Differential anti-thrombotic benefit and bleeding risk profiles of antagonists of protease-activated receptor 1 and 4 in Cynomolgus Macaques.

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.

Platelet transfusion reverses bleeding evoked by triple anti-platelet therapy including vorapaxar, a novel platelet thrombin receptor antagonist.

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.

Alpha-hydroxy amides as a novel class of bradykinin B1 selective antagonists.

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.

Determination of potato carboxypeptidase inhibitor in African Green Monkey plasma using 96-well SPE and LC-MS/MS.

Potato carboxypeptidase inhibitor (CPI), a peptide with multiple isoforms (MW>4000 Da) was determined from African Green Monkey plasma using a PE Sciex API-3000 LC-MS/MS in the positive ionization mode with the turbo ionspray interface (450 degrees C). Samples were prepared using an Oasis MCX 96-well solid phase extraction plate and chromatographed on an Allure C18 HPLC Column (50 mm x 1.0 mm, 5 microm) using gradient elution. Upon analysis of the extracts using LC-MS/MS, the concentration of CPI was calculated using a single MS/MS transition (m/z 830.5-->221.0) that was reflective of the mass concentration (microg/mL) of main the CPI isoforms present in plasma from monkeys after they were given an intravenous dose of CPI. The assay was linear for CPI over concentrations of 0.05-10 microg/mL when extracting 200-microL aliquots of African Green Monkey plasma. The assay was applied to the determination of CPI in African Green Monkey plasma samples in two separate analytical runs (correlation of standard curves, r1=0.9991 and r2=0.9953). Quality control (QC) samples were run at 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 microg/mL for each assay. Average ranges (n=12) for accuracy and precision for all concentrations of QCs during the two runs were 92.0-102.0% of expected potency and 10.4-21.8% (coefficient of variations), respectively.

9-hydroxyazafluorenes and their use in thrombin inhibitors.

Optimization of a previously reported thrombin inhibitor, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-trans-4-aminocyclohexylmethylamide (1), by replacing the aminocyclohexyl P1 group provided a new lead structure, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (2), with improved potency (K(i) = 0.49 nM for human thrombin, 2x APTT = 0.37 microM in human plasma) and pharmacokinetic properties (F = 39%, iv T(1/2) = 13 h in dogs). An effective strategy for reducing plasma protein binding of 2 and improving efficacy in an in vivo thrombosis model in rats was to replace the lipophilic fluorenyl group in P3 with an azafluorenyl group. Systematic investigation of all possible azafluorenyl P3 isomers and azafluorenyl-N-oxide analogues of 2 led to the identification of an optimal compound, 3-aza-9-hydroxyfluoren-9(R)-ylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (19b), with high potency (K(i) = 0.40 nM, 2x APTT = 0.18 microM), excellent pharmacokinetic properties (F = 55%, T(1/2) = 14 h in dogs), and complete efficacy in the in vivo thrombosis model in rats (inhibition of FeCl(3)-induced vessel occlusions in six of six rats receiving an intravenous infusion of 10 microg/kg/min of 19b). The stereochemistry of the azafluorenyl group in 19b was determined by X-ray crystallographic analysis of its N-oxide derivative (23b) bound in the active site of human thrombin.

Synthesis and evaluation of imidazole acetic acid inhibitors of activated thrombin-activatable fibrinolysis inhibitor as novel antithrombotics.

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.

An alternative method of chronic cerebrospinal fluid collection via the cisterna magna in conscious rhesus monkeys.

Models of chronic cerebrospinal fluid (CSF) collection previously have been established for nonhuman primates and canines; many of these methods implement stainless-steel cannulas into the lateral or 4th ventricles or catheters into the cerebral or spinal subarachnoid space. These models have proved successful and reliable but unfortunately require invasive techniques to pass through the skull or require a laminectomy to enter the spinal subarachnoid space, involve the use of expensive and highly specialized stereotaxic equipment for the precise placement of the implants, and may require exteriorized hardware which is cumbersome to maintain and unaesthetic. The model we developed for the rhesus monkey allows for direct access to CSF outflow from the cisterna magna by using a 3.5-French fenestrated silicone catheter which was placed 1.0 cm into the cisterna. The catheter was attached to a titanium port placed subcutaneously between the scapulae to permit easy access for sampling CSF in a conscious, chaired rhesus monkey. We currently have instrumented animals from which we have consistently collected CSF for over 18 months. This novel, economical, less-invasive method permits chronic, reliable collection of CSF in conscious rhesus monkeys and has the additional advantages that the model is easier to maintain and more aesthetic.

Metabolism-directed optimization of 3-aminopyrazinone acetamide thrombin inhibitors. Development of an orally bioavailable series containing P1 and P3 pyridines.

Recent efforts in the field of thrombin inhibitor research have focused on the identification of compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe a metabolism-based approach to the optimization of the 3-(2-phenethylamino)-6-methylpyrazinone acetamide template (e.g., 1) which resulted in the modification of each of the three principal components (i.e., P1, P2, P3) comprising this series. As a result of these studies, several potent thrombin inhibitors (e.g., 20, 24, 25) were identified which exhibit high levels of oral bioavailability and long plasma half-lives.