Endogenous Protein C Activation with AB002 Improves Outcomes in a Rat Model of Acute Ischemic Stroke

Background: The hemorrhagic side-effects of current thrombolytic agents limit their usefulness to a small subset of acute ischemic stroke patients. An early, effective, and hemostatically safe antithrombotic/neuroprotectant in addition to standard of care could improve outcomes. AB002 (E-WE thrombin) is an engineered recombinant thrombin analog (W215A/E217A) that activates cytoprotective and antithrombotic endogenous protein C on intravascular surfaces without any measurable procoagulant activity. In renal failure patients undergoing hemodialysis, AB002 treatment reduced dialyzer clotting without increasing markers of hemostasis impairment or coagulation (NCT03963895). Preclinical studies also showed that AB002 exhibits potent antithrombotic/thrombolytic effects in a non-human primate model of acute vascular graft thrombosis, as well as reduces infarct size and improves outcomes in an experimental acute myocardial ischemia-reperfusion model in mice. Aims: Here, we evaluated whether neuroprotective effects of AB002 can be observed following middle cerebral artery occlusion-recanalization (MCAO-R) in rats. Methods: The local animal care and use committee approved all studies. 8 to 9-week-old male rats (275-377 g) were anesthetized under isoflurane and a small laser-Doppler probe was affixed to the skull to monitor cortical perfusion and verify vascular occlusion and reperfusion. A silicone-coated 4-0 nylon monofilament was inserted into the right internal carotid artery (ICA) via the external carotid artery (ECA) until the laser-Doppler signal dropped to < 30 % of baseline. To evaluate the effect of AB002 on outcomes, rats (n = 4/ group) were treated with 25 µg/kg (0.25 mL/kg; i.v.) or saline placebo, administered 30 min after induction of ischemia. Since prolongation of the activated partial plasminogen time (aPTT) is a pharmacologic effect of high dose drug exposure, a 0.45 mL blood sample was collected at 20 min post treatment from the right jugular vein, processed to plasma and frozen for subsequent aPTT evaluation. At 90 min, the filament was removed, the surgical incisions were sutured closed and postoperative animals were monitored during recovery. Neurological performance scores and weight were assessed daily for 4 days. Rats were sacrificed on day 7 post-surgery and infarct size was measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining. To account for the effect of edema, the infarcted area was estimated indirectly by subtracting the non-infarcted area in the ipsilateral hemisphere from the contralateral hemisphere and expressing infarct volume as a percentage of contralateral hemisphere. APTT was measured in duplicate using a KC-4 coagulometer. All analyses were performed by researchers blinded to treatment. Results: Compared to placebo, rats that received AB002 exhibited less ischemic damage, evidenced by a statistically significant reduction in infarct volume of the cortex (32.3% versus 8.9%, P < 0.05) and hemisphere (28.0% versus 9.8%, P < 0.01). In addition, rats had better neurological scores and greater weight gains during recovery following treatment with AB002, compared to placebo. Mean aPTT values were higher in AB002 treated rats compared to placebo (34 ± 2.2 s versus 21 ± 1.6 s), confirming drug exposure. Conclusions: Treatment with AB002 during ischemia reduced infarct volume and improved outcomes in the rat experimental stroke model. Considering its neuroprotective and antithrombotic properties, we propose that early treatment with AB002 may confer outcome benefit for patients experiencing acute thrombotic emergencies.