Article pubs.acs.org/crt
Nevirapine Bioactivation and Covalent Binding in the Skin Amy M. Sharma,† Klaus Klarskov,‡ and Jack Uetrecht*,† †
Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 3M2 Department of Pharmacology, Faculty of Medicine and Health Sciences, University of Sherbrooke, Quebec J1H 5N4, Canada
‡
S Supporting Information *
ABSTRACT: Nevirapine (NVP) treatment is associated with serious skin rashes that appear to be immune-mediated. We previously developed a rat model of this skin rash that is immune-mediated and is very similar to the rash in humans. Treatment of rats with the major NVP metabolite, 12-OHNVP, also caused the rash. Most idiosyncratic drug reactions are caused by reactive metabolites; 12-OH-NVP forms a benzylic sulfate, which was detected in the blood of animals treated with NVP or 12-OH-NVP. This sulfate is presumably formed in the liver; however, the skin also has significant sulfotransferase activity. In this study, we used a serum against NVP to detect covalent binding in the skin of rats. There was a large artifact band in immunoblots of whole skin homogenates that interfered with detection of covalent binding; however, when the skin was separated into dermal and epidermal fractions, covalent binding was clearly present in the epidermis, which is also the location of sulfotransferases. In contrast to rats, treatment of mice with NVP did not result in covalent binding in the skin or skin rash. Although the reaction of 12-OHNVP sulfate with nucleophiles such as glutathione is slow, incubation of this sulfate with homogenized human and rat skin led to extensive covalent binding. Incubations of 12-OH-NVP with the soluble fraction from a 9,000g centrifugation (S9) of rat or human skin homogenate in the presence of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) produced extensive covalent binding, but no covalent binding was detected with mouse skin S9, which suggests that the reason mice do not develop a rash is that they lack the required sulfotransferase. This is the first study to report covalent binding of NVP to rat and human skin. These data provide strong evidence that covalent binding of NVP in the skin is due to 12-OH-NVP sulfate, which is likely responsible for NVP-induced skin rash. Sulfation may represent a bioactivation pathway for other drugs that cause a skin rash.
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INTRODUCTION The basic mechanisms of idiosyncratic drug reactions (IDRs) are currently not well understood. Circumstantial evidence suggests that most IDRs are caused by the formation of reactive metabolites rather than the parent drug; however, without a valid animal model, this is difficult to rigorously test. One such model that has allowed us to study the mechanism of an idiosyncratic toxicity in detail is the rat model of nevirapine (NVP)-induced skin rash.1 NVP (Viramune, Scheme 1) is a nonnucleoside reverse transcriptase inhibitor indicated for the combination treatment of HIV-1 infections. Although effective, NVP was found to induce a high incidence of skin rash or liver toxicity, and sometimes both occur in the same patient. The incidence of skin rash is approximately 9%, most of which are mild to moderate maculopapular rashes.2 However, 16% of NVP-induced rashes are very severe and life-threatening, including Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN).2 Certain risk factors for the development of the rash have been identified, such as female gender and higher pretreatment CD4+ T-cell counts.3 © 2013 American Chemical Society
Our group has developed and characterized a novel animal model of NVP-induced skin rash in female Brown Norway (BN) rats. This model shares many characteristics of the rash that occurs in humans. For example, in both humans and the rat model, evidence suggests CD4+ T-cells mediate the rash.1 Additionally, there is a delay in onset of the rash upon primary NVP treatment, but a rapid onset with secondary rechallenge in both humans and rats.1 Higher incidence in females, increased incidence of rash with increased dose, and range in severity of rash are all features shared by both the animal model and humans. Furthermore, lymphocytes taken from both patients and animals after NVP-induced skin rash produce interferon-γ.4,5 Using the BN rat model, we were able to show that the 12-hydroxylation pathway is involved in the induction of the skin rash. This is based on the observation that substitution of the NVP methyl hydrogens with deuterium markedly decreased the formation of 12-OH-NVP as well as the incidence and severity of the rash.6 Additionally, treatment with a lower dose Received: December 11, 2012 Published: February 6, 2013 410
dx.doi.org/10.1021/tx3004938 | Chem. Res. Toxicol. 2013, 26, 410−421
Chemical Research in Toxicology
Article
Scheme 1. Proposed Chemical Mechanism of NVP-Induced Skin Rash Resulting from the Covalent Binding of 12-OH-NVP Sulfate in the Skin
trypsin were purchased from Invitrogen (Carlsbad, CA). Amersham ECL Plus Western Blotting Detection System was obtained from GE Healthcare (Oakville, ON). Horseradish peroxidase-conjugated goat antirabbit IgG (H + L chains) and monoclonal GAPDH were purchased from Sigma-Aldrich (St. Louis, Mo). Normal goat serum was obtained from Invitrogen (Grand Island, NY). The synthesis of 12-OH-NVP, 12-OH-NVP sulfate, and preparation of NVP antiserum were described previously.6,9 Protein concentrations were determined using a BCA protein assay kit (Novagen, EMD Biosciences Inc.). Animal Care. Female BN rats (150−175 g; between 8 and 10 weeks of age) were obtained from Charles River (Montreal, QC). Rats were housed in pairs in standard cages in a 12:12 h light/dark cycle with access to water and Agribrands powdered lab chow diet (Leis Pet Distribution, Inc. Wellesley, ON) ad libitum. Following a 1 week acclimatization period, rats were either maintained on control chow or started on drug-containing diet (treatment groups). Drug was mixed thoroughly with powdered lab chow if it was to be administered orally. The amount of drug administered to animals was calculated based on body weight of the rats and their daily food intake. Rats were sacrificed via CO2 asphyxiation. Balb/c or C57BL/6 mice (6−8 weeks age) were obtained from Charles River (Montreal, Quebec). E3 ubiquitin ligase casitas-b-lineagelymphoma (Cbl-b−/−) knockout mice were bred in house from animals first developed by Dr. J. Penninger at the Institute of Molecular Biotechnology of the Austrian Academy of Science, Vienna, with his kind permission. Programmed cell death-1 (PD-1−/−) knockout mice were bred in house from animals first developed by Dr. Tasuku Honjo at the Department of Immunology and Genomic Medicine, Kyoto, Japan, with his kind permission. Mice were kept 4 per cage. NVP was administrated in lab chow following a 1 week acclimatization period. All animal experiments were approved by the University of Toronto Animal Care Committee in accordance with guidelines of the Canadian Council on Animal Care. Primary and Secondary Treatment of Animals with NVP or 12-OH-NVP. Female BN rats were treated orally with NVP (150 mg/ kg/day) or an equimolar dose of 12-OH-NVP (159 mg/kg/day) mixed thoroughly in rat chow for up to 21 days. For rechallenge (secondary exposure), rats treated with NVP having a moderate to severe skin rash were removed from the drug, fed a diet of rat chow for 4 weeks so that the rash resolved, and then NVP was resumed at the same dose until the animals developed a rash and systemic effects such as weight loss, which usually occurred after 7−10 days.1 Separation of Dermis and Epidermis and Preparation of Homogenates of Skin Fractions or of Whole Rat Skin. At sacrifice, hair was removed from the rats using an electric shaver, and the skin was cleaned of remaining hair using PBS (1× phosphate buffered saline, pH 7.4) and Kimwipes. Skin from the back was then
of 12-OH-NVP induced the same degree of skin rash as the treatment with NVP itself.6 Although we know that oxidation of NVP to 12-OH-NVP is required to induce the rash, it is not clear how it does so. 12-OH-NVP is not chemically reactive; therefore, if the rash is caused by a reactive metabolite, 12-OHNVP would require further bioactivation. The oxidation state of 12-OH-NVP and the quinone methide, which is the major species involved in covalent binding in the liver, is the same; therefore, the quinone methide cannot be formed by oxidation of 12-OH-NVP. 12-OH-NVP is oxidized to the corresponding carboxylic acid, which forms a glucuronide, but inhibition of this oxidation does not decrease the incidence of the rash.6 The most likely candidate is the benzylic sulfate conjugate. Sulfate is a good leaving group, and numerous sulfate metabolites are known to be reactive metabolites.7 We detected the 12-OHNVP sulfate in the blood of BN rats treated with NVP, which was presumably formed in the liver, and there are also sulfotransferases in the skin.8 In the case of the 12-OH-NVP sulfate, not only is the sulfate on a benzylic position, there is also an adjacent amide hydrogen that could be lost to form the same quinone methide as formed by direct oxidation of the methyl group without the formation of a carbocation intermediate. However, the 12-OH-NVP sulfate was synthesized and found to be less reactive than expected; specifically, it reacted only very slowly with glutathione (the reaction occurred over a period of days; unpublished results). In addition, initial attempts to detect the covalent binding of NVP in the skin of treated animals were unsuccessful. The present study was an extension of the previous studies to test the hypothesis that 12-OH-NVP sulfate is a plausible candidate for causing NVP-induced skin rashes.
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MATERIALS AND METHODS
Chemicals. NVP was kindly supplied by Boehringer-Ingelheim Pharmaceuticals Inc. (Ridgefield, CT). The majority of chemical reagents, 3′-phosphoadenosine 5′-phosphosulfate (PAPS), Tris, methanol, DMSO, PBS (pH 7.4), glycerol, silica gel, amido and black stain, were obtained from Sigma-Aldrich (Oakville, ON) unless otherwise noted. Ammonium persulfate was obtained from Fisher Scientific (Fair Lawn, NJ). SDS and Tween-20 were obtained from BioShop (Burlington, ON). Stock acrylamide/bis solution (29:1), nonfat blotting grade milk powder, and nitrocellulose membranes (pore size 0.2 μM) were purchased from Bio-Rad (Hercules, CA). Ultrapure tetramethylethylenediamine and 2.5% 411
dx.doi.org/10.1021/tx3004938 | Chem. Res. Toxicol. 2013, 26, 410−421
Chemical Research in Toxicology
Article
37 °C without the addition of any drug. All samples were stored at −80 °C until use for immunoblotting experiments. In Vitro Metabolism of 12-OH-NVP and NVP. For testing sulfation, NVP or 12-OH-NVP (50 mM stock solution in methanol) was added to Dulbecco’s PBS with MgCl2 and CaCl2 (Invitrogen, Carlsbad, CA) to a final concentration of 1 mM. Total incubation volume was 400 μL. The methanol was partially removed by nitrogen evaporation in order to limit the amount in the incubation to
