Inhibition of cytochrome P450 (CYP450) isoforms by isoniazid: potent inhibition of CYP2C19 and CYP3A

Abstract

Isoniazid (INH) remains the most safe and cost-effective drug for the treatment and prophylaxis of tuberculosis. The use of INH has increased over the past years, largely as a result of the coepidemic of human immunodeficiency virus infection. It is frequently given chronically to critically ill patients who are coprescribed multiple medications. The ability of INH to elevate the concentrations in plasma and/or toxicity of coadministered drugs, including those of narrow therapeutic range (e.g., phenytoin), has been documented in humans, but the mechanisms involved are not well understood. Using human liver microsomes (HLMs), we tested the inhibitory effect of INH on the activity of common drug-metabolizing human cytochrome P450 (CYP450) isoforms using isoform-specific substrate probe reactions. Incubation experiments were performed at a single concentration of each substrate probe at its K(m) value with a range of INH concentrations. CYP2C19 and CYP3A were inhibited potently by INH in a concentration-dependent manner. At 50 microM INH (approximately 6.86 microg/ml), the activities of these isoforms decreased by approximately 40%. INH did not show significant inhibition (<10% at 50 microM) of other isoforms (CYP2C9, CYP1A2, and CYP2D6). To accurately estimate the inhibition constants (K(i) values) for each isoform, four concentrations of INH were incubated across a range of five concentrations of specific substrate probes. The mean K(i) values (+/- standard deviation) for the inhibition of CYP2C19 by INH in HLMs and recombinant human CYP2C19 were 25.4 +/- 6.2 and 13 +/- 2.4 microM, respectively. INH showed potent noncompetitive inhibition of CYP3A (K(i) = 51.8 +/- 2.5 to 75.9 +/- 7.8 microM, depending on the substrate used). INH was a weak noncompetitive inhibitor of CYP2E1 (K(i) = 110 +/- 33 microM) and a competitive inhibitor of CYP2D6 (K(i) = 126 +/- 23 microM), but the mean K(i) values for the inhibition of CYP2C9 and CYP1A2 were above 500 microM. Inhibition of one or both CYP2C19 and CYP3A isoforms is the likely mechanism by which INH slows the elimination of coadministered drugs, including phenytoin, carbamazepine, diazepam, triazolam, and primidone. Slow acetylators of INH may be at greater risk for adverse drug interactions, as the degree of inhibition was concentration dependent. These data provide a rational basis for understanding drug interaction with INH and predict that other drugs metabolized by these two enzymes may also interact.

FIG. 1

Inhibition of CYP450 isoforms by INH at 50 μM (A) and 0 to 250 μM (B) in HLMs. The activity of each isoform was measured by isoform-specific substrate reaction probes at approximately their respective K_m values: 60 μM for phenacetin O-deethylation (CYP1A2); 25 μM for dextromethorphan (CYP2D6); 25 μM for midazolam 4-hydroxylation (CYP3A); 50 μM for omeprazole (CYP2C19); 50 μM for tolbutamide (CYP2C9); and 25 μM for chlorzoxazone (CYP2E1). Data represent averages of duplicates.

FIG. 2

Inhibitory effect of INH (0 to 250 μM) on CYP2C19-catalyzed omeprazole 5-hydroxylation in HLMs and recombinant human CYP2C19. Data represent averages of duplicate determinations.

FIG. 3

Dixon plots for the inhibition of CYP2C19-catalyzed omeprazole 5-hydroxylation by INH (0 to 250 μM) in HLMs (A) and S-mephenytoin 4-hydroxylation by INH (0 to 100 μM) in recombinant human CYP2C19 (B). Each point represents the average of duplicate determinations.

FIG. 4

Dixon plots for the inhibition of CYP3A-catalyzed omeprazole sulfone formation (A), midazolam 4-hydroxylation (B), and dextromethorphan N-demethylation (C) by INH in HLMs. The INH concentrations used were 0 to 100 μM (A and B) and 0 to 250 μM (C). Each point represents the average of duplicate determinations.

FIG. 5

Inhibitory effect of INH on CYP2C9-catalyzed 4-methylhydroxylation and flurbiprofen 4-hydroxylation. (A) Inhibition of CYP2C9 (with tolbutamide [TB] [20 and 40 μM] and flurbiprofen [FB] [13 and 25 μM] as substrates) by INH (0 to 250 μM) in HLMs; (B) Dixon plots for the inhibition by INH of CYP2C9 (with tolbutamide) in recombinant human CYP2C9; (C) inhibitory effect of INH (0 to 250 μM) on CYP2C9 (with tolbutamide [50 μM]) with preincubation (PI) or without preincubation in HLMs (see Materials and Methods for details). Data are averages for duplicate incubations.

FIG. 6

Inhibition of CYP2E1-mediated chlorzoxazone 6-hydroxylation by INH. (A) Percent inhibition of CYP2E1 activity (with chlorzoxazone [25 μM]) by INH and diethyldithiocarbamate (DEDTC) as a positive control in HLMs; (B) Representative Dixon plots for the inhibition of CYP2E1 activity by INH (25 to 250 μM) in HLMs. Data represent averages of duplicates.

FIG. 7

Inhibition of CYP2D6-catalyzed dextromethorphan O-demethylation and CYP1A2-catalyzed O-deethylation by INH in HLMs. (A) Dixon plots for the inhibition of CYP2D6 (INH, 0 to 250 μM); (B) percent inhibition of CYP1A2 (with phenacetin [25 and 50 μM] as a substrate) (INH, 0 to 250 μM). Each point represents the average of duplicate determinations.