In Vitro-In Vivo Assessment of Repaglinide Metabolism and Drug-Drug Interactions:Towards a Physiologically-Based Pharmacokinetic Model

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Abstract

Repaglinide is currently recommended as an in vivo CYP2C8 probe by the U.S. Food and Drug Administration (FDA), but the kinetic characterisation and enzymes involved in the elimination of this drug have not been fully delineated. In addition to its complex metabolism, polymorphism in the SLCO1B1 gene encoding for the hepatic uptake transporter organic anion transporting polypeptide 1B1 (OATP1B1) has been shown to impact repaglinide pharmacokinetics, further complicating the prediction of repaglinide clearance and drug-drug interactions (DDIs). The aim of this thesis was to firstly perform a systematic analysis of repaglinide metabolic pathways and thereby assess the contribution of specific enzymes to its clearance, and to secondly increase the understanding of repaglinide as a victim drug by implementing obtained in vitro metabolism data together with reported hepatic uptake parameters into a physiologically-based pharmacokinetic (PBPK) model. Furthermore, reported repaglinide DDIs, repaglinide AUC in different SLCO1B1 genotype groups and repaglinide P450 metabolite ratios were collated and critically analysed.The metabolism of repaglinide was characterised using a range of in vitro systems, namely pooled cryopreserved human hepatocytes, human liver microsomes (HLMs), human S9 fractions and recombinant P450 enzymes. The impact of in vitro systems on the analysis of repaglinide metabolic pathway was investigated and the importance of individual metabolic pathways studied. Definite differences in formation clearance ratios were found between CYP3A4 and CYP2C8 for the formation of M1 and M4 metabolites, resulting in a 60- and 0.05-fold M1:M4 ratio in recombinant CYP3A4 and CYP2C8, respectively. A major system difference was seen in clearances for the formation of M2, which is suggested to be a main metabolite of repaglinide in vivo. An approximately 7-fold higher unbound intrinsic clearance was observed in hepatocytes and S9 fractions in comparison to microsomes; the involvement of aldehyde dehydrogenase in M2 formation was shown for the first time. This systematic analysis revealed a comparable in vitro contribution from CYP2C8 and CYP3A4 to the metabolism of repaglinide (<50%), whereas the contribution of glucuronidation ranged from 2 to 20%, depending on the in vitro system and conditions used. The repaglinide M4 metabolic pathway was proposed as a specific CYP2C8 probe for the assessment of DDIs.A whole-body PBPK repaglinide model was developed in Matlab v.7.10 incorporating multiple eliminations pathways, active uptake and bidirectional passive diffusion together with drug- and system-specific parameters. A considerable clearance underprediction was initially observed after implementation of uptake parameters reported in vitro; empirical uptake scaling factors per SLCO1B1 genotype group were required for accurate prediction of repaglinide in vivo clearance. Furthermore, collated and weighted repaglinide AUC ratios in different genotype groups reflected differences seen for empirical uptake scaling factors estimated by the current PBPK model. Simulated individual differences in uptake Vmax as well as inhibition effects by gemfibrozil and its acyl-glucuronide on CYP2C8 and OATP1B1 and resulting concentration-time profiles in plasma and liver were evaluated. Incorporation of reported unbound gemfibrozil and gemfibrozil glucuronide Cmax concentrations resulted in an increase in repaglinide AUC of 6.3-fold, in comparison to the 7.0- to 8.2-fold increase reported in vivo. In contrast, the repaglinide-gemfibrozil DDI was underpredicted by approximately 2-fold when dynamic inhibitory plasma concentration-time profiles were implemented. Observed in vitro-in vivo discrepancy was further increased when repaglinide was administered ≥3 hours after the final gemfibrozil dose. On the contrary, simulated repaglinide M4 metabolite AUC ratios were predicted ≤1.4-fold of observed data independent of gemfibrozil dosing time regimen, likely reflecting the true time-dependent CYP2C8 inhibition. Further studies investigating hepatic uptake inhibition mechanisms are required for an increased understanding of the repaglinide-gemfibrozil interaction.

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drug metabolism; repaglinide

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