Abstract
Nevirapine (NVP), an antiretroviral drug, is associated with idiosyncratic hepatotoxicity and skin reactions. Metabolic pathways of haptenation and immunotoxicity mechanisms have been proposed. NVP is metabolized by liver microsomes to a reactive intermediate that binds irreversibly to protein and forms a GSH adduct. However, no reactive metabolite of NVP, trapped as stable thioether conjugates, has hitherto been identified in vivo. This study has defined the metabolism of NVP with respect to reactive intermediate formation in patients and a rat model of NVP-induced skin reactions. An integrated NMR and mass spectrometry approach has been developed to discover and quantify stable urinary metabolite biomarkers indicative of NVP bioactivation in patients. Two isomeric NVP mercapturates were identified in the urine of HIV-positive patients undergoing standard antiretroviral chemotherapy. The same conjugates were found in rat bile and urine. The mercapturates were isolated from rat bile and characterized definitively by NMR as thioethers substituted at the C-3 and exocyclic C-12 positions of the methylpyrido ring of NVP. It is proposed that NVP undergoes bioactivation to arene oxide and quinone methide intermediates. The purified major mercapturate was quantified by NMR and used to calibrate a mass spectrometric assay of the corresponding metabolite in patient urine. This is the first evidence for metabolic activation of NVP in humans, and only the second minimum estimate in patients of bioactivation of a widely prescribed drug associated with idiosyncratic toxicities. The method can be used as a template for comparative estimations of bioactivation of any drug in patients.
Footnotes
This study was supported in part by the Wellcome Trust [Grant GR078857MA] (clinical recruitment); the MRC Centre for Drug Safety Science; and the Leche Trust and World Friendship Fund (to A.S).
M.C. is a Wellcome Trust Clinical Training Fellow.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.028688
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
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- NVP
- nevirapine
- NAC
- N-acetylcysteine
- OH NVP
- hydroxynevirapine
- 4-carboxy NVP
- 4-carboxynevirapine
- BN
- Brown Norway
- P450
- cytochrome P450
- HLM
- human liver microsomes
- RLM
- rat liver microsomes
- LC/MS/MS
- liquid chromatography/tandem mass spectrometry
- HPLC
- high-performance liquid chromatography
- LC/MS
- liquid chromatography/mass spectrometry
- MRM
- multiple reaction monitoring
- NVP-M1
- minor nevirapine mercapturate
- SPE
- solid-phase extraction
- NVP-G2
- major nevirapine GSH conjugate
- NVP-M2
- major nevirapine mercapturate
- DQFCOSY
- double quantum-filtered correlation spectroscopy
- NOESY
- nuclear Overhauser enhancement spectroscopy
- APAP
- acetaminophen
- amu
- atomic mass unit
- NVP-G1
- minor nevirapine GSH conjugate
- 2D
- two-dimensional
- NOE
- nuclear Overhauser effect
- 1D
- one-dimensional.
- Received June 1, 2009.
- Accepted September 29, 2009.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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