elevation of the level of reticulocytes, which could represent an additional risk for MI patients. Therefore, it would be advantageous to have a compound that would have tissue protective properties of EPO without its erythropoietic effect. The purpose of the current studies was to investigate the possible therapeutic effects of a small molecule designed by Warren Pharmaceutical on the basis of EPO structure, a pyroglutamate helix B surface peptide (pHBP) that includes only a part of the EPO molecule that does not bind to EPO receptor and thus, is not erythropoietic, but retains tissue protective properties of EPO. This work had been done in collaboration with researchers from Warren Pharmaceutical and with Drs. Sollott and Boheler of the LCS. We compared the ability of pHBP and EPO to protect cardiac myocytes from oxidative stress in vitro and cardiac tissue from ischemic damage in vivo. HBP, similar to EPO, increased the reactive oxygen species (ROS) threshold for induction of the mitochondrial permeability transition by 40%. In an experimental model of myocardial infarction induced by permanent ligation of a coronary artery in rats, a single bolus injection of 60 g/kg of pHBP immediately after coronary ligation, similar to EPO, reduced apoptosis in the myocardial area at risk, examined 24 h later, by 80% and inflammation by 34%. Myocardial infarction (MI) measured 24 h after coronary ligation was similarly reduced by 50% in both pHBP- and EPO-treated rats. Two wks after surgery, left ventricular remodeling and functional decline assessed via echocardiography were significantly and similarly attenuated in pHBP- and EPO-treated rats, and MI size was reduced by 25%. The effect was retained during the 6-wk follow-up. A single bolus injection of pHBP immediately after coronary ligation was effective in reduction of MI size in a dose as low as 1 g/kg, but was ineffective at a 60 g/kg dose if administered 24 h after MI induction. We conclude that pHBP is equally cardioprotective with EPO and deserves further consideration as a safer alternative to rhEPO in the search for therapeutic options to reduce myocardial damage following blockade of the coronary circulation. Despite positive outcomes observed in the vast majority of animal experiments demonstrating strong cardioprotective properties of rhEPO in the models of myocardial infarction, the results of several phase II clinical trials in humans concluded recently were far less encouraging. As a result, the enthusiasm for this new possible therapeutic intervention was greatly diminished. We postulated that time that elapsed between symptoms onset and rhEPO administration in negative clinical trials was much longer that in successful animal experiments. To test a hypothesis that in negative clinical trials of erythropoietin in patients with acute myocardial infarction (MI) the erythropoietin (rhEPO) could be administered outside the narrow therapeutic window, we designed an animal experiment that imitated that general design of clinical trials. MI was induced in rats either by a permanent ligation of a descending coronary artery or by a 2-hr occlusion followed by a reperfusion. rhEPO, 3000 IU/kg, was administered intraperitoneally at the time of reperfusion, 4 hrs after beginning of reperfusion, or 6 hrs after permanent occlusion. MI size was measured histologically 24 hrs after coronary occlusion. The area of myocardium at risk was similar among groups. The MI size in untreated rats averaged 42% of area at risk, or 24% of left ventricle, and was reduced by more than 50% (p<0.001) in rats treated with rhEPO at the time of reperfusion. The MI size was not affected by treatment administered 4 hrs after reperfusion or 6 hrs after permanent coronary occlusion. Therefore, our study in a rat experimental model of MI demonstrates that rhEPO administered within 2 hrs of a coronary occlusion effectively reduces MI size, but when rhEPO was administered following a delay similar to that encountered in clinical trials, it had no effect on MI size. Results of our experiment strongly indicate that clinical trials that failed to demonstrate rhEPO efficacy in patients with MI may have missed a narrow therapeutic window defined in animal experiments.
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