Umbelina Caixas

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.

Intra-Individual Variability in Efavirenz Plasma Concentrations Supports Therapeutic Drug Monitoring Based on Quarterly Sampling in the First Year of Therapy

Intrapatient variability in drug plasma concentrations is critical to the use of therapeutic drug monitoring with efavirenz, a non-nucleoside reverse-transcriptase inhibitor. Marked intrapatient variability, particularly for concentrations near the minimal therapeutic concentration, could be a predictor of virologic failure, meaning that a single concentration is of limited value. Previous reports on efavirenz intra-individual variability were obtained only in follow-up periods of 3 to 12 months and do not provide a rationale for the periodicity of sample measurements needed in long-term therapy to identify patients with a large variability and increased risk of therapeutic failure. The aim of this work was to investigate intra-individual variability in efavirenz plasma concentrations over a long-term follow-up period to support therapeutic drug monitoring. In a case series study, clinical and laboratory data were collected from all HIV-positive adults at the immunodeficiency outpatient clinic who were on regimens containing efavirenz in 2002 and who gave their informed consent (n = 31). Efavirenz plasma concentrations were measured throughout a 3 year period, without dose adjustments. For each patient, 6 to 12 samples were obtained over the follow-up period with an interval of at least 3 months between each sample. Mean plasma concentrations (mg/L) in the first, second, and third year of follow-up were 2.20 ± 0.64, 2.17 ± 0.68, and 2.31 ± 0.57. Mean intra-individual variability throughout the first, second, and third year of study was 27%, 31%, and 25%, ranging from 12% to 63%. No differences in intrapatient variability in efavirenz plasma concentrations were found between females and males, HBV/HCV+ and HBV/HCV− patients, or age above/below 40 years. Mean values (intra-individual variability) in plasma concentrations (mg/L) found in 3 of 31 patients who experienced virologic failure were 1.78 (42%), 1.52 (16%), and 1.68 (45%). The high interindividual variability and low maintained values of intrapatient variability in plasma concentrations support therapeutic drug monitoring, which could be based on measurements taken quarterly during the first year of therapeutics. In patients presenting high values of intra-individual variability (eg, >40%) associated with low plasma concentrations (eg, <2 mg/L), more frequent measurements over longer periods (more than 1 yr) of controlled concentrations might be recommended, but this requires further investigation.

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.

Efavirenz concentrations in HIV-infected patients with and without viral hepatitis

AIMS Data on efavirenz in HIV/viral hepatitis co-infected patients is non-consensual, probably due to liver function heterogeneity in the patients included. METHODS A case control study was performed on 27 HIV-infected patients, with controlled and homogenous markers of hepatic function, either mono-infected or co-infected with HBV/HCV, to ascertain the influence of viral hepatitis on efavirenz concentrations over a 2-year follow-up period. RESULTS No differences were found in efavirenz concentrations between groups both during and at the end of the follow-up period: control (2.43 +/- 1.91 mg l(-1)) vs. co-infected individuals (2.37 +/- 0.37 mg l(-1)). CONCLUSION It was concluded that HBV/HCV infections in themselves do not predispose to an overexposure to efavirenz.

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

Efavirenz biotransformation as an up-stream event of mood changes in HIV-infected patients.

Efavirenz is a drug of choice for adults and children infected with the human immunodeficiency virus. Notably, up to 35% of patients on efavirenz suffer from mood changes. This work aimed to investigate efavirenz biotransformation into 8-hydroxy-efavirenz as an up-stream event of mood changes and to evaluate the suitability of 8-hydroxy-efavirenz biomonitoring for the minimization of these manifestations. A case-control study with two age-matched groups of HIV-infected male patients was performed in a group without adverse central nervous system complaints (28 patients) and a group presenting mood changes (14 patients). The plasma concentration of non-conjugated 8-hydroxy-efavirenz was higher in patients with mood changes (p=0.020). An association between efavirenz and 8-hydroxy-efavirenz-glucuronide was found (Spearman r=0.414, p<0.010), only within therapeutic efavirenz concentrations. This correlation was not observed in patients with toxic (>4mg/L) plasma concentrations of the parent drug. We conclude that metabolism to 8-hydroxy-efavirenz is associated with efavirenz-related mood changes, which suggests that the concentration of this metabolite is a suitable parameter for therapeutic drug monitoring aimed at controlling these manifestations. Moreover, our data suggest that 8-hydroxy-efavirenz is able to cross the blood-brain barrier and that the peripheral detoxification of 8-hydroxy-efavirenz by glucuronidation may be inhibited by toxic efavirenz concentrations.

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.

HIV and antioxidant lipoprotein-associated effect. Is there a correlation?

Purpose of the study HIV-patients often develop long-term pro-atherogenic metabolic alterations. HDL-cholesterol (C) concentration is known to be decreased in HIV-infected patients. Lipoprotein metabolism changes occurring as host response to infection include an antioxidant effect that is part of the defense system. Paraoxonase (PON) are HDL-associated enzymes that, due to their antioxidant properties, have been considered the main factor responsible for the HDL anti-atherogenic role. Higher HDL concentrations have been associated with a better disease course in HIV patients undergoing antiretroviral treatment. The aim of this study was to determine relationship between HIVinfection in treatment-naïve patients and activity of PON1 and lipoprotein concentrations.