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. 2014 Jul;58(7):4145-52.
doi: 10.1128/AAC.02532-14. Epub 2014 May 12.

Isoniazid mediates the CYP2B6*6 genotype-dependent interaction between efavirenz and antituberculosis drug therapy through mechanism-based inactivation of CYP2A6

Michael H Court  1 , Fawziah E Almutairi  2 , David J Greenblatt  3 , Suwagmani Hazarika  3 , Hongyan Sheng  4 , Kathrin Klein  5 , Ulrich M Zanger  5 , Joanne Bourgea  6 , Christopher J Patten  6 , Awewura Kwara  7
Affiliations

Isoniazid mediates the CYP2B6*6 genotype-dependent interaction between efavirenz and antituberculosis drug therapy through mechanism-based inactivation of CYP2A6

Michael H Court et al. Antimicrob Agents Chemother. 2014 Jul.

Abstract

Efavirenz is commonly used to treat patients coinfected with human immunodeficiency virus and tuberculosis. Previous clinical studies have observed paradoxically elevated efavirenz plasma concentrations in patients with the CYP2B6*6/*6 genotype (but not the CYP2B6*1/*1 genotype) during coadministration with the commonly used four-drug antituberculosis therapy. This study sought to elucidate the mechanism underlying this genotype-dependent drug-drug interaction. In vitro studies were conducted to determine whether one or more of the antituberculosis drugs (rifampin, isoniazid, pyrazinamide, or ethambutol) potently inhibit efavirenz 8-hydroxylation by CYP2B6 or efavirenz 7-hydroxylation by CYP2A6, the main mechanisms of efavirenz clearance. Time- and concentration-dependent kinetics of inhibition by the antituberculosis drugs were determined using genotyped human liver microsomes (HLMs) and recombinant CYP2A6, CYP2B6.1, and CYP2B6.6 enzymes. Although none of the antituberculosis drugs evaluated at up to 10 times clinical plasma concentrations were found to inhibit efavirenz 8-hydroxylation by HLMs, both rifampin (apparent inhibition constant [Ki] = 368 μM) and pyrazinamide (Ki = 637 μM) showed relatively weak inhibition of efavirenz 7-hydroxylation. Importantly, isoniazid demonstrated potent time-dependent inhibition of efavirenz 7-hydroxylation in both HLMs (inhibitor concentration required for half-maximal inactivation [KI] = 30 μM; maximal rate constant of inactivation [kinact] = 0.023 min(-1)) and recombinant CYP2A6 (KI = 15 μM; kinact = 0.024 min(-1)) and also formed a metabolite intermediate complex consistent with mechanism-based inhibition. Selective inhibition of the CYP2B6.6 allozyme could not be demonstrated for any of the antituberculosis drugs using either recombinant enzymes or CYP2B6*6 genotype HLMs. In conclusion, the results of this study identify isoniazid as the most likely perpetrator of this clinically important drug-drug interaction through mechanism-based inactivation of CYP2A6.

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Figures

FIG 1
FIG 1
The effect of antituberculosis drugs on 8-hydroxyefavirenz (A) and 7-hydroxyefavirenz (B) formation in human liver microsomes (n = 10 pooled). Antituberculosis drugs evaluated included ethambutol (100 μM), pyrazinamide (1,000 μM) rifampin (100 μM), isoniazid (100 μM), and their combination (EMB+PZA+RIF+INH). Clopidogrel (0.5 μM) and 8-methoxypsoralen (0.5 μM) were included as positive-control selective inhibitors of CYP2B6 and CYP2A6, respectively. Efavirenz concentration was 30 μM. Inhibitors were either preincubated for 20 min with microsomes and NADPH and transferred to a tube containing efavirenz or added directly to a tube containing efavirenz. Results are the means and standard deviations from triplicate determinations of pooled microsomes and are expressed as a percentage of control reactions performed without any inhibitor. Also shown are the P values for pairwise comparisons (analysis of variance [ANOVA] with Student-Newman-Keuls test) evaluating effects of preincubation for all inhibitors that decreased activity by more than 50%.
FIG 2
FIG 2
Time- and concentration-dependent kinetics for inhibition by isoniazid (10 to 250 μM) of the 7-hydroxylation of efavirenz (100 μM) in pooled (n = 10) human liver microsomes (A) and recombinant CYP2A6 (B). Kitz-Wilson plots of these data are shown in panels C and D. Inactivation half-life values were obtained by linear regression of the log-linear plots (see fitted curves in panels A and B) and plotted against the reciprocal of inhibitor concentration in panels C and D. Estimates of the maximal rate constant of inactivation (kinact) and the inhibitor concentration required for half-maximal inactivation (KI) were derived by linear regression of Kitz-Wilson plot data.
FIG 3
FIG 3
Effect of increasing isoniazid concentration on 8-hydroxyefavirenz formation by CYP2B6.1 (wild-type enzyme) and CYP2B6.6 (H172 and R262 variant allozyme) either with or without 20 min of preincubation of isoniazid with enzyme and NADPH.
FIG 4
FIG 4
UV absorbance spectral scans (400 to 500 nm) showing the effect of NADPH and 15 min of incubation on metabolite intermediate complex formation of isoniazid (250 μM) with pooled (n = 10) human liver microsomes (A), recombinant CYP2A6 (B), and recombinant CYP2B6.1 and CYP2B6.6 (C).
FIG 5
FIG 5
The effect of antituberculosis drugs on 8-hydroxyefavirenz (A) and 7-hydroxyefavirenz (B) formation in human liver microsomes with either CYP2B6*1/*1 (n = 7 individuals) or CYP2B6*6/*6 (n = 8 individuals) genotypes. Clopidogrel (1 μM) and 8-methoxypsoralen (0.5 μM) were included as positive-control selective inhibitors of CYP2B6 and CYP2A6, respectively. Efavirenz concentration was 30 μM. Results are the means and standard deviations from activities determined for individual liver microsomes expressed as a percentage of control reactions performed without any inhibitor. Also shown are the P values for pairwise comparisons (ANOVA with Student-Newman-Keuls test) evaluating effects of the CYP2B6 genotype for all inhibitors that decreased activity by more than 50%.
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