Mitigation of reactive metabolite formation for a series of 3-amino-2-pyridone inhibitors of Bruton's tyrosine kinase (BTK).

Reactive metabolites have been putatively linked to many adverse drug reactions including idiosyncratic toxicities for a number of drugs with black box warnings or withdrawn from the market. Therefore, it is desirable to minimize the risk of reactive metabolite formation for lead molecules in optimization, in particular for non-life threatening chronic disease, to maximize benefit to risk ratio. This article describes our effort in addressing reactive metabolite issues for a series of 3-amino-2-pyridone inhibitors of BTK, e.g. compound 1 has a value of 459pmol/mg protein in the microsomal covalent binding assay. Parallel approaches were taken to successfully resolve the issues: establishment of a predictive screening assay with correlation association of covalent binding assay, identification of the origin of reactive metabolite formation using MS/MS analysis of HLM as well as isolation and characterization of GSH adducts. This ultimately led to the discovery of compound 7 (RN941) with significantly reduced covalent binding of 26pmol/mg protein.

Development and Validation of an Analytical Method to Quantitate Hydroxycitric Acid, the Key Constituent in Garcinia cambogia Extract, in Rodent Plasma and Fetus.

Garcinia cambogia extract (GCE) is a popular botanical supplement used in weight loss products. Hydroxycitric acid (HCA) is the principal component in GCE. Due to lack of adequate toxicity data to assess the safe use of GCE, the National Toxicology Program is testing GCE in Hsd:Sprague Dawley® SD® rats following perinatal exposure and in adult B6C3F1/N mice. We report a validated method utilizing sample clean up with ultrafiltration followed by liquid chromatography-tandem mass spectrometry analysis to quantify HCA in rat plasma over the concentration range of 20 to 800 ng/mL. The method was linear (r2 ≥ 0.99) with the limits of quantitation (LOQ) and detection (LOD) of 20.0 and 3.9 ng/mL plasma, respectively. The accuracy (determined as relative error, RE) and precision (determined as relative standard deviation, RSD) using Quality Control standards analyzed over multiple days were ≤ ± 7.5% and ≤ 9.5%, respectively. The method can be applied to quantify HCA in study matrices (RE ≤ ± 23.0%; RSD ≤ 6.0) except gestational day (GD)18 fetus. The method was partially validated in GD18 fetal homogenate over the concentration range 60-3000 ng/g (r2 ≥ 0.99, RE ≤ ± 11.9%, and RSD ≤ 5.5%; LOQ 60.0 ng/g; LOD 7.77 ng/g). The standards as high as 20,000 ng/mL (plasma) and 502,000 ng/g (fetus) can successfully be quantified after diluting into the validated range (RE ≤ ± 2.6%; RSD ≤ 5.2%). These data demonstrate that the method is suitable to quantify HCA in rodent matrices and can be adapted to other biological matrices.