Quantitative analyses of CTP-499 and five major metabolites by core-structure analysis

CTP-499 is a novel oral multi-subtype selective inhibitor of PDEs that is currently in clinical testing, in combination with angiotensin modulators, as a potentially first-in-class treatment for diabetic kidney disease. The compound was discovered and developed by using Concertʹs proprietary DCE Platform® in which deuterium was incorporated at select positions of 1-((S)-5-hydroxyhexyl)-3,7-dimethylxanthine (HDX). CTP-499 metabolizes to five major metabolites: C-21256, D-M2, D-M3, D-M4 and M5, of which all contains deuterium except M5. During in vivo metabolism, however, H/D exchange takes place. As a result, each analyte, except M5, has multiple molecular masses. To accurately quantify the analytes, we developed an LC–MS/MS method focusing on the core structures of the molecules, termed “core-structure analyses”. The core-structure analyses method was then validated under GLP guidance in dog, rat and rabbit plasma, with a sample volume of 50 μL. Results demonstrated that this approach accurately quantifies each of the six analytes despite partial exchange of deuterium with hydrogen atoms in the in vivo samples. The validation parameters included accuracy, precision, sensitivity, stability, dilution integrity, hemolysis, matrix effect, selectivity, and recovery. Acceptable intra-run and inter-run assay precision (%CV ≤ 5.5%) and accuracy (90.1–106.7%) were achieved over a linear range of 10–5000 ng/mL of each analyte. Various stability tests, including bench-top, freeze/thaw, stock solution, and long-term storage, were also performed. All stability results met acceptance criteria. The robustness of the methods was demonstrated by the incurred sample reproducibility (ISR) tests. After validation, the method was successfully used in support of multiple toxicological studies of CTP-499.