Haptenation of the antiretroviral drug nevirapine upon bioactivation in man

AT Marinho1, AMM Antunes2, U Caixas1,3, T Branco4, ALA Godinho1,2, MC Oliveira2, MM Marques2, EC Monteiro1, SA Pereira1. 1Centro de Estudos de Doenças Crónicas (CEDOC), Faculdade de Ciências Médicas, Universidade NOVA, 1169-056, Portugal, 2Centro de Química Estrutural (CQE), Instituto Superior Técnico, Universidade Técnica de Lisboa, 1049-001, Portugal, 3Centro Hospitalar de Lisboa Central (CHLC), EPE, 1150-199, Portugal, 4Hospital Prof. Doutor Fernando Fonseca, EPE, 2720-276, Portugal

Chronic treatment of HIV-infection with combined antiretroviral therapy (cART) is currently unavoidable. Nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor (NNRTI) (1) is globally one of the most prescribed antiretrovirals (2), since it is available as a generic drug and, among other advantages, enables the prevention of mother-to-child HIV transmission (3). However, its association with skin and hepatic toxicity (4), which granted an EMEA warning, is a major drawback of NVP use. In addition, NNRTIs have been associated with increased incidences of non-AIDS-defining cancers, raising concerns about the chronic use of this class of anti-HIV drugs. The mechanisms underlying NVP toxicity are not fully known but drug bioactivation to reactive intermediates capable of forming stable protein and DNA adducts is thought to be involved (5).

The aim of the present work was to identify NVP-derived hemoglobin (Hb) adducts in HIV-infected individuals as biomarkers of NVP haptenation ability following bioactivation of its phase I metabolite 12-hydroxy-NVP. All included patients were adults on NVP-containing cART for at least 2 weeks, regardless of previous therapeutic regimens. The protocol received prior approval from the Hospital Ethics Committees, patients gave their written informed consent, and adherence was controlled through a questionnaire. Hb was precipitated from erythrocytes and then subjected to N-alkyl Edman degradation, leading to the specific detachment of NVP adducts with the N-terminal valine residues from the protein (Edman adducts). 12-Hydroxy-NVP-derived Edman adducts were detected in samples from 92% of the patients. The adducts were analyzed by an established LC-ESI-MS/MS protocol (6) and characterized on the basis of retention time and MS fragmentation pattern by comparison with synthetic standards. A wide inter-patient variability (coefficient of variation of 79%) was observed in the adduct MS/MS signal areas. This work represents the first evidence of NVP-derived protein adduct formation in man. Moreover, the consistent occurrence of NVP bioactivation, combined with the high inter-individual variability found in adduct formation, provides clues to the molecular mechanisms underlying NVP-induced toxic reactions.

(1) FDA approves nevirapine to treat HIV. News release June 24 1996, T96-44; (2) Jackson, J. B. et al. Lancet 2003; 362: 859–868; (3) Marseille, E., et al. Lancet 1999; 354: 803–809; (4) Sha, B. E., Proia, L. A., and Kessler, H. A. J. Am. Med. Assoc. 2000; 284: 2723. (5) Pereira, S.A. et al., in: Berhardt, L.V. (Ed.), Advances in Medicine and Biology, Vol. 44, NovaPublisher, 2012; (6) Antunes, A. M. M. et al. Chem Res Toxicol. 2010; 23:1714-1725.

Acknowledgments Thanks are due to the Portuguese NMR and MS Networks (IST-UTL Center) for providing access to the facilities and to Inês Faustino (FCM) for technical support. This work was supported by research grants and pluriannual funds from FCT, Portugal (PTDC/SAU-TOX/111663/2009, PTDC/QUI-QUI/113910/2009; PEst-OE/QUI/UI0100/2011).