No abstract

A higher-throughput bioanalytical method based on fast-gradient (1 min run time) high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) was developed for screen-type analyses of plasma samples from early drug discovery studies in support of exploratory pharmacodynamic studies. The HPLC system equipped with minibore column was interfaced with either atmospheric pressure chemical ionization (APCI) or electrospray (ESI) ionization techniques. The matrix ion suppression effect of both quantitative HPLC/MS/MS analyses was compared using the post-column infusion system. The use of the described methods provided advantages such as a shorter chromatographic region of ion suppression, less solvent consumption and shorter run times in comparison with standard analytical column HPLC/MS/MS methods. The analytical results obtained by both HPLC/MS/MS methods were in good agreement (within 15% of error) and displayed a good correlation with the pharmacodynamic outcome.

Antiplatelet agents are a cornerstone of therapy for atherothrombotic disease. However, despite the proven efficacy of agents targeting the thromboxane A2 (aspirin) and P2Y12 ADP (clopidogrel, prasugrel) platelet activation pathways, the residual risk for ischemic events remains considerable. Binding of thrombin to the platelet PAR-1 is an important platelet activation pathway not targeted by existing agents. Inhibition of PAR-1 may thus provide incremental clinical benefits over aspirin and ADP receptor antagonists. PAR-1 receptors expressed on endothelial cells, smooth muscle cells, monocytes and neutrophils have been reported to mediate the pro-inflammatory and chemotactic responses to thrombin. In the present study, we report pharmacologic characterization of SCH 530348, a novel thrombin receptor antagonist (TRA) selective for PAR-1, using in vitro assays with human platelet membranes and cultured human coronary artery smooth muscle cells (HCASMC), and ex vivo platelet aggregation assays in cynomolgus monkeys. The affinity of SCH 530348 for the PAR-1 receptor was determined in human platelet membranes. Functional studies involving calcium transients, thymidine incorporation, and receptor kinetics were performed in HCASMC. The oral antiplatelet effects of SCH 530348 in cynomolgus monkeys were evaluated in ex vivo platelet aggregation assays. SCH 530348 exhibited high affinity for PAR-1 receptor. SCH 530348 potently inhibited thrombin-stimulated calcium transients and thymidine incorporation in HCASMC, and displayed slow dissociation from PAR-1 receptor. In cynomolgus monkeys, SCH 530348 administered orally at doses ranging from 0.1 mg/kg to 3 mg/kg, provided rapid, complete, and sustained inhibition of thrombin receptor agonist peptide (TRAP)-induced ex vivo platelet aggregation for 24 hours. Significant inhibition of TRAP-induced platelet aggregation was maintained at 48 hours after dosing of SCH 530348. SCH 530348 is a highly selective, potent, and orally active PAR-1 antagonist. Inhibition of PAR-1 by SCH 530348 may translate into beneficial clinical effects in patients with atherothrombotic disease, and this hypothesis is currently being evaluated in 2 large trials.