Bryan A. Ward

OBJECTIVES: To determine the influence of cytochrome P450 2B6 (CYP2B6) genotype on the rate of oxidative efavirenz metabolism in human liver microsomes. MATERIALS & METHODS: Formation rates of 8-hydroxyefavirenz, 7-hydroxyefavirenz and 8,14-dihydroxyefavirenz were determined in vitro with efavirenz as a substrate (10 microM) in a large panel of human liver microsomes (n = 87) that were genotyped for variants of the CYP2B6 gene and phenotyped for CYP2B6 protein expression and bupropion hydroxylation. RESULTS: Efavirenz 8-hydroxylation, the major route of efavirenz clearance, was detected in all samples, exhibiting an overall interindividual variability of 44.7-fold; 8,14-dihydroxyefavirenz and 7-hydroxyefavirenz were also detected in most samples. The formation rate of 8-hydroxyefavirenz correlated significantly with CYP2B6 protein (Spearman's r(S) = 0.54; p < 0.0001) and bupropion hydroxylase activity (r(S) = 0.73; p < 0.0001). Compared with the *1/*1 genotype, efavirenz 8-hydroxylation was significantly lower in samples with *1/*6 and *6/*6 genotype, which also had significantly decreased CYP2B6 protein (Mann-Whitney test, p < 0.05). A decrease in CYP2B6 protein was also observed in samples with *1/*5 and *5/*6 genotypes, but this did not result in significant reduction of efavirenz metabolism, probably due to differences in specific activity of the protein variants. Lower CYP2B6 protein and activity, as well as efavirenz 8-hydroxylation was also found in several samples with rarer genotypes. We found no effect of gender and age on any of the phenotypes tested, but prior exposure to carbamazepine markedly increased CYP2B6 protein expression and activity as well as efavirenz 8-hydroxylation. CONCLUSIONS: We have shown that CYP2B6 genetic polymorphism markedly influences the metabolism of efavirenz in human liver microsomes. Importantly, the CYP2B6*6 allele harboring the SNPs c.516G>T [Q172H] and c.785A>G [K262R] was significantly associated with a pronounced decrease in CYP2B6 expression and activity, as well as a low rate of efavirenz 8-hydroxylation. These results represent a first step towards elucidating the mechanism by which this allele identifies patients exhibiting very high efavirenz plasma concentrations.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Anastrozole is primarily cleared by hepatic metabolism via oxidative and conjugating enzymes. WHAT THIS STUDY ADDS • Anastrozole is oxidized to hydroxyanastrozole mainly by CYP3A4/5 and glucuronidated to anastrozole glucuronide predominantly by UGT1A4 in vitro . • Hydroxyanastrozole glucuronide and hydroxyanastrozole were quantified as the major metabolites of anastrozole in plasma of breast cancer patients. • This study describes for the first time anastrozole metabolic pathways and the enzymes involved, which may serve as the scientific basis for pharmacogenetic and drug‐interaction assessments. AIMS Little information is available regarding the metabolic routes of anastrozole and the specific enzymes involved. We characterized anastrozole oxidative and conjugation metabolism in vitro and in vivo . METHODS A sensitive LC‐MS/MS method was developed to measure anastrozole and its metabolites in vitro and in vivo . Anastrozole metabolism was characterized using human liver microsomes (HLMs), expressed cytochrome P450s (CYPs) and UDP‐glucuronosyltransferases (UGTs). RESULTS Hydroxyanastrozole and anastrozole glucuronide were identified as the main oxidative and conjugated metabolites of anastrozole in vitro , respectively. Formation of hydroxyanastrozole from anastrozole was markedly inhibited by CYP3A selective chemical inhibitors (by &gt;90%) and significantly correlated with CYP3A activity in a panel of HLMs ( r = 0.96, P = 0.0005) and mainly catalyzed by expressed CYP3A4 and CYP3A5. The K m values obtained from HLMs were also close to those from CYP3A4 and CYP3A5. Formation of anastrozole glucuronide in a bank of HLMs was correlated strongly with imipramine N ‐glucuronide, a marker of UGT1A4 ( r = 0.72, P &lt; 0.0001), while expressed UGT1A4 catalyzed its formation at the highest rate. Hydroxyanastrozole (mainly as a glucuronide) and anastrozole were quantified in plasma of breast cancer patients taking anastrozole (1 mg day −1 ); anastrozole glucuronide was less apparent. CONCLUSION Anastrozole is oxidized to hydroxyanastrozole mainly by CYP3A4 (and to some extent by CYP3A5 and CYP2C8). Once formed, this metabolite undergoes glucuronidation. Variable activity of CYP3A4 (and probably UGT1A4), possibly due to genetic polymorphisms and drug interactions, may alter anastrozole disposition and its effects in vivo .