Differential Mechanism-Based Inhibition of Cyp3a4 and Cyp3a5 by Verapamil

Abstract

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...

Cytochrome P450 3A-Dependent Metabolism of a Potent and Selective γ-Aminobutyric AcidAα2/3 Receptor Agonist in Vitro: Involvement of Cytochrome P450 3A5 Displaying Biphasic Kinetics

“…In recent years, several in vitro approaches have been used to estimate the clinical relevance of CYP3A5, including the use of inhibitors displaying isoform selectivity (Khan et al, 2002;Patki et al, 2003;Galetin et al, 2004;Huang et al, 2004). Although differential inhibition of CYP3A4 and CYP3A5 has been observed for many compounds (Khan et al, 2002;Patki et al, 2003;McConn et al, 2004;Wang et al, 2005;Granfors et al, 2006), only mifepristone appears to exhibit suitable isoform selectivity for reaction-phenotyping experiments. On pretreatment of mifepristone, the residual activity of t-butyl hydroxylation and N-deethylation was indicative of the expression level of CYP3A5 in microsomal preparations used.…”

Effects of Spice Constituents on P-Glycoprotein-Mediated Transport and CYP3A4-Mediated Metabolism in Vitro

“…Ketoconazole (1 M) served as the negative control (Perloff et al, 2005) and verapamil (50 M) a mechanism-based inhibitor of CYP3A4 (Wang et al, 2005) as the positive control.…”

“…Ketoconazole, a known reversible inhibitor of CYP3A4 (Perloff et al, 2005), was used as a negative control and verapamil, a mechanism-based inhibitor of CYP3A (Wang et al, 2005), as the positive control. As expected, the inhibitory effect of ketoconazole on CYP3A4-mediated MDZ metabolism did not increase with prolonged preincubation, whereas that of verapamil increased with prolonged preincubation (Fig.…”

Iatrogenic heart block during treatment of a patient with Cushing’s syndrome: report of a case

“…Hypertension is a presenting feature of Cushing's syndrome and requires, not infrequently, high doses of antihypertensives to achieve blood pressure control. Verapamil is also a CYP3A4-based inhibitor and is classified as a moderate CYP3A4 inhibitor in the FDA guidance [3]. In our patient, ketoconazole might have increased the levels of verapamil in the blood to toxic levels and caused the heart block.…”