Ridong Chen

Optimizing human apyrase to treat arterial thrombosis and limit reperfusion injury without increasing bleeding risk

APT102, an optimized human apyrase designed for treatment of myocardial infarction, improves recanalization of occluded arteries and decreases heart muscle damage, without an increased bleeding risk. A Better Antithrombotic, Without Bleeding The drugs that prevent blood clots—the cause of heart attacks and strokes—almost always also increase the risk of dangerous bleeding. Now, Moeckel et al. report that the enzyme apyrase can be engineered to degrade the prothrombotic molecule adenosine diphosphate (ADP) faster and longer than the native version, thereby preventing clots without increasing bleeding. Delivered to dogs or mice with heart attacks, the optimized apyrase kept the concentration of ADP low, averting the blood cell aggregation that initiates a clot. At the highest dose tested, the animals showed less heart damage but did not bleed. Clopidogrel, a common drug used currently in this situation, was less effective in minimizing heart damage and caused bleeding. In patients with acute myocardial infarction undergoing reperfusion therapy to restore blood flow through blocked arteries, simultaneous inhibition of platelet P2Y12 receptors with the current standard of care neither completely prevents recurrent thrombosis nor provides satisfactory protection against reperfusion injury. Additionally, these antiplatelet drugs increase the risk of bleeding. To devise a different strategy, we engineered and optimized the apyrase activity of human nucleoside triphosphate diphosphohydrolase-3 (CD39L3) to enhance scavenging of extracellular adenosine diphosphate, a predominant ligand of P2Y12 receptors. The resulting recombinant protein, APT102, exhibited greater than four times higher adenosine diphosphatase activity and a 50 times longer plasma half-life than did native apyrase. Treatment with APT102 before coronary fibrinolysis with intravenous recombinant human tissue-type plasminogen activator in conscious dogs completely prevented thrombotic reocclusion and significantly decreased infarction size by 81% without increasing bleeding time. In contrast, clopidogrel did not prevent coronary reocclusion and increased bleeding time. In a murine model of myocardial reperfusion injury caused by transient coronary artery occlusion, APT102 also decreased infarct size by 51%, whereas clopidogrel was not effective. These preclinical data suggest that APT102 should be tested for its ability to safely and effectively maximize the benefits of myocardial reperfusion therapy in patients with arterial thrombosis.

Combination treatment of r-tPA and an optimized human apyrase reduces mortality rate and hemorrhagic transformation 6h after ischemic stroke in aged female rats.

Recombinant tissue plasminogen activator (r-tPA) is the only FDA-approved drug treatment for ischemic stroke and must be used within 4.5h. Thrombolytic treatment with r-tPA has deleterious effects on the neurovascular unit that substantially increases the risk of intracerebral hemorrhage if administered too late. These therapeutic shortcomings necessitate additional investigation into agents that can extend the therapeutic window for safe use of thrombolytics. In this study, combination of r-tPA and APT102, a novel form of human apyrase/ADPase, was investigated in a clinically-relevant aged-female rat embolic ischemic stroke model. We propose that successfully extending the therapeutic window of r-tPA administration would represent a significant advance in the treatment of ischemic stroke due to a significant increase in the number of patients eligible for treatment. Results of our study showed significantly reduced mortality from 47% with r-tPA alone to 16% with co-administration of APT102 and r-tPA. Co-administration decreased cortical (47 ± 5% vs. 29 ± 5%), striatal (50 ± 2%, vs. 40 ± 3%) and total (48 ± 3%vs. 33 ± 4%) hemispheric infarct volume compared to r-tPA alone. APT102 improved neurological outcome (8.9±0.6, vs. 6.8 ± 0.8) and decreased hemoglobin extravasation in cortical tissue (1.9 ± 0.1mg/dl vs. 1.4 ± 0.1mg/dl) striatal tissue (2.1 ± 0.3mg/dl vs. 1.4 ± 0.1mg/dl) and whole brain tissue (2.0 ± 0.2mg/dl vs. 1.4 ± 0.1mg/dl). These data suggest that APT102 can safely extend the therapeutic window for r-tPA mediated reperfusion to 6h following experimental stroke without increased hemorrhagic transformation. APT102 offers to be a viable adjunct therapeutic option to increase the number of clinical patients eligible for thrombolytic treatment after ischemic stroke.

A non-contrast CMR index for assessing myocardial fibrosis

PURPOSE Safe, sensitive, and non-invasive imaging methods to assess the presence, extent, and turnover of myocardial fibrosis are needed for early stratification of risk in patients who might develop heart failure after myocardial infarction. We describe a non-contrast cardiac magnetic resonance (CMR) approach for sensitive detection of myocardial fibrosis using a canine model of myocardial infarction and reperfusion. METHODS Seven dogs had coronary thrombotic occlusion of the left anterior descending coronary arteries followed by fibrinolytic reperfusion. CMR studies were performed at 7days after reperfusion. A CMR spin-locking T1ρ mapping sequence was used to acquire T1ρ dispersion data with spin-lock frequencies of 0 and 511Hz. A fibrosis index map was derived on a pixel-by-pixel basis. CMR native T1 mapping, first-pass myocardial perfusion imaging, and post-contrast late gadolinium enhancement imaging were also performed for assessing myocardial ischemia and fibrosis. Hearts were dissected after CMR for histopathological staining and two myocardial tissue segments from the septal regions of adjacent left ventricular slices were qualitatively assessed to grade the extent of myocardial fibrosis. RESULTS Histopathology of 14 myocardial tissue segments from septal regions was graded as grade 1 (fibrosis area, <20% of a low power field, n=9), grade 2 (fibrosis area, 20-50% of field, n=4), or grade 3 (fibrosis area, >50% of field, n=1). A dramatic difference in fibrosis index (183%, P<0.001) was observed by CMR from grade 1 to 2, whereas differences were much smaller for T1ρ (9%, P=0.14), native T1 (5.5%, P=0.12), and perfusion (-21%, P=0.05). CONCLUSION A non-contrast CMR index based on T1ρ dispersion contrast was shown in preliminary studies to detect and correlate with the extent of myocardial fibrosis identified histopathologically. A non-contrast approach may have important implications for managing cardiac patients with heart failure, particularly in the presence of impaired renal function.