Induction of CYP2A6 by Metronidazole in Primary Human Hepatocytes

Abstract

BackgroundMetronidazole is the drug of choice for treating sexually transmitted infections caused by T. vaginalis. Although metronidazole remains effective in treating trichomoniasis, treatment failures occur and resistance is rising. Metronidazole is converted to its primary metabolite, 2‐hydroxymetronidazole, by CYP2A6. CYP2A6 expression is induced by estrogen and some studies suggest that nicotine also affects CYP2A6 activity. These data indicate the potential for drug‐drug interactions leading to altered therapeutic concentrations of metronidazole, possibly contributing to therapeutic failure in patients that are pregnant, taking estrogen‐containing contraceptives, or that smoke. In addition, the effects of metronidazole on its own biotransformation via CYP2A6 are unknown. The objective of this study was to explore the effects of metronidazole, estradiol, and nicotine on CYP2A6 expression and activity in vitro.MethodsPooled, cryopreserved, primary human hepatocytes (N=10) were treated with metronidazole (100 & 300 μM), estradiol (0.3 & 1 μM), nicotine (0.3 & 1 μM), a combination of estradiol + nicotine (both 0.37 μM), CITCO (positive control) or vehicle (DMSO) for 72 hours. These concentrations reflect either average exposure (nicotine) or therapeutic plasma concentrations of compounds. After 72 hours, compound exposure was terminated by removing media and washing cells. Cells were then exposed to metronidazole 100 μM in fresh media to assess CYP2A6 activity in situ via formation of 2‐hydroxymetronidazole. Aliquots of media were removed over an 8 hour time course and concentrations of 2‐hydroxymetronidazole present in media were determined by LC/MS/MS. Cells were then lysed and RNA was extracted for qRT‐PCR analysis of CYP2A6 gene expression.ResultsMetronidazole (300 μM) led to a 2.1‐fold increase in CYP2A6 mRNA expression and a 5.2‐fold increase in activity over vehicle control (p<0.01). Estradiol (1 μM) treatment caused a 3.8‐fold increase in gene expression (p<0.01) and 3.5‐fold increase in activity (p<0.05) over vehicle control. Nicotine alone failed to induce CYP2A6 mRNA expression or activity. Combination estradiol + nicotine led to a 1.7‐fold increase in gene expression (n.s.); however, no increase in activity was evident with combination treatment.ConclusionsTherapeutic concentrations of metronidazole (300 μM) and physiologic concentrations of estradiol (1 μM, as seen in pregnancy) are capable of inducing CYP2A6 mRNA expression and activity in pooled, primary human hepatocytes. However, at concentrations relevant to use of estrogen containing contraceptives (0.3 μM), estradiol did not induce CYP2A6. Nicotine alone also did not induce CYP2A6. Further studies are needed to characterize interactions that may lead to compromised metronidazole efficacy.Support or Funding InformationYoung Investigator Award, The Children's Mercy Hospital; Division of Adolescent Medicine and Division of Clinical Pharmacology, Toxicology and Therapeutic Innovation, The Children's Mercy Hospital