Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus type-1 (HIV-1), mostly to prevent mother-to-child transmission of the virus in developing countries. However, reports of severe NVP-induced hepatotoxicity and serious adverse cutaneous effects have raised concerns about its use. NVP metabolism involves oxidation of the 4-methyl substituent to 4-hydroxymethyl-NVP (12-hydroxy-NVP) and the formation of phenolic derivatives. Further metabolism, through either oxidation to quinoid derivatives or phase II esterification, may produce electrophilic derivatives capable of reacting with bionucleophiles to yield covalent adducts. These adducts could potentially be involved in the initiation of toxic responses. To gain insight into potentially reactive sites in proteins and prepare reliable and fully characterized NVP-amino acid adduct standards for subsequent assessment as biomarkers of NVP toxicity, we have used the model electrophile, 12-mesyloxy-NVP, as a synthetic surrogate for the NVP metabolite, 12-sulfoxy-NVP. Reactions of this model ester were conducted with glutathione and the nucleophilic amino acids arginine, cysteine, histidine, and tryptophan. Moreover, because adducts through the N-terminal valine of hemoglobin are convenient biomarkers of exposure to electrophilic toxicants, we also investigated the reaction with valine. We obtained very efficient (>80%) binding through the sulfur of both glutathione and N-acetylcysteine and moderate yields (10-14%) for binding through C2 of the indole ring of tryptophan and N1 of the imidazole ring of histidine. Reaction with arginine occurred through the alpha-amino group, possibly due to the high basicity of the guanidino group in the side chain. Reaction at the alpha-amino group of valine occurred to a significant extent (33%); the resulting adduct was converted to a thiohydantoin derivative, to obtain a standard useful for prospective biomonitoring studies. All adducts were characterized by a combination of (1)H and (13)C NMR spectroscopy and mass spectrometry techniques. The NVP conjugates with glutathione and N-acetylcysteine identified in this work were previously reported to be formed in vivo, although the corresponding structures were not fully characterized. Our results support the validity of 12-mesyloxy-NVP as a surrogate for 12-sulfoxy-NVP and suggest that NVP metabolism to 12-hydroxy-NVP, and subsequent esterification, could potentially be a factor in NVP toxicity. They further imply that multiple sites in proteins may be targets for modification by 12-hydroxy-NVP-derived electrophiles in vivo. Additionally, we obtained reliable, fully characterized standards for the assessment of protein modification by NVP in vivo, which should help clarify the potential role of metabolism in NVP-induced toxicity.

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus type-1 (HIV-1), mostly to prevent mother-to-child HIV-1 transmission in developing countries. Despite its clinical efficacy, NVP administration is associated with a variety of toxic responses that include hepatotoxicity and skin rash. Although the reasons for the adverse effects of NVP administration are still unclear, increasing evidence supports the involvement of metabolic activation to reactive electrophiles. In particular, Phase II activation of the NVP metabolite 12-hydroxy-NVP is thought to mediate NVP binding to bionucleophiles, which may be at the onset of toxicity. In the present study, we investigated the nature and specific locations of the covalent adducts produced in human serum albumin and human hemoglobin by reaction in vitro with the synthetic model electrophile 12-mesyloxy-NVP, used as a surrogate for the Phase II metabolite 12-sulfoxy-NVP. Multiple sites of modification were identified by two different mass spectrometry-based methodologies, liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-TOF-TOF-MS). These two distinct methodologies, which in some instances afforded complementary information, allowed the identification of multiple adducts involving cysteine, lysine, tryptophan, histidine, serine, and the N-terminal valine of hemoglobin. Tryptophan, which is not a common site of covalent protein modification, was the NVP-modified amino acid residue detected in the two proteins and consistently identified by both LC-ESI-MS/ MS and MALDI-TOF-TOF-MS. The propensity of tryptophan to react with the NVP-derived electrophile is further emphasized by the fact that human serum albumin possesses a single tryptophan residue, which suggests a remarkable selectivity that may be useful for biomonitoring purposes. Likewise, the NVP adduct with the terminal valine of hemoglobin, detected by LC-ESI-MS/MS after N-alkyl Edman degradation, appears as an easily assessed marker of NVP binding to proteins. Our results demonstrate the merits and complementarity of the two MS-based methodologies for the characterization of protein binding by NVP and suggest a series of plausible biomarkers of NVP toxicity, that should be useful in the monitoring of toxicity effects in patients administered NVP.

Abacavir is a nucleoside reverse transcriptase inhibitor marketed since 1999 for the treatment of infection with the human immunodeficiency virus type 1 (HIV). Despite its clinical efficacy, abacavir administration has been associated with serious and sometimes fatal toxic events. Abacavir has been reported to undergo bioactivation in vitro, yielding reactive species that bind covalently to human serum albumin, but the haptenation mechanism and its significance to the toxic events induced by this anti-HIV drug have yet to be elucidated. Abacavir is extensively metabolized in the liver, resulting in inactive glucuronide and carboxylate metabolites. The metabolism of abacavir to the carboxylate involves a two-step oxidation via an unconjugated aldehyde, which under dehydrogenase activity isomerizes to a conjugated aldehyde. Concurrently with metabolic oxidation, the two putative aldehyde metabolites may be trapped by nucleophilic side groups in proteins yielding covalent adducts, which can be at the onset of the toxic events associated with abacavir. To gain insight into the role of aldehyde metabolites in abacavir-induced toxicity and with the ultimate goal of preparing reliable and fully characterized prospective biomarkers of exposure to the drug, we synthesized the two putative abacavir aldehyde metabolites and investigated their reaction with the α-amino group of valine. The resulting adducts were subsequently stabilized by reduction with sodium cyanoborohydride and derivatized with phenyl isothiocyanate, leading in both instances to the formation of the same phenylthiohydantoin, which was fully characterized by NMR and MS. These results suggest that the unconjugated aldehyde, initially formed in vivo, rapidly isomerizes to the thermodynamically more stable conjugated aldehyde, which is the electrophilic intermediate mainly involved in reaction with bionucleophiles. Moreover, we demonstrated that the reaction of the conjugated aldehyde with nitrogen bionucleophiles occurs exclusively via Schiff base formation, whereas soft sulfur nucleophiles react by Michael-type 1,4-addition to the α,β-unsaturated system. The synthetic phenylthiohydantoin adduct was subsequently used as standard for LC-ESI-MS monitoring of N-terminal valine adduct formation, upon modification of human hemoglobin in vitro with the conjugated abacavir aldehyde, followed by reduction and Edman degradation. The same postmodification strategy was applied to investigate the products formed by incubation of abacavir with rat liver cytosol, followed by trapping with ethyl valinate. In both instances, the major adduct detected corresponded to the synthetic phenylthiohydantoin standard. These results suggest that abacavir metabolism to the carboxylate(s) via aldehyde intermediate(s) could be a factor in the toxic events elicited by abacavir administration. Furthermore, the availability of a reliable and fully characterized synthetic standard of the abacavir adduct with the N-terminal valine of hemoglobin and its easy detection in the model hemoglobin ...

a b s t r a c tDespite its efficacy, including in the prevention of vertical transmission, the antiretroviral nevirapine is associated with severe idiosyncratic hepatotoxicity and skin rash. The mechanisms underlying nevirapine toxicity are not fully understood, but drug bioactivation to reactive metabolites capable of forming stable protein adducts is thought to be involved. This hypothesis is based on the paradigm that drug reactive metabolites have the potential to bind to self-proteins, which results in drug-modified proteins being perceived as foreign by the immune system. The aim of the present work was to identify hemoglobin adducts in HIV patients as biomarkers of nevirapine haptenation upon bioactivation. The ultimate goal is to develop diagnostic methods for predicting the onset of nevirapine-induced toxic reactions.All included subjects were adults on nevirapine-containing antiretroviral therapy for at least 1 month. The protocol received prior approval from the Hospital Ethics Committees and patients gave their written informed consent. Nevirapine-derived adducts with the N-terminal valine of hemoglobin were analyzed by an established liquid chromatography-electrospray ionization-tandem mass spectrometry method and characterized on the basis of retention time and mass spectrometric fragmentation pattern by comparison with adduct standards prepared synthetically. The nevirapine adducts were detected in 12/13 patient samples, and quantified in 11/12 samples (2.58 ± 0.8 fmol/g of hemoglobin).This work represents the first evidence of nevirapine-protein adduct formation in man and confirms the ability of nevirapine to modify self-proteins, thus providing clues to the molecular mechanisms underlying nevirapine toxicity. Moreover, the possibility of assessing nevirapine-protein adduct levels has the potential to become useful for predicting the onset of nevirapine-induced adverse reactions.© 2012 Published by Elsevier Ireland Ltd.Abbreviations: cART, combined antiretroviral therapy; CID, collision-induced dissociation; GSH, glutathione; Hb, hemoglobin; HIV, human immunodeficiency virus; HLA, human leukocyte antigen; 12-OH-NVP, 12-hydroxy-nevirapine; LC-ESI-MS/MS, liquid chromatography-electrospray ionization-tandem mass spectrometry; MHC, major histocompatibility complex; NNRTI, non-nucleoside reverse transcriptase inhibitor; NVP, nevirapine.* Corresponding author.