Quantifying the metabolic activation of nevirapine in patients by integrated applications of NMR and mass spectrometries.
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.
MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, School of Biomedical Sciences, the University of Liverpool, Liverpool, United Kingdom.
Abhishek
A
Srivastava
Lu-Yun
LY
Lian
James L
JL
Maggs
Masautso
M
Chaponda
Munir
M
Pirmohamed
Dominic P
DP
Williams
B Kevin
BK
Park
D000111:Acetylcysteine / Q000378:metabolism; D000328:Adult; D000818:Animals; D001646:Bile / Q000737:chemistry; D015415:Biomarkers / Q000032:analysis / Q000652:urine; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D003907:Dexamethasone / Q000494:pharmacology; D005260:Female; D020723:Glucuronic Acid / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000201:enzymology; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008862:Microsomes, Liver / Q000201:enzymology; D008875:Middle Aged; D015394:Molecular Structure; D019829:Nevirapine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics*; D051381:Rats; D011914:Rats, Inbred BN; D017208:Rats, Wistar; D014556:Urine / Q000737:chemistry
D015415:Biomarkers; D003907:Dexamethasone; D020723:Glucuronic Acid; D019829:Nevirapine; D005978:Glutathione; D000111:Acetylcysteine