Development and application of sensitive HPLC assays for NK3 antagonists in rat plasma

CAM 5500 and CAM 5187 are new nonpeptide tachykinin NK3 receptor antagonists with different lipophilicity and solubility. We have developed and validated two separate, simple HPLC methods for quantitation of these two compounds in plasma to support oral pharmacokinetic/bioavailability studies in rats. The two compounds in plasma were extracted on cyano SPE cartridges with different washing schemes to optimize extraction efficiency and chromatographic specificity. The analytes and internal standard in the resulting extracts were chromatographed on a C18 HPLC column, using mobile phases containing different phosphate buffer strengths and acetonitrile concentrations. Both compounds were detected using UV, Peak area ratios were proportional over the concentration range of 50-3000 ng ml-1 for CAM 5500, and 100-1500 ng ml-1 for CAM 5187. Stability profiles of both drugs and internal standard in rat plasma at 37 degrees C and in injection solvent at ambient temperature were good. Assay precision, based on quality controls, was < 5.6% and 13.4% (%RSD) for CAM 5500 and CAM 5187, respectively. Similarly, assay accuracy for both compounds was within +/- 7.1% and +/- 6.0% (%RE), respectively. The HPLC methods were successfully applied to assay samples from two oral bioavailability studies. Oral bioavailability studies were conducted for each compound in rats receiving a PO dose of 20 mg kg-1 or an i.v. dose of 5 mg kg-1. Despite their difference in lipophilicity and solubility, the absolute oral bioavailability of CAM 5500 (5.3 +/- 4.8%) is similar to that of CAM 5187 (8.8% +/-3.2%).