Nevirapine Metabolites Research Articles

The development and characterization of a CRISPR/Cas9-mediated PD-1 functional knockout rat as a tool to study idiosyncratic drug reactions.

Idiosyncratic drug reactions are rare but serious adverse drug reactions unrelated to the known therapeutic properties of the drug and manifest in only a small percentage of the treated population. Animal models play an important role in advancing mechanistic studies examining idiosyncratic drug reactions. However, to be useful, they must possess similarities to those seen clinically. Although mice currently represent the dominant mammalian genetic model, rats are advantageous in many areas of pharmacologic study where their physiology can be examined in greater detail and is more akin to that seen in humans. In the area of immunology, this includes autoimmune responses and susceptibility to diabetes, in which rats more accurately mimic disease states in humans compared with mice. For example, oral nevirapine treatment can induce an immune-mediated skin rash in humans and rats, but not in mice due to the absence of the sulfotransferase required to form reactive metabolites of nevirapine within the skin. Using CRISPR-mediated gene editing, we developed a modified line of transgenic rats in which a segment of IgG-like ectodomain containing the core PD-1 interaction motif containing the native ligand and therapeutic antibody domain in exon 2 was deleted. Removal of this region critical for mediating PD-1/PD-L1 interactions resulted in animals with an increased immune response resulting in liver injury when treated with amodiaquine.

Consideration of Nevirapine Analogs To Reduce Metabolically Linked Hepatotoxicity: A Cautionary Tale of the Deuteration Approach.

Idiosyncratic drug reactions (IDRs) in their most deleterious form can lead to serious medical complications and potentially fatal events. Nevirapine (NVP), still widely used in developing countries for combinatorial antiretroviral and prophylactic therapies against HIV infection, represents a prototypical example of IDRs causing severe skin rashes and hepatotoxicity. Complex metabolic pathways accompanied by production of multiple reactive metabolites often complicate our understanding of IDR's origin. While assessment of NVP analogs has helped characterize the pathways involved in IDRs for NVP, which are largely driven by metabolism at the 12-methyl position, it has yet to be investigated if some of these analogs could be valuable replacement drugs with reduced reactive metabolite properties and drug-drug interaction (DDI) risks. Here, we evaluated a set of eight NVP analogs, including the deuterated 12-d3-NVP and two NVP metabolites, for their efficacy and inhibitory potencies against HIV reverse transcriptase (HIV-RT). A subset of three analogs, demonstrating >85% inhibition for HIV-RT, was further assessed for their hepatic CYP induction-driven DDI risks. This led to a closer investigation of the inactivation properties of 12-d3-NVP for hepatic CYP3A4 and a comparison of its propensity in generating reactive metabolite species. The metabolic shift triggered with 12-d3-NVP, increasing formation of the 2-hydroxy and glutathione metabolites, emphasized the importance of the dynamic balance between induction and metabolism-dependent inactivation of CYP3A4 and its impact on clearance of NVP during treatment. Unfortunately, the strategy of incorporating deuterium to reduce NVP metabolism and production of the electrophile species elicited opposite results, illustrating the great challenges involved in tackling IDRs through deuteration.

Sulfation of 12-hydroxy-nevirapine by human SULTs and the effects of genetic polymorphisms of SULT1A1 and SULT2A1

Nevirapine (NVP) is an effective drug for the treatment of HIV infections, but its use is limited by a high incidence of severe skin rash and liver injury. 12-Hydroxynevirapine (12-OH-NVP) is the major metabolite of nevirapine. There is strong evidence that the sulfate of 12-OH-NVP is responsible for the skin rash. While several cytosolic sulfotransferases (SULTs) have been shown to be capable of sulfating 12-OH-NVP, the exact mechanism of sulfation in vivo is unclear. The current study aimed to clarify human SULT(s) and human organs that are capable of sulfating 12-OH-NVP and investigate the metabolic sulfation of 12-OH-NVP using cultured HepG2 human hepatoma cells. Enzymatic assays revealed that of the thirteen human SULTs, SULT1A1 and SULT2A1 displayed strong 12-OH-NVP-sulfating activity. 1-Phenyl-1-hexanol (PHHX), which applied topically prevents the skin rash in rats, inhibited 12-OH-NVP sulfation by SULT1A1 and SULT2A1, implying the involvement of these two enzymes in the sulfation of 12-OH-NVP in vivo. Among five human organ cytosols analyzed, liver cytosol displayed the strongest 12-OH-NVP-sulfating activity, while a low but significant activity was detected with skin cytosol. Cultured HepG2 cells were shown to be capable of sulfating 12-OH-NVP. The effects of genetic polymorphisms of SULT1A1 and SULT2A1 genes on the sulfation of 12-OH-NVP by SULT1A1 and SULT2A1 allozymes were investigated. Two SULT1A1 allozymes, Arg37Asp and Met223Val, showed no detectable 12-OH-NVP-sulfating activity, while a SULT2A1 allozyme, Met57Thr, displayed significantly higher 12-OH-NVP-sulfating activity compared with the wild-type enzyme. Collectively, these results contribute to a better understanding of the involvement of sulfation in NVP-induced skin rash and provide clues to the possible role of SULT genetic polymorphisms in the risk of this adverse reaction.

Ultra-high pressure liquid chromatography coupled to travelling wave ion mobility-time of flight mass spectrometry for the screening of pharmaceutical metabolites in wastewater samples: Application to antiretrovirals

The presence of pharmaceutical compounds in the aquatic environment is a significant environmental health concern, which is exacerbated by recent evidence of the contribution of drug metabolites to the overall pharmaceutical load. In light of a recent report of the occurrence of metabolites of antiretroviral drugs (ARVDs) in wastewater, we investigate in the present work the occurrence of further ARVD metabolites in samples obtained from a domestic wastewater treatment plant in the Western Cape, South Africa. Pharmacokinetic data indicate that ARVDs are biotransformed into several positional isomeric metabolites, only two of which have been reported wastewater samples. Given the challenges associated with the separation and identification of isomeric species in complex wastewater samples, a method based on liquid chromatography hyphenated to ion mobility spectrometry-high resolution mass spectrometry (LC-IMS-HR-MS) was implemented. Gradient LC separation was achieved on a sub-2 µm reversed phase column, while the quadrupole-time-of-flight MS was operated in data independent acquisition (DIA) mode to increase spectral coverage of detected features. A mass defect filter (MDF) template was implemented to detect ARVD metabolites with known phase I and phase II mass shifts and fractional mass differences and to filter out potential interferents. IMS proved particularly useful in filtering the MS data for co-eluting species according to arrival time to provide cleaner mass spectra. This approach allowed us to confirm the presence of two known hydroxylated efavirenz and nevirapine metabolites using authentic standards, and to tentatively identify a carboxylate metabolite of abacavir previously reported in literature. Furthermore, three hydroxylated-, two sulphated and one glucuronidated metabolite of efavirenz, two hydroxylated metabolites of nevirapine and one hydroxylated metabolite of ritonavir were tentatively or putatively identified in wastewater samples for the first time. Assignment of the metabolites is discussed in terms of high resolution fragmentation data, while collisional cross section (CCS) values measured for the detected analytes are reported to facilitate further work in this area.

The 2-hydroxy-nevirapine metabolite as a candidate for boosting apolipoprotein A1 and for modulating anti-HDL antibodies

The antiretroviral nevirapine (NVP) is associated to a reduction of atherosclerotic lesions and increases in high-density lipoprotein (HDL)-cholesterol. Despite being a hepatotoxic drug, which forbids its re-purposing to other therapeutic areas, not all NVP metabolites have the same potential to induce toxicity. Our aim was to investigate the effects of NVP and its metabolites in an exploratory study, towards the identification of a candidate to boost HDL. A pilot prospective (n = 11) and a cross-sectional (n = 332) clinical study were performed with the following endpoints: HDL-cholesterol and apolipoprotein A1 (ApoA1) levels, anti-HDL and anti-ApoA1 antibodies titers, paraoxonase, arylesterase and lactonase activities of paraoxonase-1, and NVP’s metabolite profile. NVP treatment increased HDL-cholesterol, ApoA1 and paraoxonase-1 activities, and lowered anti-HDL and anti-ApoA1 titers. In the prospective study, the temporal modulation induced by NVP was different for each HDL-related endpoint. The first observation was a decrease in the anti-HDL antibodies titers. In the cross-sectional study, the lower titers of anti-HDL antibodies were associated to the proportion of 2-hydroxy-NVP (p = 0.03). In vitro models of hepatocytes were employed to clarify the individual contribution of NVP’s metabolites for ApoA1 modulation. Long-term incubations of NVP and 2-hydroxy-NVP in the metabolically competent 3D model caused an increase in ApoA1 reaching 43 % (p < 0.05) and 86 % (p < 0.001), respectively. These results support the contribution of drug biotransformation for NVP-induced HDL modulation, highlighting the role of 2-hydroxy-NVP as ApoA1 booster and its association to lower anti-HDL titers. This biotransformation-guided approach allowed us to identify a non-toxic NVP metabolite as a candidate for targeting HDL.

Nevirapine Biotransformation Insights: An Integrated In Vitro Approach Unveils the Biocompetence and Glutathiolomic Profile of a Human Hepatocyte-Like Cell 3D Model.

The need for competent in vitro liver models for toxicological assessment persists. The differentiation of stem cells into hepatocyte-like cells (HLC) has been adopted due to its human origin and availability. Our aim was to study the usefulness of an in vitro 3D model of mesenchymal stem cell-derived HLCs. 3D spheroids (3D-HLC) or monolayer (2D-HLC) cultures of HLCs were treated with the hepatotoxic drug nevirapine (NVP) for 3 and 10 days followed by analyses of Phase I and II metabolites, biotransformation enzymes and drug transporters involved in NVP disposition. To ascertain the toxic effects of NVP and its major metabolites, the changes in the glutathione net flux were also investigated. Phase I enzymes were induced in both systems yielding all known correspondent NVP metabolites. However, 3D-HLCs showed higher biocompetence in producing Phase II NVP metabolites and upregulating Phase II enzymes and MRP7. Accordingly, NVP-exposure led to decreased glutathione availability and alterations in the intracellular dynamics disfavoring free reduced glutathione and glutathionylated protein pools. Overall, these results demonstrate the adequacy of the 3D-HLC model for studying the bioactivation/metabolism of NVP representing a further step to unveil toxicity mechanisms associated with glutathione net flux changes.

Synthesis and evaluation of nevirapine analogs to study the metabolic activation of nevirapine

Nevirapine (NVP) is widely used as a non-nucleoside reverse transcriptase inhibitor of HIV-1, however, it is associated with severe skin and liver injury. The mechanisms of these adverse reactions are not yet clear, but the metabolic activation of NVP is thought to be related to the injury process. Until now, several metabolic activation pathways of NVP have been reported. In this study, in order to identify the reactive metabolite of NVP mainly responsible for CYP inhibition and liver injury, we synthesized five NVP analogs designed to avoid the proposed bioactivation pathway and evaluated their metabolic stabilities, CYP3A4 time-dependent inhibitory activities, and cytotoxicity. As a result, only a pyrimidine analog of NVP, which could avoid the formation of a reactive epoxide intermediate, did not inhibit CYP3A4. Outside of this compound, the other synthesized compounds, which could avoid the generation of a reactive quinone-methide intermediate, inhibited CYP3A4 equal to or stronger than NVP. The pyrimidine analog of NVP did not induce cytotoxicity in HepG2 and transchromosomic HepG2 cells, expressing major four CYP enzymes and CYP oxidoreductase. These results indicated that the epoxide intermediate of NVP might play an important role in NVP-induced liver injury.

Simultaneous quantification of commonly prescribed antiretroviral drugs and their selected metabolites in aqueous environmental samples by direct injection and solid phase extraction liquid chromatography - tandem mass spectrometry

The widespread implementation of antiretroviral therapy medication has made antiretroviral drugs (ARVDs) a significant pharmaceutical class in regions of high HIV infection rates. However, relatively little is known regarding the environmental occurrence of these emerging contaminants, and this is especially true for their metabolites. In this work, we report analytical methods to study the simultaneous occurrence of a range of common ARVDs and some of their known metabolites in surface water and wastewater. A novel direct injection liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is reported for the analysis of ARVDs of different therapeutic classes and their selected metabolites in wastewater samples. In addition, a solid phase extraction (SPE) procedure was developed for pre-concentration of ARVs and metabolites from surface water samples. The respective methods proved suitable for the quantitative analysis of six parent ARVDs from three ARV classes, as well as three metabolites. Method validation showed average recoveries of 86% for the direct injection method, and 64% for the SPE method. With the exception of Zidovudine and the metabolites of Zidovudine and Ritonavir, all target ARVDs were detected in wastewater samples from two wastewater treatment plants in the Western Cape, South Africa. Higher concentrations were generally measured in influent compared to effluent samples, in the dry compared to the wet season as well as in chlorinated compared to uv-irradiated effluents. This study contributes for the first time quantitative data on the environmental occurrence of the known metabolites of Nevirapine (12-hydroxy-Nevirapine) and Efavirenz (8,14-dihydroxy-Efavirenz).

In vitro assessment of competitive and time-dependent inhibition of the nevirapine metabolism by nortriptyline in rats

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1 (HIV-1) widely used as a component of High Active Antiretroviral Therapy (HAART) since it is inexpensive, readily absorbed after oral administration and non-teratogenic. In the present work, the mechanism of a previously described pharmacokinetic interaction between NVP and the antidepressant drug nortriptyline (NT) was studied using rat hepatic microsomes. The obtained results showed a competitive inhibition of the NVP metabolism by NT. The three main NVP metabolites (2-OH-NVP, 3-OH-NVP and 12-OH-NVP) where competitively inhibited with similar inhibitory constant values (Ki = 4.01, 3.97 and 4.40 μM, respectively). Time-dependent inhibition of the NVP metabolism was also detected, with a 2.5-fold reduction in the IC50 values of NT for 2-, 3-, and 12-OH-NVP formation when NT was preincubated with the microsomal suspension in the presence of an NADPH-generating system. A concentration-dependent inhibition of the formation of NVP metabolites by the main NT metabolite (10-OH-NT) was also observed, however, the inhibitory potency of 10-OH-NT was much lower than that of the parent drug. The apparent hepatic intrinsic clearance of NVP determined in these in vitro experiments was used to predict the in vivo clearance of NVP using the “well-stirred” and the “parallel-tube” models, resulting in values close to those previously observed in vivo clearance. Finally, a good prediction of the increase in the plasma concentrations of NVP when co-administered with NT was obtained employing the inhibitory constant of NT determined in vitro and the estimated plasma concentration of NT entering the liver.

Site‐Specific Dueteration Decreases Cytrochrome P450 Production of 12‐Hydroxy‐Nevirapine through the Kinetic Isotope Effect

The non‐nucleoside reverse transcriptase inhibitor nevirapine (NVP), classified as an essential medication by the World Health Organization, has been shown to cause life‐threatening hepatotoxicity and skin rash in patients. The 12‐hydroxyNVP (12‐OHNVP) metabolite of NVP has been implicated in these toxicities and here we aimed to gain a detailed understanding of utility of introducing deuteriums at the 12‐position of NVP as an approach to decreasing the formation of this metabolite. To do so, an ultra‐high performance liquid chromatograph tandem high‐resolution mass spectrometry assay was developed and employed to detect 12‐OHNVP formation following incubation of NVP with human liver microsomes and individual cDNA‐expressed enzymes. Through this work, we found that formation of 12‐OHNVP was most abundant following incubation with cytochrome P450 (P450; CYP) 2C19 although each of the P450s tested, with the exception of CYP3A5, catalyzed the formation of 12‐OHNVP to some extent. In line with this, formation of 12‐OHNVP by human liver microsomes was not abrogated in the presence of any established small molecule inhibitors of P450 activity including ketoconazole (CYP3A inhibitor). In order to test whether the deuterium kinetic isotope effect could be employed to control 12‐OHNVP production by all P450s we investigated the impact of 12th position deuteration of NVP on the formation of this metabolite. Human liver microsomes were incubated with 10 μM NVP or 10 μM 12‐d3NVP for 30 minute and the production of the 12‐OHNVP was measured. The abundance of 12‐OHNVP was 6.8‐fold lower when human liver microsomes were incubated with 12‐d3NVP, as compared to undeuterated NVP incubations. Of note, no difference in the formation of other P450‐dependent metabolites of NVP was observed. In order to test the impact of deuterium kinetic isotope effects on the metabolism of NVP in a whole cell system, we measured metabolite levels in the medium of primary mouse hepatocytes treated with 10 μM NVP and 12‐d3NVP for 24 hours. A 4.7‐fold decrease was observed in 12‐OHNVP production in the medium of cells treated with deuterated NVP as compared to undeuterated NVP. As with the human liver microsome experiments, no significant difference was observed in the production of other P450‐dependent metabolites between treatments. These results demonstrate that dueteration at the 12th position can markedly decrease cytochrome P450 production of 12‐OHNVP through the kinetic isotope effect. The impact of this controlled metabolism on NVP toxicity will be a subject of further research. This work was funded by NSF GRFP DGE‐1232825 awarded to CJSH and NIH R01 GM103853.Support or Funding InformationThis work was funded by NSF GRFP DGE‐1232825 awarded to CJSH and NIH R01 GM103853.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The impact of a Wastewater Treatment Works in Southern Gauteng, South Africa on efavirenz and nevirapine discharges into the aquatic environment

Abstract There has been growing concern regarding the pollution of the aquatic environment with synthetic organic chemicals. Antiretroviral drugs, such as efavirenz and nevirapine, are pharmaceutical drugs and are referred to as emerging contaminants. Such drugs can be environmentally persistent and may be expected to pose potential risks to drinking water supplies. Sources of pharmaceutical drugs include effluents from Wastewater Treatment Works (WWTPs), hospital and pharmaceutical production facilities and the incorrect disposal of unused and expired medicines. Currently there are no monitoring programs and legislative guidelines for their regulations in South Africa. The aims of this study were firstly to develop a semi-quantitative method to extract and analyse efavirenz and nevirapine in the primary settling tank sludge. Secondly to use that method, and an existing method for liquid wastewater samples, to monitor the concentrations of efavirenz and nevirapine as the wastewater passes through the different stages of purification (anoxic; aerobic; pre and post chlorination) in the WWTP. This was repeated weekly over a period of 4 weeks. Thirdly, to determine if binding of efavirenz and nevirapine to the solids in the WWTP played a role in the removal of these compounds from the WWTP liquid phase. No references to the analysis of ARVDs in WWTP sludge were found in the literature. Grab samples of wastewater and sludge samples were collected from a WWTP (activated sludge treatment process) weekly for 4 weeks. Liquid samples were extracted solid phase extraction, solid samples were extracted using sonication followed by a QuEChERs clean-up. Sample extracts were then subjected to gas chromatography-time of flight mass spectrometry for analyte determination. Efavirenz concentrations entering the WWTP ranged between 5500 to almost 14 000 ng/L. The removal of efavirenz by the WWTP ranged between 27 and 71%. The largest removal occurred in the anoxic zone, smaller amounts were removed in the aerators. Slight increases in efavirenz concentrations were found after chlorination and the final effluent into the river post maturation ponds again were slightly lower. Solids were found to contain efavirenz at concentrations between 17 and 43 mg/kg dried primary settling tank sludge and it is proposed that this binding to the solids is the main mechanism of removal of efavirenz from the wastewater stream as it passes through the WWTP. Although an order of magnitude lower nevirapine concentrations displayed the opposite behaviour and gradually increased through the various stages of purification in the WWTP. Minor fluctuations occurred but the concentrations of nevirapine were higher at the effluent (between 92 and 473 ng/L) than those entering the WWTP. No nevirapine was detected in the PST sludge. The increase in nevirapine concentrations are likely to be the result of the de-conjugation of the hydroxylated metabolites of nevirapine in the WWTP, its resistance to degradation and the lack of binding of the nevirapine to the PST sludge.

Different Reactive Metabolites of Nevirapine Require Distinct Glutathione S-Transferase Isoforms for Bioinactivation.

Nevirapine (NVP) is a non-nucleoside reverse transcriptase-inhibitor, which is associated with severe idiosyncratic skin rash and hepatotoxicity. These adverse drug reactions are believed to be mediated by the formation of epoxides and/or quinone methide formed by oxidative metabolism by P450s and 12-sulfoxyl-NVP formed by sequential 12-hydroxylation and O-sulfonation. Although different GSH-conjugates and corresponding mercapturic acids have been demonstrated previously in vitro and in vivo, the role of the glutathione S-transferases in the inactivation of the different reactive metabolites has not been studied so far. In the present study the activity of 10 recombinant human glutathione S-transferases (GSTs) in the detoxification of the different reactive metabolites of NVP was studied. The results show that GSTP1-1 is a highly active catalyst of GSH-conjugation of the oxidative metabolites of NVP, even at high GSH-concentration. Experiments with trideuterated NVP suggest involvement of a reactive epoxide rather than quinone methide in the formation of the GSH-conjugate formed after oxidative bioactivation. GSH-conjugation of 12-sulfoxyl-NVP forming NVP-12-GSH was only catalyzed by GSTM1-1, GSTA1-1, and GSTA3-3. Although the exact expression levels of these enzymes in the skin is unknown, the relatively low activity of this catalysis makes it unlikely that GSTs can provide significant protection against this metabolite. However, since NVP-12-GSH is specifically formed via the 12-sulfoxyl-NVP, its corresponding urinary mercapturic acid can be considered as a biomarker for recent internal exposure to this protein-reactive sulfate. However, it has to be taken into account that 12-sulfoxyl-NVP is not completely trapped by GSH and that rates of bioinactivation will differ between patients due to variability in expression of GSTM1, GSTA1, and GSTA3.

Hepatocyte spheroids as a competent in vitro system for drug biotransformation studies: nevirapine as a bioactivation case study.

The development of metabolically competent in vitro models is of utmost importance for predicting adverse drug reactions, thereby preventing attrition-related economical and clinical burdens. Using the antiretroviral drug nevirapine (NVP) as a model, this work aimed to validate rat hepatocyte 3D spheroid cultures as competent in vitro systems to assess drug metabolism and bioactivation. Hepatocyte spheroids were cultured for 12days in a stirred tank system (3D cultures) and exposed to equimolar dosages of NVP and its two major Phase I metabolites, 12-OH-NVP and 2-OH-NVP. Phase I NVP metabolites were detected in the 3D cultures during the whole culture time in the same relative proportions reported in in vivo studies. Moreover, the modulation of SULT1A1 activity by NVP and 2-OH-NVP was observed for the first time, pointing their synergistic effect as a key factor in the formation of the toxic metabolite (12-sulfoxy-NVP). Covalent adducts formed by reactive NVP metabolites with N-acetyl-L-cysteine and bovine serum albumin were also detected by high-resolution mass spectrometry, providing new evidence on the relative role of the reactive NVP metabolites, 12-sulfoxy-NVP, and NVP quinone methide, in toxicity versus excretion pathways. In conclusion, these results demonstrate the validity of the 3D culture system to evaluate drug bioactivation, enabling the identification of potential biomarkers of bioactivation/toxicity, and providing new evidence to the mechanisms underlying NVP-induced toxic events. This model, integrated with the analytical strategies described herein, is of anticipated usefulness to the pharmaceutical industry, as an upstream methodology for flagging drug safety alerts in early stages of drug development.

Enzymatic bioactivation of the phenolic nevirapine metabolites 2-hydroxy- and 3-hydroxy-nevirapine to quinoid intermediates: Covalent adduct formation with nitrogen-based amino acids

Enzymatic bioactivation of the phenolic nevirapine metabolites 2-hydroxy- and 3-hydroxy-nevirapine to quinoid intermediates: Covalent adduct formation with nitrogen-based amino acids

Quinoid derivatives of the nevirapine metabolites 2-hydroxy- and 3-hydroxy-nevirapine: activation pathway to amino acid adducts

Quinoid electrophiles from the nevirapine metabolites, 2-OH- and 3-OH-nevirapine, react with nitrogen-based bionucleophiles yielding covalent adducts.

Human sulfotransferase 1A1-dependent mutagenicity of 12-hydroxy-nevirapine: the missing link?

Nevirapine (NVP) is a frequently used anti-HIV drug. Despite its efficacy, NVP has been associated with serious skin and liver injuries in exposed patients and with increased incidences of hepatoneoplasias in rodents. Current evidence supports the involvement of reactive metabolites in the skin and liver toxicities of NVP, formed by cytochrome P450-mediated oxidations and/or subsequent phase II sulfonation. However, to date, standard in vitro genotoxicity tests have provided no evidence that NVP is either mutagenic or clastogenic. The human sulfotransferase 1A1-dependent mutagenicity of 12-hydroxy-NVP, one of the major metabolites of NVP, is demonstrated here.

Development and validation of an HPLC-UV method for quantifying nevirapine and its main phase I metabolites in human blood

Nevirapine (NVP) is a widely used drug for the treatment of HIV-infection, which is associated with severe toxic events dependent on its biotransformation. Thus, the availability of analytical methods for the biomonitoring of NVP and its main metabolites is of utmost importance. In this paper we report the development and validation of a reversed phase HPLC-UV method for the quantification of NVP and its main phase I metabolites in human plasma. The method was validated over a range of 10–2500 ng mL−1 for the phase I metabolites of NVP and 10–10 000 ng mL−1 for NVP. The coefficients of variation (CV) of the average back-calculated concentrations were lower than 9% and the LLOQ was 10 ng mL−1 for each analyte. The accuracy and precision of the method were acceptable. There was no significant interference from plasma components or from other typically co-administered antiretroviral drugs and the mean recovery was 94%. This method represents an inexpensive, sensitive, accurate and precise alternative to mass spectrometry-based biomonitoring of NVP.

Differences in nevirapine biotransformation as a factor for its sex-dependent dimorphic profile of adverse drug reactions

Nevirapine is widely used for the treatment of HIV-1 infection; however, its chronic use has been associated with severe liver and skin toxicity. Women are at increased risk for these toxic events, but the reasons for the sex-related differences are unclear. Disparities in the biotransformation of nevirapine and the generation of toxic metabolites between men and women might be the underlying cause. The present work aimed to explore sex differences in nevirapine biotransformation as a potential factor in nevirapine-induced toxicity. All included subjects were adults who had been receiving 400 mg of nevirapine once daily for at least 1 month. Blood samples were collected and the levels of nevirapine and its phase I metabolites were quantified by HPLC. Anthropometric and clinical data, and nevirapine metabolite profiles, were assessed for sex-related differences. A total of 52 patients were included (63% were men). Body weight was lower in women (P = 0.028) and female sex was associated with higher alkaline phosphatase (P = 0.036) and lactate dehydrogenase (P = 0.037) levels. The plasma concentrations of nevirapine (P = 0.030) and the metabolite 3-hydroxy-nevirapine (P = 0.035), as well as the proportions of the metabolites 12-hydroxy-nevirapine (P = 0.037) and 3-hydroxy-nevirapine (P = 0.001), were higher in women, when adjusted for body weight. There was a sex-dependent variation in nevirapine biotransformation, particularly in the generation of the 12-hydroxy-nevirapine and 3-hydroxy-nevirapine metabolites. These data are consistent with the sex-dependent formation of toxic reactive metabolites, which may contribute to the sex-dependent dimorphic profile of nevirapine toxicity.

Identification of Danger Signals in Nevirapine-Induced Skin Rash

Nevirapine (NVP) can cause serious skin rashes and hepatotoxicity. It also causes an immune-mediated skin rash in rats but not hepatotoxicity. This rash is caused by a metabolite of NVP; specifically, NVP is oxidized in the liver to a benzylic alcohol (12-OH-NVP), which travels to the skin where it forms a reactive benzylic sulfate. This could both act as a hapten and induce a danger signal. In contrast, most of the covalent binding in the liver involves oxidation of the methyl group leading to a reactive quinone methide. In this study, we examined the effects of NVP and 12-OH-NVP on gene expression in the liver and skin. Both NVP and 12-OH-NVP induced changes in the liver, but the list of genes was different, presumably reflecting different bioactivation pathways. In contrast, many more genes were up-regulated in the skin by 12-OH-NVP than by NVP, which is consistent with the fact that 12-OH-NVP is an obligate intermediate in the formation of the reactive sulfate in the skin. Genes up-regulated by 12-OH-NVP in the skin included TRIM63 (18-fold increase), S100a7a (7-fold increase), IL22-RA2 (4-fold increase), and DAPK1 (3-fold increase). TRIM63 acts as a ubiquitin ligase, which is consistent with protein damage leading to an increase in protein turnover. In addition, TRIM proteins are involved in inflammasome activation, and it appears that inflammasome activation is an essential step in the induction of NVP-induced skin rash. S100A7 is considered a danger signal, and its upregulation supports the danger hypothesis. Upregulation of the IL-22 RA2 gene marks an immune response. DAPK1 is involved with inflammasome assembly through binding directly to NLRP3, a NOD-like receptor expressed in keratinocytes. These results provide important clues to how NVP causes the induction of an immune response, in this case leading to skin rash.

Detection of Drug Bioactivation in Vivo: Mechanism of Nevirapine–Albumin Conjugate Formation in Patients

The non-nucleoside reverse transcriptase inhibitor nevirapine (NVP) is widely used for the treatment of human immunodeficiency virus type 1 (HIV-1), particularly in developing countries. Despite its therapeutic benefits, NVP has been associated with skin and liver injury in exposed patients. Although the mechanism of the tissue injury is not yet clear, it has been suggested that reactive metabolites of NVP may be involved. The detection of NVP mercapturate in the urine of patients undergoing standard antiretroviral chemotherapy indicates that NVP undergoes bioactivation in vivo. However, covalent binding of drug to protein in patients remains to be determined. In this study, we investigate the chemical basis of NVP protein adduct formation by using human serum albumin (HSA) and glutathione S-transferase pi (GSTP) as model proteins in vitro. In addition, HSA was isolated from serum samples of HIV-1 patients undergoing NVP therapy to measure NVP haptenation. Mass spectrometric analysis of 12-sulfoxyl-NVP-treated HSA revealed that the drug bound selectively to histidine (His146, His242, and His338) and a cysteine residue (Cys34). The reaction proceeds most likely by a concerted elimination-addition mechanism. This pathway was further confirmed by the observation of NVP-modified Cys47 in GSTP. Importantly, the same adduct (His146) was detected in HSA isolated from the blood of patients receiving NVP, providing direct evidence that NVP modifies protein in vivo, via the formation of a reactive metabolite.