This article is available online at http://dmd.aspetjournals.org ABSTRACT:Verapamil inhibition of CYP3A activity results in many drug-drug interactions with CYP3A substrates, but the mechanism of inhibition is unclear. The present study showed that verapamil enantiomers and their major metabolites

ABSTRACT:The genetic basis for polymorphic expression of CYP3A5 has been recently identified, but the significance of CYP3A5 expression is unclear. The purpose of this study is to quantify the capability of verapamil, a mechanism-based inhibitor of CYP3A, and its metabolites to inhibit the activities of CYP3A4 and CYP3A5, and to determine whether CYP3A5 expression in human liver microsomes alters the inhibitory potency of verapamil. Testosterone 6␤-hydroxylation or midazolam 1-hydroxylation was used to quantify CYP3A activity. The possibility that verapamil and its metabolites form metabolic-intermediate complex (MIC) with CYP3A was assessed using dual beam spectrophotometry. Verapamil and Ndesalkylverapamil (D617) were found to have little inhibitory effect on cDNA-expressed CYP3A5 activity and did not form a MIC with cDNA-expressed CYP3A5 as indicated by the appearance of the CYP3A4 and CYP3A5 are the most abundant CYP3A enzymes in adult human liver and small intestine, and they share 83% identity in amino acid sequence (Aoyama et al., 1989). These two CYP3A enzymes have similar substrate specificity and are responsible for metabolism of more than 50% of administered drugs that are eliminated through the liver (Thummel and Wilkinson, 1998). CYP3A substrates show large interindividual variations in oral bioavailability and systemic clearance. These variations are thought to be contributed by interindividual differences in CYP3A activities and their expression.In addition to inducers and inhibitors, genetic variation is considered to be one of the major factors that contribute to interindividual variability (Ozdemir et al., 2000). CYP3A5 has been found in approximately 10 to 30% of livers from white adults, and the genetic basis for CYP3A5 polymorphic expression has been identified (Wrighton et al., 1989;Kuehl et al., 2001). An A3 G substitution within intron 3 of the CYP3A5 gene accounts for much of the variability in CYP3A5 content found in the human liver and intestine (Kuehl et al., 2001). Individuals who have at least one CYP3A5*1 allele, the wild-type allele, produce high levels of CYP3A5 protein in their livers and intestines, whereas those who are homozygous for the CYP3A5*3 allele, the variant allele, produce very low or no CYP3A5 protein. The significance of CYP3A5 phenotypes is unclear because CYP3A4 and CYP3A5 have similar substrate specificity and no CYP3A5-specific probe substrates or inhibitors are available. One study found that, for individuals who have at least one CYP3A5*1 allele, CYP3A5 represents at least 50% of the total hepatic CYP3A content, whereas others found that CYP3A5 represents only 15 to 30% of the total CYP3A protein (Wrighton et al., 1989;Lin et al., 2002;Westlind-Johnsson et al., 2003). It has been suggested that individual differences in CYP3A5 may contribute to the interindividual variability in CYP3A activity and their susceptibility to drug interactions (Gibbs et al., 1999;Xie et al., 2004).Verapamil is a calcium channel blocker widely used in the treatment of angina pector...

Baseline intestinal activity of CYP3A4 was a key determinant of variability of the inhibitory effect of clarithromycin among individuals. CYP3A5*1 alleles were associated with greater baseline intestinal CYP3A activity and, therefore, greater extent of inhibition. The primary in vivo mechanism was not rapidly reversible competitive or irreversible inhibition but was likely formation of metabolic intermediate complexes.

1. Preliminary studies have identified cytochrome P450 (CYP) 3A4 and CYP1B1 as the human CYPs inhibited by tamoxifen. To quantify the inhibitory potency of tamoxifen and its major metabolites, the metabolism of three substrates of CYP3A, midazolam, diltiazem and testosterone, and 7-ethoxyresorufin as a substrate of CYP1B1 were examined in catalytic assays carried out using human liver microsomes and cDNA-expression systems. 2. Tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and 3-hydroxytamoxifen reversibly inhibited midazolam 1'-hydroxylation, diltiazem N-demethylation and testosterone 6beta-hydroxylation with K(i) ranging from 3 to 37 micro M in human liver microsomes. Tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and 3-hydroxytamoxifen also reversibly inhibited the activity of cDNA-expressed CYP3A4, CYP3A5 and CYP1B1. 3. Tamoxifen and N-desmethyltamoxifen exhibited time-dependent inactivation of testosterone 6beta-hydroxylation by cDNA-expressed CYP3A4 (+ cytochrome b5) yielding k(inact) and K(i) of 0.04 min(-1) and 0.2 micro M for tamoxifen and 0.08 min(-1) and 2.6 micro M for N-desmethyltamoxifen. A metabolic intermediate complex (MIC) was also formed by tamoxifen and N-desmethyltamoxifen with CYP3A4 (+ cytochrome b5) and CYP3A4 but not with CYP3A5 or CYP3A7. Pre-incubation with 4-hydroxytamoxifen and 3-hydroxytamoxifen did not result in any CYP3A inactivation or detectable MIC formation. There was no detectable time-dependent inactivation or MIC formation with tamoxifen or metabolites with CYP1B1. 4. These data indicate that tamoxifen and its three major metabolites are effective inhibitors of CYP3A in vitro and that tamoxifen and N-desmethyltamoxifen are effective mechanism-based inhibitors. Thus, caution should be exercised when tamoxifen is coadministered with other CYP3A substrates.