Sofia A. Pereira

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.

Reactive aldehyde metabolites from the anti-HIV drug abacavir: amino acid adducts as possible factors in abacavir toxicity.

Abacavir is a nucleoside reverse transcriptase inhibitor marketed since 1999 for the treatment of infection with the human immunodeficiency virus type 1 (HIV). Despite its clinical efficacy, abacavir administration has been associated with serious and sometimes fatal toxic events. Abacavir has been reported to undergo bioactivation in vitro, yielding reactive species that bind covalently to human serum albumin, but the haptenation mechanism and its significance to the toxic events induced by this anti-HIV drug have yet to be elucidated. Abacavir is extensively metabolized in the liver, resulting in inactive glucuronide and carboxylate metabolites. The metabolism of abacavir to the carboxylate involves a two-step oxidation via an unconjugated aldehyde, which under dehydrogenase activity isomerizes to a conjugated aldehyde. Concurrently with metabolic oxidation, the two putative aldehyde metabolites may be trapped by nucleophilic side groups in proteins yielding covalent adducts, which can be at the onset of the toxic events associated with abacavir. To gain insight into the role of aldehyde metabolites in abacavir-induced toxicity and with the ultimate goal of preparing reliable and fully characterized prospective biomarkers of exposure to the drug, we synthesized the two putative abacavir aldehyde metabolites and investigated their reaction with the α-amino group of valine. The resulting adducts were subsequently stabilized by reduction with sodium cyanoborohydride and derivatized with phenyl isothiocyanate, leading in both instances to the formation of the same phenylthiohydantoin, which was fully characterized by NMR and MS. These results suggest that the unconjugated aldehyde, initially formed in vivo, rapidly isomerizes to the thermodynamically more stable conjugated aldehyde, which is the electrophilic intermediate mainly involved in reaction with bionucleophiles. Moreover, we demonstrated that the reaction of the conjugated aldehyde with nitrogen bionucleophiles occurs exclusively via Schiff base formation, whereas soft sulfur nucleophiles react by Michael-type 1,4-addition to the α,β-unsaturated system. The synthetic phenylthiohydantoin adduct was subsequently used as standard for LC-ESI-MS monitoring of N-terminal valine adduct formation, upon modification of human hemoglobin in vitro with the conjugated abacavir aldehyde, followed by reduction and Edman degradation. The same postmodification strategy was applied to investigate the products formed by incubation of abacavir with rat liver cytosol, followed by trapping with ethyl valinate. In both instances, the major adduct detected corresponded to the synthetic phenylthiohydantoin standard. These results suggest that abacavir metabolism to the carboxylate(s) via aldehyde intermediate(s) could be a factor in the toxic events elicited by abacavir administration. Furthermore, the availability of a reliable and fully characterized synthetic standard of the abacavir adduct with the N-terminal valine of hemoglobin and its easy detection in the model hemoglobin modifications support the usefulness of this adduct as a prospective biomarker of abacavir toxicity in humans.

The role of competitive binding to human serum albumin on efavirenz-warfarin interaction: a nuclear magnetic resonance study

The potential for co-prescription of the anti-human immunodeficiency virus (anti-HIV) drug efavirenz (EFV) and the oral anticoagulant warfarin (WAR) is currently high as EFV is a drug of choice for HIV type 1 infection and because cardiovascular disease is increasing among HIV-infected individuals. However, clinical reports of EFV-WAR interaction, leading to WAR overdosing, call for elucidation of the mechanisms involved in this drug-drug interaction. Here we present the first report demonstrating competition of the two drugs for the same binding site of human serum albumin. Using ligand-based nuclear magnetic resonance experiments, this study proves that EFV has an effect on the concentration of free WAR. This previously unidentified EFV-WAR interaction represents a potential risk factor that should be taken into account when considering treatment options.

HNF1β drives glutathione (GSH) synthesis underlying intrinsic carboplatin resistance of ovarian clear cell carcinoma (OCCC).

Chemoresistance to platinum-based antineoplastic agents is a consistent feature among ovarian carcinomas; however, whereas high-grade serous carcinoma (OSC) acquires resistance during chemotherapy, ovarian clear cell carcinoma (OCCC) is intrinsically resistant. The main objective of this study was to explore, in vitro and in vivo, if hepatocyte nuclear factor 1β (HNF1β) and glutaminolysis contribute for the resistance of OCCC to carboplatin through the intrinsically increased GSH bioavailability. To disclose the role of HNF1β, experiments were also performed in an OSC cell line, which does not express HNF1β. Metabolic profiles, GSH quantification, HNF1β, and γ-glutamylcysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) expression, cell cycle, and death were assessed in ES2 cell line (OCCC) and OVCAR3 cell line (OSC); HNF1β knockdown was performed in ES2 and murine model of subcutaneous and peritoneal OCCC tumors was established to test buthionine sulphoxamine (BSO), as a sensitizer to carboplatin. Glutaminolysis is activated in ES2 and OVCAR3, though ES2 exclusively synthesizes amino acids and GSH. ES2 cells are more resistant to carboplatin than OVCAR3 and the abrogation of GSH production by BSO sensitizes ES2 to carboplatin. HNF1β regulates the expression of GCLC, but not GCLM, and consequently GSH production in ES2. In vivo, BSO prior to carboplatin reduces dramatically subcutaneous tumor size and GSH levels, as well as peritoneal dissemination. Our study discloses HNF1β as the mediator of intrinsic OCCC chemoresistance and sheds a light to re-explore a cancer adjuvant therapeutic approach using BSO to overcome the lack of efficient therapy in OCCC.

Efficacy of carvedilol in reversing hypertension induced by chronic intermittent hypoxia in rats

Animal models of chronic intermittent hypoxia (CIH) mimic the hypertension observed in patients with obstructive sleep apnoea. Antihypertensive drugs were applied to these animal models to address the physiological mechanism but not to revert established hypertension. We aimed to investigate the efficacy of carvedilol (CVDL), an unselective beta-blocker that exhibits intrinsic anti-α1-adrenergic and antioxidant activities in a rat model of CIH-induced hypertension. The variability of CVDL enantiomers in plasma concentrations was also evaluated. Wistar rats with indwelling blood pressure telemeters were exposed during their sleep period to 5.6 CIH cycles/h, 10.5 h/day, for 60 days. CVDL was administered by gavage beginning on Day 36 of the CIH period and was continued for 25 days. R-(+)-CVDL and S-(-)-CVDL plasma concentrations were monitored by HPLC. CIH significantly increased diastolic and systolic blood pressure by 25.7 and 21.6 mm Hg respectively, while no effect was observed on the heart rate (HR). CVDL administration at 10, 30 and 50 mg/kg/day promoted a significant reduction in HR but did not affect arterial pressure. The S/(R+S) ratio of CVDL enantiomers was lower in rats exposed to CIH. The blockade of the sympathetic nervous system together with the putative pleiotropic effects of CVDL did not alter the CIH-induced hypertension. Although CIH induced pharmacokinetic changes in the R/(R+S) ratio, these effects do not appear to be responsible for the inability of CVDL to reverse this particular type of hypertension.

Bioactivation to an aldehyde metabolite—Possible role in the onset of toxicity induced by the anti-HIV drug abacavir

Aldehydes are highly reactive molecules, which can be generated during numerous physiological processes, including the biotransformation of drugs. Several non-P450 enzymes participate in their metabolism albeit alcohol dehydrogenase and aldehyde dehydrogenase are the ones most frequently involved in this process. Endogenous and exogenous aldehydes have been strongly implicated in multiple human pathologies. Their ability to react with biomacromolecules (e.g. proteins) yielding covalent adducts is suggested to be the common primary mechanism underlying the toxicity of these reactive species. Abacavir is one of the options for combined anti-HIV therapy. Although individual susceptibilities to adverse effects differ among patients, abacavir is associated with idiosyncratic hypersensitivity drug reactions and an increased risk of cardiac dysfunction. This review highlights the current knowledge on abacavir metabolism and discusses the potential role of bioactivation to an aldehyde metabolite, capable of forming protein adducts, in the onset of abacavir-induced toxic outcomes.

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.