Aline T. Marinho

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

Despite 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 1month. 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.

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

OBJECTIVES 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Quantification of the arylesterase activity of paraoxonase-1 in human blood

Paraoxonase-1 (PON1) is known as a free-radical scavenging system associated with circulating serum high-density lipoprotein (HDL). PON1 catalyzes the hydrolysis of multiple compounds such as arylesters, lactones and hydroperoxides. The arylesterase (AREase) activity of PON1 is involved in the detoxification of lipid peroxides, which are related to several clinical conditions. Therefore, the possibility of measuring the AREase activity in routine clinical studies would be advantageous. The AREase activity was obtained by monitoring the formation of acetic acid, upon the hydrolysis of phenyl acetate, using 10 μL of sample. The method accuracy was higher than 90% and intra-assay and inter-assay precisions were 96% and 95%, respectively. The method validation supported that this analytical procedure is suitable for use in human serum and heparinized plasma samples, while ethylenediaminetetra-acetic acid (EDTA)-containing samples should be avoided. The methodology herein described constitutes an easy, fast and reliable method for assessing the AREase activity of PON1. This method can be easily implemented as a clinical analytical tool and is also suitable for research purposes.

Long-term maraviroc use as salvage therapy in HIV-2 infection

References 1 Obeid S, Printsevskaya SS, Olsufyeva EN et al. Inhibition of hepatitis C virus replication by semi-synthetic derivatives of glycopeptide antibiotics. J Antimicrob Chemother 2011; 66: 1287–94. 2 Balzarini J, Keyaerts E, Vijgen L et al. Inhibition of feline (FIPV) and human (SARS) coronavirus by semisynthetic derivatives of glycopeptide antibiotics. Antiviral Res 2006; 72: 20–33. 3 Preobrazhenskaya MN, Olsufyeva EN. Polycyclic peptide and glycopeptide antibiotics and their derivatives as inhibitors of HIV entry. Antiviral Res 2006; 71: 227–36. 4 Barcia-Macay M, Seral C, Mingeot-Leclercq MP et al. Pharmacodynamic evaluation of the intracellular activities of antibiotics against Staphylococcus aureus in a model of THP-1 macrophages. Antimicrob Agents Chemother 2006; 50: 841–51. 5 Nagy PD, Pogany J. The dependence of viral RNA replication on co-opted host factors. Nat Rev Microbiol 2011; 10: 137–49. 6 Welsh RM, Che JW, Brehm MA et al. Heterologous immunity between viruses. Immunol Rev 2010; 235: 244–66. 7 Wedemeyer H, Mizukoshi E, Davis AR et al. Cross-reactivity between hepatitis C virus and influenza A virus determinant-specific cytotoxic T cells. J Virol 2001; 75: 11392–400. J Antimicrob Chemother 2012 doi:10.1093/jac/dks240 Advance Access publication 22 June 2012

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.

Sex differences in hepatic and intestinal contributions to nevirapine biotransformation in rats

The understanding of the intestine contribution to drug biotransformation improved significantly in recent years. However, the sources of inter-individual variability in intestinal drug biotransformation, namely sex-differences, are still elusive. Nevirapine (NVP) is an orally taken anti-HIV drug associated with severe idiosyncratic reactions elicited by toxic metabolites, with women at increased risk. As such, NVP is a good model to assess sex-dimorphic metabolism. The aim of this study was to perform a comparative profiling of NVP biotransformation in rat intestine and liver and evaluate whether or not it is organ- and sex-dependent. Therefore, nevirapine-containing solutions were perfused through the intestine, in a specially designed chamber, or incubated with liver slices, from male and female Wistar rats. The levels of NVP and its Phase I metabolites were quantified by HPLC-UV. Liver incubation experiments yielded the metabolites 2-, 3-, 8-, and 12-OH-NVP, being 12-OH-NVP and 2-OH-NVP the major metabolites in males and females, respectively. Inter-sex differences in the metabolic profile were also detected in the intestine perfusion experiments. Herein, the metabolites 3- and 12-OH-NVP were only found in male rats, whereas 2-OH-NVP levels were higher in females, both in extraluminal (p<0.01) and intraluminal media. The metabolite 8-OH-NVP was not detected in the intraluminal media from either males or females. In this study, important inter-sex differences were detected in both organs, providing further clues to the sex-dimorphic profile of NVP toxicity. Moreover, an extra-hepatic contribution to NVP biotransformation was observed, strengthening the relevance of the intestinal contribution in the biotransformation of orally taken-drugs.

Nevirapine modulation of paraoxonase-1 in the liver: An in vitro three-model approach.

INTRODUCTION Nevirapine is associated with severe hepatotoxicity, through the formation of reactive metabolites. Paraoxonase-1 (PON-1) is a promiscuous enzyme involved in the metabolism of xeno- and endobiotics and proposed as a biomarker of hepatotoxicity. The aim of this work was to explore the effects of nevirapine and its phase I metabolites, 2-hydroxy-nevirapine and 12-hydroxy-nevirapine, on PON-1 activities. MATERIAL AND METHODS 2D and 3D primary cultures of rat hepatocytes, and also HepG2 2D cell cultures, were exposed to nevirapine, 2-hydroxy-nevirapine, and 12-hydroxy-nevirapine. The paraoxonase (POase), arylesterase (AREase) and lactonase (LACase) activities of PON-1 were quantified. RESULTS Effects of nevirapine and its metabolites were only observed in the 3D cell model. Both nevirapine and 12-hydroxy-nevirapine increased POase (p<0.05, p<0.01) and LACase activities (p<0.05, p<0.001). The AREase activity was increased only upon 12-hydroxy-nevirapine exposure (p<0.01). These modulatory effects were observed at 300μM concentrations of nevirapine and 12-hydroxy-nevirapine. CONCLUSIONS The formation of 12-hydroxy-nevirapine seems to be the main factor responsible for the increase of PON-1 activities induced by nevirapine exposure. This effect was only observed in the 3D model, suggesting that an in vivo-like system is necessary for this modulation to occur. The present data suggest that the 3D model is a more suitable in vitro model than the conventional ones to explore drug effects on PON-1.

Monitoring of the lactonase activity of paraoxonase-1 enzyme in HIV-1-infection

Paraoxonase‐1 (PON1) is a high‐density lipoprotein (HDL)‐associated enzyme known as a free radical scavenging system ( 1 ). PON‐1 has three main activities, responsible for its antioxidant and anti‐inflammatory potential: paraoxonase, arylesterase and lactonase (LACase), the latest to be discovered and pointed out to be its native activity ( 2 ). Among other physiological roles, the LACase might minimize the deleterious effects of hyperhomocysteinaemia in infection, by detoxifying the highly reactive metabolite homocysteine‐thiolactone (HcyTL) ( 3 ), 4 . In the present work, we have developed and applied a method to quantify LACase activity and to explore the role of this enzyme in HIV‐infection and virological response. The LACase activity was monitored in a cohort of HIV‐1‐infected patients, through the titration of 3‐(o‐hydroxyphenyl) propionic acid, formed upon the LACase‐mediated hydrolysis of the substrate dihydrocoumarin. The study protocol was approved by the Ethics Committee of Centro Hospitalar de Lisboa Central and Hospital Prof. Doutor Fernando Fonseca. All patients gave their written informed consent and were adults with documented HIV‐1‐infection, regardless of combined antiretroviral therapy (cART) use. Naïve patients and patients who had received continuous antiretroviral treatment for more than one month were included. A total of 179 HIV‐1‐infected patients were included on this study (51% Men, 39% non‐Caucasian, 45±13 years old). Patients with non‐suppressed viraemia, either from the non‐cART (n=89, 12±4 kU/L, p<0.01) or from the cART with detectable viral load (n=11, 10±5 kU/L, p<0.05) groups, had lower activity than the cART with suppressed viraemia (n=79, 15±7 kU/L) (Kruskal–Wallis test). Among naïve patients, higher viral load (> 31,500 cps/mL, Spearman r=−0.535, p=0.003) and lower CD4+ T‐cells count (< 500 cell/mm3, Pearson r=0.326, p=0.024) were associated with the LACase activity. The present study suggests that lower LACase activity is associated with uncontrolled HIV‐1‐infection, particularly with non‐suppressed viraemia, despite of cART. This data seems to point to LACase role in HIV‐infection, probably reflecting an increased formation of HcyTL deleterious species. A better knowledge of the LACase and its role in HcyTL pathophysiology might identify new therapeutic targets in HIV‐1‐infected patients.

Sex differences in apolipoprotein A1 and nevirapine-induced toxicity.

Nevirapine (NVP) is associated with severe liver and skin toxicity through sulfotransferase (SULT) bioactivation of the phase I metabolite 12‐hydroxy‐NVP [ 1 – 3 ]. The female sex, a well‐known risk factor for NVP‐induced toxicity, is associated with higher SULT expression [ 4 ] and lower plasma levels of 12‐hydroxy‐NVP [ 3 ]. Interestingly, apolipoprotein A1 (ApoA1) increases SULT2B1 activity and ApoA1 synthesis is increased by NVP [ 5 , 6 ]. Herein, we explore the effect of ApoA1 levels on NVP metabolism and liver function. The study protocol was firstly approved by the hospitals’ Ethics Committees. All included individuals were HIV‐infected patients treated with NVP for at least one month. The plasma concentrations of NVP and its phase I metabolites were quantified by HPLC [ 7 ]. ApoA1 levels were assessed by an immunoturbidimetric assay. Forty‐nine HIV‐infected patients on NVP were included (53% men, 59% Caucasian). NVP plasma levels were correlated with HDL‐cholesterol (Spearman r=0.2631; p=0.0441) and ApoA1 (Spearman r=0.3907; p=0.0115). Women had higher ApoA1 levels than men (Students t Test; p=0.0051). In both sexes, 12‐hydroxy‐NVP levels were negatively correlated with ApoA1 (male: Spearman r=−0.3810; p=0.0499 female: Spearman r=−0.5944; p=0.0415). In men, ApoA1 was positively correlated with aspartate aminotransferase (AST, Spearman r=0.5507; p=0.0413), while in women ApoA1 was associated (Spearman r=0.6408; p=0.0056) with alanine aminotransferase (ALT). These results show sex differences in NVP‐induced ApoA1 synthesis. The higher ApoA1 levels in women might stabilize SULT2B1 [ 6 ]. This would explain the lower levels of 12‐hydroxy‐NVP [ 3 ] and the higher hepatotoxicity found in women, due to increased sulfonation of this metabolite. These data support a role for ApoA1 in the sex dimorphic mechanism leading to NVP‐induced toxicity.