Platelet activation plays a crucial role in hemostasis and thrombosis. Thrombin, the most potent stimulus of platelet activation, mediates platelet activation via the protease activated receptors (PARs). The platelet PAR repertoire in mediating thrombin's action differs across species. Only nonhuman primate (NHP) platelet activation is known to be similar to humans, mediated by PAR1 and PAR4, hence limiting translational in vivo studies of PAR's role in thrombosis and hemostasis to NHPs. Earlier studies have demonstrated a range of distinct in vitro activities of PAR1 and 4 in platelet activation yet the implications of these events in vivo is unclear. The objective of this study is to investigate and compare the roles of PAR1 and PAR4 in hemostasis and thrombosis in a relevant animal species. NHP models for pharmacokinetic, ex vivo platelet aggregation responses, FeCI3 injury-mediated arterial thrombosis and template bleeding were developed in Cynomolgus Macaques. Potent and selective small molecule antagonists of PAR1 and PAR4 were characterized in an array of in vitro assays, and subsequently examined head-to-head in the NHP models. Treatment of NHPs with antagonists of PAR1 or PAR4 both resulted in strong inhibition of ex vivo platelet aggregation. At doses that led to similar inhibition of platelet aggregation, animals treated with the PAR4 antagonist showed similar levels of anti-thrombotic efficacy, but longer bleeding times, compared to animals treated with the PAR1 antagonist. These findings suggest that PAR1 antagonism will likely produce a larger therapeutic index (ie. a larger anti-thrombotic efficacy over bleeding risk margin) than PAR4 antagonism.

Vorapaxar is a novel protease-activated receptor-1 (PAR-1) antagonist recently approved for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. Patients who received vorapaxar in addition to standard of care antiplatelet therapy had an increased incidence of major bleeding events compared with placebo. To assess whether platelet transfusion can restore hemostasis in primates on triple antiplatelet therapy, template bleeding times were assessed concurrently in the buccal mucosa, finger pad, and distolateral tail of anesthetized cynomolgus macaques to evaluate bleeding with vorapaxar as either monotherapy or in combination with aspirin or aspirin and clopidogrel. Aspirin (5mg/kg, IV) or vorapaxar (1mg/kg, PO) alone had no significant effect on bleeding times in the three vascular beds examined. A modest (<2-fold) increase in bleeding time was achieved in the three beds with the dual combination of aspirin and vorapaxar. Major increases in bleeding time were achieved in the three beds with the triple combination of aspirin (5mg/kg, IV), vorapaxar (1mg/kg, PO), and clopidogrel (1mg/kg, PO). Transfusion of fresh human platelet rich plasma, but not platelet poor plasma, reversed the increase in bleeding time in the triple therapy group. Transfusion of human platelets may be a viable approach in situations requiring a rapid reversal of platelet function in individuals treated with triple anti-platelet therapy that includes vorapaxar.

Antagonism of the bradykinin B(1) receptor represents a potential treatment for chronic pain and inflammation. Novel antagonists incorporating alpha-hydroxy amides were designed that display low-nanomolar affinity for the human bradykinin B(1) receptor and good bioavailability in the rat and dog. In addition, these functionally active compounds show high passive permeability and low susceptibility to phosphoglycoprotein mediated efflux, predictive of good CNS exposure.

Alzheimer's Disease is characterized neuropathologically by the presence of amyloid β–peptide–containing plaques along with neurofibrillary tangles in the brain. A novel animal model was established to allow chronic, simultaneous sampling of CSF and plasma from conscious nonhuman primates. The model was validated with an extremely potent γ–secretase inhibitor (GSiA) which showed clear dose–related effects from minimal to near complete reduction of CSF and plasma Aβ40. A dynamic colony of cisterna magna ported (CMP) rhesus monkeys is maintained and used extensively to evaluate the potential of multimechanistic agents to alter CSF and plasma Aβ40 and Aβ42 with simultaneous plasma and CSF compound concentrations. This model allowed the ability to compare and contrast the effects of γ–secretase inhibition vs. modulation of the γ–secretase cleavage site. Crossover studies were conducted with γ–secretase inhibitors of high (GSiB) and moderate (GSiC) potency and a γ–secretase modulator (GSm) compound in an optimized, standard protocol. Baseline CSF and plasma samples were taken prior to single oral dose administration followed by sampling at multiple postdose timepoints over 7 days. Samples were assayed for CSF Aβ40, CSF Aβ42, and plasma Aβ40. Effects on CSF amyloid were seen as dose–related reduction of Aβ40 and Aβ42 by γ–secretase inhibitors and selective reduction of Aβ42 by the γ–secretase modulator. Comparison of these different mechanisms of targeting CNS γ–secretase, showed 25–30% reduction of CSF Aβ42 by all compounds that correlated to 30–40% reduction of CSF Aβ40 with inhibitors and, as predicted, no inhibitory effect on CSF Aβ40 with the modulator. Plasma Aβ40 was affected by γ–secretase inhibitors and was dependent on compound potency, dose and time after dosing; γ–secretase modulator had no effect on plasma Aβ40. As has been observed in humans and other preclinical species, plasma Aβ40 elevated above baseline after γ–secretase inhibition; time and extent of ‘overshoot‘ was dependent upon potency and dose. These comparative results highlight the opportunity to evaluate the amyloid hypothesis with varied mechanisms. CMP rhesus monkey studies are ongoing to address scientific (CSF Proteomics) and development (formulations, dosing regimes) questions with diverse compounds and to establish proof of concept with novel targets.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is an important regulator of fibrinolysis, and inhibitors of this enzyme have potential use in antithrombotic and thrombolytic therapy. Appropriately substituted imidazole acetic acids such as 10j were found to be potent inhibitors of activated TAFI and selective versus the related carboxypeptidases CPA, CPN, and CPM but not CPB. Further, 10j accelerated clot lysis in vitro and was shown to be efficacious in a primate model of thrombosis.