Investigations of Bi-phasic Drug Depletion in Liver Microsomes and Hepatocytes in Metabolic Stability Studies

Abstract

Development of a new drug is a long and costly process and to avoid attritions due to inadequate metabolic and pharmacokinetic data it is important to determine these early by the use of in vitro systems. The intrinsic clearance (CLint) is calculated from the disappearance of test compound in the incubation plate over time and for most compounds this disappearance is linear, however, some compounds show a bi-phasic depletion profile which can cause under predictions of in vivo clearance when using the same calculation model. To improve the quality of the assay for these compounds the reasons behind the bi-phasic depletion profile were investigated. A set of 26 carboxylic acids were incubated with rat hepatocytes and human liver microsomes (HLM) on robot and the disappearance was determined by LC-MSMS on HSS T3, CSH and BEH C18 columns. The incubations with HLM suggest that conjugative metabolism dominated for this set of compounds. Viability and matrix matched zero time point had no impact on the depletion curve. Mass/chromatographic interferences from co-elution of metabolites were observed for two of the compounds which likely had an impact on the response but this could not explain the bi-phasic profile. The identities of the hydroxy and acyl glucuronide metabolites were confirmed on quadrupole time-of-flight (Q-TOF). A correlation was found between high LogD7.4 and a more bi-phasic appearance at higher cell concentrations. The depletion was modelled with a bi-exponential model that produced unbiased CLint values that were less prone to cause under prediction of in vivo clearance