Impact of Intracellular Concentrations on Metabolic Drug-Drug Interaction Studies

Abstract

Accurate prediction of drug-drug interactions (DDI) is a challenging task in drug discovery and development. It requires determination of enzyme inhibition in vitro which is highly system-dependent for many compounds. The aim of this study was to investigate whether the determination of intracellular unbound concentrations in primary human hepatocytes can be used to bridge discrepancies between results obtained using human liver microsomes and hepatocytes. Specifically, we investigated if Kp uu could reconcile differences in CYP enzyme inhibition values (K i or IC 50 ). Firstly, our methodology for determination of Kp uu was optimized for human hepatocytes, using four well-studied reference compounds. Secondly, the methodology was applied to a series of structurally related CYP2C9 inhibitors from a Roche discovery project. Lastly, the Kp uu values of three commonly used CYP3A4 inhibitors—ketoconazole, itraconazole, and posaconazole—were determined and compared to compound-specific hepatic enrichment factors obtained from physiologically based modeling of clinical DDI studies with these three compounds. Kp uu obtained in suspended human hepatocytes gave good predictions of system-dependent differences in vitro . The Kp uu was also in fair agreement with the compound-specific hepatic enrichment factors in DDI models and can therefore be used to improve estimations of enrichment factors in physiologically based pharmacokinetic modeling. Electronic supplementary material The online version of this article (10.1208/s12248-019-0344-8) contains supplementary material, which is available to authorized users.

Influence of Proteome Profiles and Intracellular Drug Exposure on Differences in CYP Activity in Donor-Matched Human Liver Microsomes and Hepatocytes

“…Intracellular unbound drug concentration has previously been used in: bridging differences between biochemical and cellular potency assays (IC 50 ); 21 predicting time-dependent CYP inhibition; 22 and explaining differences in CYP enzyme inhibition in microsomes and hepatocytes. 23 We therefore investigated whether Kp uu could also explain the observed system-dependent differences in metabolic CL int of the five substrates. The hypothesis was that active transport and/or metabolic processes in intact hepatocytes could result in non-unity Kp uu , that is, that more or less compound is available for metabolism in HH than in HLM.…”

“… 22 Furthermore, the intracellular unbound concentration can be used as a scaling factor to explain differences in CYP enzyme inhibition in both microsomes and hepatocytes. 23 …”

Primary Human Hepatocyte Spheroid Model as a 3D In Vitro Platform for Metabolism Studies

“…Primary human hepatocytes (PHH) in suspension or in shortterm monolayer cultures, are well-established in vitro screening tools routinely used in drug discovery for hepatic metabolism studies. In addition to determining drug clearance, 1e3 they are used for metabolite identification, 4,5 enzyme inhibition, 6,7 and enzyme induction studies. 8,9 PHH can also be used as a model to study the effect on the drug target and drug safety.…”

HLM-beads: Rapid Assessment of Nonspecific Binding to Human Liver Microsomes Using Magnetizable Beads

“…For compounds possessing Log D 7.4 values greater than 3.5, f u,mic values determined by equilibrium dialysis were consistently greater than 2-fold lower than those values determined using the intrinsic clearance method. This suggests that the 2-to 3-fold higher f u,mic values determined for itraconazole (cLogD 7.4 = 7.31) (Treyer et al, 2019) and BIRT2584 (cLogD 7.4 = 4.4) using HLM-beads may be a more accurate assessments of the free concentration of compound in each HLM incubation. Although not assessed in this work, it should be relatively easy to simultaneously assess f u,mic values using the intrinsic clearance method and the HLM-bead method as long as the test compounds exhibit sufficient turnover at various concentrations of HLM.…”