Evidence for nevirapine bioactivation in man: Searching for the first step in the mechanism of nevirapine toxicity
Introduction
Since 1996, the inception of combined antiretroviral therapy (cART) has changed the prognosis of human immunodeficiency virus (HIV) infection from a lethal disease to a chronic condition in properly medicated patients. However, HIV-positive individuals still face obstacles associated with chronic treatment, including adherence to a daily administration schedule, loss of therapeutic efficacy and drug-induced toxicity. Moreover, increased concerns are emerging, regarding the long-term adverse effects of cART (Powles et al., 2009), that have been linked to the premature onset of aging observed in HIV-infected patients (Deeks, 2009). Powles et al. (2009) showed an epidemiologic association between the chronic treatment with non-nucleoside reverse transcriptase inhibitors (NNRTI) and a higher incidence of non-AIDS-defining cancers.
Nevirapine (NVP, Fig. 1) was the first NNRTI approved by the US Food and Drug Administration for the treatment of HIV type-1 infection, as part of cART (Food and Drug Administration, 1996). Currently, NVP is still the most prescribed NNRTI in the world, and remains the most prescribed antiretroviral in countries with limited economic resources, partly due to its low cost (Ades et al., 2000, Lockman et al., 2007). The favorable metabolic profile is one of the therapeutic advantages of NVP (Ruiz et al., 2001, Clotet et al., 2003), rendering it suitable for use in patients with diabetes, dyslipidemia or metabolic syndrome comorbidities. Moreover, the low incidence of adverse drug reactions in the central nervous system (Medrano et al., 2008), allows NVP use in the context of psychiatric disorders or addiction to narcotic drugs. Furthermore, one of the most relevant benefits of NVP is its efficacy in the prevention of mother-to-child transmission of the HIV-1 infection, with the drug being commonly prescribed to pregnant women and their children (Ades et al., 2000, Medrano et al., 2008, Perinatal HIV Guidelines Working Group, 2009). However, NVP use has been associated with restrictive idiosyncratic hepatotoxicity and cutaneous hypersensitivity (Taiwo, 2006, Medrano et al., 2008, De Lazzari et al., 2008). Severe, life-threatening, and even fatal cases of hepatotoxicity (Cattelan et al., 1999) have been described.
Concern about NVP adverse reactions arose following case reports of liver failure in individuals on post-exposure prophylaxis (Johnson and Baraboutis, 2000, CDC, 2001) and in asymptomatic HIV-infected patients with well preserved immunity, administered NVP-containing first line cART (Cattelan et al., 1999, Stern et al., 2003). These adverse reactions are more frequent during the first 6 weeks of treatment and women (including those who are pregnant and of Asian ethnicity) seem to be at increased risk of developing NVP-related toxicities (Ho et al., 1998, Antinori et al., 2001, Bersoff-Matcha et al., 2001). Given that immunocompetence is regarded as an additional risk factor for the development of these reactions (De Lazzari et al., 2008), it is recommended that the drug should be initiated in cART-naïve-women with a CD4 cell count below 250 cells/mm3 and below 400 cells/mm3 in men (Thompson et al., 2010). For the same reason, NVP is not recommended as part of post-exposure prophylaxis (Patel et al., 2004). This association with high CD4 T cell counts stems from an immune response at the onset of NVP-induced toxicity (Yuan et al., 2011). 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, Antunes et al., 2010b) 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., 2010a, Antunes et al., 2010b, Chen et al., 2008, Pereira et al., 2012).
Typically, electrophilic drug metabolites are short-lived species in vivo, and thus extremely difficult to detect in humans or experimental animals; however, their propensity to react with bionucleophiles (e.g., amino acids and DNA bases), yielding stable covalent adducts, provides an adequate means for their indirect detection and quantification in body fluids and tissues (Park et al., 2005). Electrophilic metabolites can react with glutathione (GSH), potentially leading to its depletion, and with biomacromolecules (proteins, lipids, and nucleic acids) which can trigger an immune response (Russmann et al., 2009, Park et al., 2011) (Fig. 1). Besides providing clues about the molecular mechanisms underlying drug toxicity, the covalent adducts formed upon reaction with bionucleophiles can be useful markers of toxicity for biomonitoring purposes, especially when well-characterized standards are available (Törnqvist et al., 2002, Angerer et al., 2007, Rubino et al., 2009).
Srivastava et al. (2010) have provided evidence for covalent binding of reactive NVP metabolites to amino acids and peptides (presumably GSH) in vivo, by identifying two NVP mercapturate conjugates in the urine of HIV-positive individuals. However, evidence for NVP metabolism to reactive derivatives with haptenation ability in humans, yielding stable protein adducts, has yet to be provided. Toward this goal, the aim of this work was to investigate whether metabolism to reactive 12-OH-NVP derivatives resulted in detectable adducts with the N-terminal valine of hemoglobin (Hb) in HIV-infected patients on NVP-containing cART. Herein is reported the identification of NVP-protein adducts in the blood samples of HIV-infected individuals, thus demonstrating the ability of NVP to modify self-proteins.
Section snippets
Chemicals and standards
NaCl was purchased from Merck KGaA (Darmstadt, Germany) and water was filtered using a Millipore Milli-Q Water Purification System (Billerica, MA). 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).
Study design
The protocol received prior approval from the Ethics Committees of Centro Hospitalar de Lisboa Central, EPE and Hospital Prof. Doutor Fernando Fonseca, EPE
Results
Thirteen HIV-positive patients [nine men and four women; 54 ± 11 years old (mean ± standard deviation)] were included in the present study. Four of the patients were cART-naïve. A tenofovir + emtricitabine combination was the cART-backbone in 7 of the patients; the other cART backbones were equally distributed between lamivudine + zidovudine and lamivudine + abacavir combinations; all regimens included NVP. The patients’ demographic and therapeutic data are summarized in Table 1, along with the detected
Discussion
Increasing evidence shows that allergic idiosyncratic NVP reactions, characterized by the presence of typical symptoms and signs of adaptive immune responses, including hepatotoxicity, fever, skin reactions and eosinophilia, are genetically determined. It seems that NVP-specific antigens may trigger an immunological response, as shown by screening of human leukocyte antigen (HLA) markers [located within the class I and class II regions of the major histocompatibility complex (MHC)] (Gangar et
Conclusion
The present manuscript provides the first evidence for the ability of 12-OH-NVP-derived reactive metabolites to form adducts with proteins in humans. The consistent detection of this adduct in the patient samples provides unequivocal proof of NVP bioactivation, via the 12-OH-NVP pathway, and may be a relevant clue to the molecular mechanisms underlying NVP-related adverse reactions. This approach can lead to a better understanding of the long-term risks of NVP bioactivation in chronic therapies
Role of the funding source
This work was supported by Fundação para a Ciência e a Tecnologia (FCT, Portugal), through research grants (PTDC/SAU-TOX/111663/2009 and PTDC/QUI-QUI/113910/2009) and pluriannual funds to Centro de Química Estrutural (PEstOE/QUI/UI0100/2011).
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
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.
References (54)
- et al.
Human biomonitoring: state of the art
Int. J. Hyg. Environ. Health
(2007) - et al.
N-terminal globin adducts as biomarkers for formation of butadiene derived epoxides
Chem. Biol. Interact.
(2007) - et al.
Analysis of hemoglobin adducts of acrylamide and glycidamide by liquid chromatography–electrospray ionization tandem mass spectrometry, as exposure biomarkers in French population
J. Chromatogr. A
(2007) - et al.
Association between presence of HLA-B*5701, HLA-DR7, and HLA-DQ3 and hypersensitivity to HIV-1 reverse-transcriptase inhibitor abacavir
Lancet
(2002) - et al.
Drug bioactivation and protein adduct formation in the pathogenesis of drug-induced toxicity
Chem. Biol. Interact.
(2011) - et al.
Protein adducts: quantitative and qualitative aspects of their formation, analysis and application
J. Chromatogr. B
(2002) - et al.
The importance of glutathione in human disease
Biomed. Pharmacother.
(2003) - et al.
Economic issues in the prevention of vertical transmission of HIV
Pharmacoeconomics
(2000) - et al.
Female sex and the use of anti-allergic agents increase the risk of developing cutaneous rash associated with nevirapine therapy
AIDS
(2001) - et al.
Synthesis and characterization of DNA adducts from the HIV reverse transcriptase inhibitor nevirapine
Chem. Res. Toxicol.
(2008)