title: Impact of Probe Substrate Selection on Cytochrome P450 Reaction Phenotyping Using the Relative Activity Factor.
abstract: Accurately assessing the contribution of cytochrome P450 (P450) isoforms to overall metabolic clearance is important for prediction of clinical drug-drug interactions (DDIs). The relative activity factor (RAF) approach in P450 reaction phenotyping assumes that the interaction between P450-selective probes and testing systems is the same as the interaction of drug candidate with those systems. To test this assumption, an intersystem clearance ratio (ICR) was created to evaluate the difference in values between RAF-scaled intrinsic clearance (CLint) and measured CLintin human liver microsomes (HLMs). The RAF value for CYP3A4 or CYP2C9 derived from a particular P450-selective probe reaction was applied to calculate RAF-scaled CLintfor other probe reactions of the same P450 isoform in a crossover manner and compared with the measured HLM CLintWhen RAF derived from midazolam or nifedipine was used for CYP3A4, the ICR for testosterone 6β-hydroxylation was 31 and 25, respectively, suggesting significantly diverse interactions of CYP3A4 probes with the testing systems. Such ICR differences were less profound among probes for CYP2C9. In addition, these RAF values were applied to losartan and meloxicam, whose metabolism is mostly CYP2C9 mediated. Only using the RAF derived from testosterone for CYP3A4 produced the expected CYP2C9 contribution of 72%-87% and 47%-69% for metabolism of losartan and meloxicam, respectively. RAF derived from other CYP3A4 probes would have attributed predominantly to CYP3A4 and led to incorrect prediction of DDIs. Our study demonstrates a significant impact of probe substrate selection on P450 phenotyping using the RAF approach, and the ICR may provide a potential solution.
authors.affiliation: Drug Metabolism and Pharmacokinetics Department, Biopharmaceutical Assessments, Eisai Inc., Andover, Massachusetts amy_siu@eisai.com george_lai@eisai.com.
authors.forename: Y Amy
authors.initials: YA
authors.lastname: Siu
authors.affiliation: Drug Metabolism and Pharmacokinetics Department, Biopharmaceutical Assessments, Eisai Inc., Andover, Massachusetts amy_siu@eisai.com george_lai@eisai.com.
authors.forename: W George
authors.initials: WG
authors.lastname: Lai
mesh_terms: D018592:Cross-Over Studies; D065729:Cytochrome P-450 CYP2C9 / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D004347:Drug Interactions; D006801:Humans; D066298:In Vitro Techniques; D008657:Metabolic Clearance Rate; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D008954:Models, Biological; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D004364:Pharmaceutical Preparations; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human