Amino Acid Adduct Formation by the Nevirapine Metabolite, 12-Hydroxynevirapine—A Possible Factor in Nevirapine Toxicity

Abstract

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.

Evidence for nevirapine bioactivation in man: Searching for the first step in the mechanism of nevirapine toxicity

“…2). Identification of the NVP-valine Edman adducts in the patient samples was based upon undistinguishable mass spectra and identical retention times when compared with the corresponding synthetic standard, prepared and fully characterized as described in Antunes et al (2010a). Specifically, the samples were subjected to LC-ESI-MS/MS analysis and the MS 3 transition from the protonated molecule of the NVP-valine Edman adduct (m/z 499) to the product ion at m/z 265, formed by loss of the hydantoin moiety from the protonated molecule, was followed (Fig.…”

“…The current work took advantage of the availability, within our research team, of a synthetic adduct standard prepared through reaction of 12-mesyloxy-NVP (a synthetic surrogate for the Phase II metabolite, 12-sulfoxy-NVP) with ethyl valinate. Following N-alkyl Edman degradation, the NVP-valine Edman adduct standard was isolated and fully characterized by mass spectrometry and nuclear magnetic resonance (Antunes et al, 2010a). The availability of this adduct standard allowed the development of a suitable mass spectrometry-based analytical tool, used in the current work to assess this potential biomarker of NVP toxicity in vivo.…”

“…All other reagents were purchased from Sigma-Aldrich Química S.A. (Madrid, Spain) and used as received. The NVP-valine Edman adduct standard was synthesized as described in Antunes et al (2010a).…”

“…The adducts were identified on the basis of retention time and MS fragmentation pattern by comparison with an NVP-valine Edman adduct standard prepared synthetically (Antunes et al, 2010a). Quantification was based upon calibration with this synthetic standard.…”

“…Although, the specific mechanisms underlying the idiosyncratic toxicity of NVP are still uncertain, several approaches in vitro (Antunes et al, 2008(Antunes et al, , 2010a and in animal models in vivo (Shenton et al, 2003;Chen et al, 2008) have suggested that bioactivation of the Phase I NVP metabolite, 12-hydroxy-NVP (12-OH-NVP), to reactive electrophiles (e.g., 12-sulfoxy-NVP, Fig. 1) is involved (Antunes et al, 2008(Antunes et al, , 2010aChen et al, 2008;Pereira et al, 2012).…”