gms | German Medical Science

Aim: The pharmacokinetics of many drugs is markedly influenced by intestinal and hepatic biotransformation enzymes such as cytochrome P450 (CYP450) enzymes and UDP-glucuronosyltransferases (UGT). In order to predict their impact on drug disposition as well as on drug-drug interactions, data on their absolute intestinal and hepatic abundance are required. Therefore, it was the aim of this study to develop and comprehensively validate LC-MS/MS methods for the absolute quantification of clinically relevant CYP and UGT enzymes.

Method: LC-MS/MS methods were developed for nine CYP (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) and four UGT enzymes (UGT1A1, UGT1A3, UGT2B7 and UGT2B15) that have been shown to be of clinical relevance in human drug metabolism. Protein quantification was performed by targeted proteomics using liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based determination of enzyme specific peptides after tryptic digestion using stable isotope-labelled peptides as internal standard. The assays were validated according to current bioanalytical guidelines with respect to specificity, linearity, within-day and between-day accuracy and precision, digestion efficiency as well as stability. Finally, the developed method was applied to determine the CYP and UGT protein amount in human liver (HLM) and intestinal microsomes (HIM).

Results: A LC-MS/MS method was developed that allowed the simultaneous quantification of all aforementioned enzymes in one analytical run (60 min). The method was shown to be selective for the respective enzymes and the analytical range was in each case 0.25–50 nmol/l. Within-day (intra-day) as well as between-day (inter-day) accuracy was between -13.1–12.5% (relative error) and precision 1.1–14.8% (coefficient of variation). All peptides were shown to be stable during preparation, storage in the autosampler (24 h at 4°C), during overnight digestion (16 h at 37°C) and during up to three freeze-thaw cycles. Significant matrix effects could not be observed. Using this validated method, the following relative percentage contribution of metabolizing enzymes was observed in pooled HLM: CYP2E1, 24%; CYP2C9, 13.8%; CYP3A4, 12.1%; UGT2B15, 10.3%; CYP2C8, 10%; UGT2B7, 8.9%; UGT1A1, 7.5%; CYP1A2, 4.7%; CYP2B6 & CYP2D6, 3.5%; CYP2C19, 0.8%; CYP3A5, 0.7% and UGT1A3, 0.2%. In HIM it was: CYP3A4, 47.1%; UGT1A1, 19.7%; CYP2C9, 10.8%; UGT1A3, 8.3%; UGT2B7, 7.1% and CYP2C19, 7%.

Conclusion: The developed method was shown to possess sufficient specificity, sensitivity, accuracy, precision and stability to measure clinically relevant metabolizing enzymes in human tissue preparations. These absolute expression data may allow more precise prediction of drug disposition using PBPK modelling-based approaches.