12-OH-Nevirapine Sulfate, Formed in the Skin, Is Responsible for

Apr 16, 2013 - We have shown that the sulfate of a major oxidative metabolite,. 12-OH-NVP .... persulfate was obtained from Fisher Scientific (Fair La...
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12-OH-Nevirapine Sulfate, Formed in the Skin, Is Responsible for Nevirapine-Induced Skin Rash Amy M. Sharma,† Maria Novalen,† Tadatoshi Tanino,‡ and Jack P. Uetrecht*,† †

Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 3M2, Canada Faculty of Pharmacy, Kinki University, 3-4-1 Kowakae, Higashiosaka City, Osaka 577-8502, Japan

‡

S Supporting Information *

ABSTRACT: Nevirapine (NVP) treatment is associated with a significant incidence of skin rash in humans, and it also causes a similar immune-mediated skin rash in Brown Norway (BN) rats. We have shown that the sulfate of a major oxidative metabolite, 12-OH-NVP, covalently binds in the skin. The fact that the sulfate metabolite is responsible for covalent binding in the skin does not prove that it is responsible for the rash. We used various inhibitors of sulfation to test whether this reactive sulfate is responsible for the skin rash. Salicylamide (SA), which depletes 3′-phosphoadenosine-5′-phosphosulfate (PAPS) in the liver, significantly decreased 12-OH-NVP sulfate in the blood, but it did not prevent covalent binding in the skin or the rash. Topical application of 1-phenyl-1-hexanol, a sulfotransferase inhibitor, prevented covalent binding in the skin as well as the rash, but only where it was applied. In vitro incubations of 12-OH-NVP with PAPS and cytosolic fractions from the skin of rats or from human skin also led to covalent binding that was inhibited by 1-phenyl-1-hexanol. Incubation of 12-OH-NVP with PAPS and sulfotransferase 1A1*1, a human isoform that is present in the skin, also led to covalent binding, and this binding was also inhibited by 1-phenyl-1-hexanol. We conclude that salicylamide did not deplete PAPS in the skin and was unable to prevent covalent binding or the rash, while topical 1-phenyl-1-hexanol inhibited sulfation of 12-OH-NVP in the skin and did prevent covalent binding and the rash. These results provide definitive evidence that 12-OH-NVP sulfate formed in skin is responsible for NVP-induced skin rashes. Sulfotransferase is one of the few metabolic enzymes with significant activity in the skin, and it may be responsible for the bioactivation of other drugs that cause skin rashes.

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P450 are very low.4 At one point early in the NVP studies, we thought that the p-i hypothesis might be relevant for NVPinduced skin rash because we had eliminated several possible reactive metabolites; however, we used the NVP animal model to show that the basis for the p-i hypothesis is false, and certainly, it is not the mechanism of NVP-induced skin rash.5 That does not mean that the hypothesis itself is false; the p-i hypothesis probably is relevant for some compounds, especially small peptidomimetic drugs such as ximelagatran.6 We have developed an animal model of nevirapine (NVP, Viramune; see the graphical abstract)-induced skin rash, which is clearly immunemediated and has characteristics very similar to those of the rash that occurs in humans.7 These characteristics include a similar

INTRODUCTION Idiosyncratic drug reactions (IDRs) are unpredictable adverse events that significantly impact drug development and use. Many drugs that cause IDRs form reactive metabolites, and it is usually assumed that these reactive metabolites are responsible for the IDR associated with the drug involved.1,2 However, IDRs are difficult to study, and it has not been possible to definitively demonstrate that reactive metabolites are causal. In addition, many drugs form several reactive metabolites, and it is very difficult to test which, if any, is responsible for a specific IDR. Furthermore, Pichler has proposed the p-i hypothesis in which a reversible interaction between the T cell receptor and the major histocompatibility complex is sufficient to trigger an IDR without the formation of a reactive metabolite.3 This is especially attractive for skin rashes because the skin has very limited drug metabolism capacity; specifically, the levels of cytochromes © 2013 American Chemical Society

Received: March 8, 2013 Published: April 16, 2013 817

dx.doi.org/10.1021/tx400098z | Chem. Res. Toxicol. 2013, 26, 817−827

Chemical Research in Toxicology

Article

PCCA (London, ON). The syntheses of 12-OH-NVP, 12-OH-NVP sulfate, 4-carboxy-NVP (4-COOH-NVP), and the NVP antiserum were described previously.9,10 Human liver cytosol (pool of 10, mixed gender) or a 9,000 X supernatant (S9) fraction containing cytosol and microsomes (pool of 50; mixed gender); rat liver cytosol (pool of 115; female Sprague−Dawley rats) or rat skin cytosol (pool of 50; female); and recombinant human sulfotransferase (SULT) 1A1*1 expressed in Escherichia coli (E. coli) were purchased from XenoTech LLC (Lexena, KS). Animal Care. Female BN rats (150−175 g; 8 to 10 weeks of age) were age-matched and 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 a drug-containing diet (treatment groups). For chronic experiments, the drug was mixed thoroughly with powdered lab chow to produce a NVP dose of 150 mg/kg/day or an equimolar dose of 12-OH-NVP (159 mg/kg/day) for a maximum of 21 days. The amount of drug administered to rats was calculated based on their body weight and daily food intake. For experiments examining blood metabolite levels, drugs were ground to obtain fine particles, and NVP was gavaged at a dose of 100 mg/kg/day in 0.5% methyl cellulose. The dose was scaled up from 50 mg/kg/day over a period of 3−5 days to avoid central nervous system toxicity associated with high peak plasma levels of NVP. Rats were sacrificed via CO2 asphyxiation. Animal experiments were approved by the University of Toronto Animal Care Committee in accordance with Guidelines of the Canadian Council on Animal Care. Quantification of NVP, 12-OH-NVP, 12-OH-NVP Sulfate, and 4-COOH-NVP in Plasma. Plasma (50 μL) was mixed with internal standard (ethyl-NVP, 5.4 μg/mL, 50 μL) and concentrated with a Strata solid phase extraction column (C18-E, 100 mg, Phenomenex, Torrance, CA). The column was washed with 1 mL of water and the metabolites eluted with 1 mL of methanol. The methanol was collected, dried, and reconstituted with 50 μL of the HPLC mobile phase. The samples were separated on HPLC and analyzed by mass spectrometry. The separation was carried out on an Ultracarb C18 30 × 2.0 mm, 5 μm column (Phenomenex) under isocratic conditions with a mobile phase consisting of 16% acetonitrile and 84% water with 2 mM ammonium acetate and 1% acetic acid and a flow rate of 0.2 mL/min. Mass spectrometry was carried out using a PE Sciex API 3000 quadrupole system with an electrospray ionizing source. The ion pairs used for quantitation in the multiple reaction monitoring/positive ion mode were 267.0/226.1 for NVP, 283.1/223.1 for 12-OH-NVP, 297.1/210.1 for 4-COOH-NVP, 283.1/161.0 for 2-OH-NVP, 283.1/214.0 for 3-OH-NVP, and 255.1/227.2 for ethyl-NVP. Standard curves prepared for 2-OH-NVP (0.43−102.9 μg/mL), 3-OH-NVP (0.36−86.8 μg/mL), 12-OH-NVP (0.38−91.0 μg/mL), 4-COOH-NVP (0.26−61.8 μg/mL), and NVP (0.74−176.9 μg/mL) had R2 values of >0.99. Quantification of 12-OH-NVP sulfate was done in a similar manner, the major difference being that it was performed in the negative ion mode. Because of this, the internal standard was changed to naproxen (2.5 μg/mL, 50 μL added to the plasma). The ion pairs used for the analysis were 361.0/96.0 for 12-OH-NVP sulfate and 229.0/169.8 for naproxen. The HPLC column used for the separation was an Ultracarb C18 column (100 × 2 mm, 5 μm, Phenomenex) with a gradient elution of 20 → 80% acetonitrile over a period of 10 min. The second solvent was water with 2 mM ammonium acetate and 1% acetic acid. The flow rate was 0.2 mL/min. Standard curves prepared for 12-OH-NVP sulfate (0.28−14.0 μg/mL) had R2 values of >0.99.

time to onset, higher incidence in females, and the observation that a low CD4+ T cell count decreases rash incidence. We used this model to test the involvement of a reactive metabolite in the mechanism of NVP-induced skin rash. NVP, a non-nucleoside reverse transcriptase inhibitor indicated for the treatment of HIV-1 infections, causes idiosyncratic hepatotoxicity and mild-to-severe skin rashes.8 We have demonstrated that NVP is oxidized to a reactive quinone methide, which covalently binds in the liver.9 However, we have not been able to demonstrate that this reactive metabolite is responsible for the idiosyncratic liver injury caused by NVP because we were not able to produce liver injury in animals with characteristics similar to those of the liver injury that occurs in humans. In contrast, we identified that the 12-hydroxylation pathway to form 12-OH-NVP is responsible for the induction of the skin rash because substitution of the methyl hydrogens of NVP with deuterium significantly decreased the incidence and severity of the rash, and treatment with 12-OH-NVP also caused a skin rash.10 Because 12-OH-NVP is the same oxidation state as the quinone methide species responsible for covalent binding in the liver, a quinone methide species could not be produced by oxidation of 12-OH-NVP. In a recent paper, we demonstrated that covalent binding of NVP in the skin is mediated by a benzylic sulfate formed by first oxidation of NVP in the liver to 12-OH-NVP followed by the formation of a benzylic sulfate (12-OH-NVP sulfate), which has sufficient chemical reactivity to covalently bind to proteins.11 Both the liver and skin contain sulfotransferases, and although chemically reactive, we were able to detect 12-OH-NVP sulfate in the blood of rats treated with NVP. It remained to be determined if 12-OH-NVP sulfate is responsible for NVP-induced skin rash, and if so, whether it is sulfation in the liver or skin that is most important. We used our animal model and various inhibitors of sulfation to answer this question.

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MATERIALS AND METHODS

Chemical Materials and Reagents. NVP and ethyl-NVP (a NVP analogue where the cyclopropyl group has been replaced by an ethyl group) were kindly supplied by Boehringer-Ingelheim Pharmaceuticals Inc. (Ridgefield, CT). Common chemical reagents (3′-phosphoadenosine 5′-phosphosulfate (PAPS), β-glucuronidase type IX-A, Tris, methanol, DMSO, PBS (phosphate buffered saline, 1.47 mM KH2PO4, and 8.06 mM Na2HPO4-7H2O, pH 7.4), glycerol, silica gel, etc.) were obtained from Sigma-Aldrich (Oakville, ON) unless otherwise noted in the Materials and Methods section. Ammonium persulfate was obtained from Fisher Scientific (Fair Lawn, NJ). SDS and Tween-20 were obtained from BioShop (Burlington, ON). Stock 30% acrylamide/bisacrylamide solution (29:1), nonfat blotting grade milk powder, and nitrocellulose membrane (0.2 μM) were purchased from Bio-Rad (Hercules, CA). Ultra pure tetramethylethylenediamine, frozen 2.5% trypsin, and normal goat serum 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) was purchased from Sigma-Aldrich (St. Louis, Mo). 1-Phenyl-1-hexanol was obtained from Tokyo Chemical Industry (Toshima, Japan), and micronized dehydroepiandrosterone (DHEA) was obtained from

Table 1. Inhibitors of Sulfation and Dosing Method inhibitor name

dose

vehicle

application method

salicylamide DHEA 1-phenyl-1-hexanol

274 mg/kg/day 20 mg/kg/day topical; 50 or 100 mg/kg/day oral 20 mg/kg/day topical, or 20 mg/kg/day oral

0.5% methyl cellulose 50:50 oil:acetone (topical); 0.5% methyl cellulose (oral) 50:50 oil/acetone (topical); 0.5% methyl cellulose (oral)

oral; 1×/day after 5 p.m. topical or oral gavage; 1×/day topical or oral gavage; 1×/day

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Figure 1. (A) Incidence of skin rash and (B) plasma concentrations of NVP, (C) 12-OH-NVP, and (D) 12-OH-NVP sulfate in female Brown Norway rats treated with NVP only (100 mg/kg/day, n = 4), in combination with oral DHEA (50 and 100 mg/kg/day), or in combination with oral salicylamide (274 mg/kg/day). Sulfation Inhibition Studies. The effects of 3 sulfation inhibitors on covalent binding and the resulting skin rash were studied (Table 1); see Results for further information on specific experiments. For topical inhibition studies, the desired area of skin was shaved using an electric shaver, and administration of the inhibitors was started 3 days after shaving to allow the skin barrier to heal from any nicks that might have occurred. The skin was painted with either DHEA or 1-phenyl-1-hexanol using a 200 μL pipet. For oral administration, inhibitors were given as a cotreatment with NVP. Controls were either fed standard lab chow or administered vehicle by gavage if they were controls for the salicylamide cotreatment groups. For systemic inhibition studies, female BN rats were treated with NVP together with salicylamide, DHEA, or 1-phenyl-1-hexanol. All drugs were ground, suspended in 0.5% methylcellulose, and administered by gavage with minimal time between NVP and the inhibitor. If NVP was to be given by gavage, the dose was escalated during the first 4 to 5 days of the study from 50 mg/kg/day to the full dose of 100 mg/kg/day (see Results for specific details). The lower dose was given in the beginning of the study to avoid the central nervous system toxicity associated with high peak plasma levels of NVP.

12-OH-NVP was coadministered at a dose of 100 mg/kg/day by oral gavage with DHEA at doses of 25, 50, or 100 mg/kg/day by oral gavage. 1-Phenyl-1-hexanol was administered orally by gavage, while NVP was fed in food at 150 mg/kg/day. All inhibition studies were carried out for a maximum of 28 days. Separation of Skin Dermis and Epidermis and Preparation of Homogenates. At sacrifice, hair was removed from the rats using an electric shaver, and the skin was cleaned of remaining hair using PBS and Kimwipes. Skin from the back was then excised and placed on dry ice over aluminum foil. Care was taken to remove all hypodermis and connective tissue using blunt-tip forceps. Sections (∼200 mg) of whole skin were stretched using sharp-tipped forceps on clean Petri dishes for a maximum of 2 h at 4 °C. Once stretched, skins were floated individually overnight at 4 °C in a solution of 0.25% trypsin (approximately 50 mL per skin section). On day 2, the epidermis was lifted off of the dermis, the dermis was wiped clean of any residual epidermis using Kimwipes, and each fraction was homogenized separately in cell lysis buffer (Cell Signaling Technologies, Pickering, ON) with protease inhibitor (HALT Protease Inhibitor Cocktail, Pierce, Rockford, IL) in a 10:1 ratio using a Polytron 2100 homogenizer (∼1.5 mL working cell lysis 819

dx.doi.org/10.1021/tx400098z | Chem. Res. Toxicol. 2013, 26, 817−827

Chemical Research in Toxicology

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Figure 2. (A) Immunoblot of the epidermis comparing individual 12-OH-NVP-treated rats to NVP + oral salicylamide cotreated rats (N+Sal) or NVP only treated rats, against 0.5% methyl cellulose gavaged controls. Protein loading was 15 μg/lane. (B) Skin histology of NVP + oral salicylamide cotreated rats, n = 4. (C) Skin histology compared between various treatment groups: normal and gavaged controls are normal without a cellular infiltrate in the dermis, while NVP, 12-OH-NVP, and NVP + oral salicylamide treated rats display keratinocyte necrosis within the epidermis, with marked inflammatory infiltrate at the dermal−epidermal junction. A representative photo from one of four animals per group is shown. All rats represented in this figure were treated for 21 days. Magnification is 20× for all slides in this figure. Preparation of Human Skin Dermatome. Human skin (9 g) was obtained from XenoTech LLC (Lenexa, KS, USA) from the abdomen of a 56 year old Caucasian male 9.1 h after death due to heart disease without any skin or infectious diseases. Skin was prepared to contain the epidermal layers only. Dermatomed skin was then prepared by chopping into fine pieces and immersing in cell lysis buffer with protease inhibitor for ∼15 min on ice, followed by homogenization via a Polytron 2100 homogenizer. Skin was clarified of debris via centrifugation as described above. The supernatant was separated and stored at −80 °C.

buffer per fraction). In order to clarify samples, dermal and epidermal fractions were centrifuged at 13,000 rpm each for 2 min. The supernatant was separated and stored at −80 °C. The protein concentration of the prepared homogenates was quantified using a BCA protein assay kit (Novagen, EMD Biosciences Inc., Mississauga, ON); bovine serum albumin was used as the standard. Whole rat skin tissue was prepared using the same method except that dermal and epidermal layers were not separated, and skin was not floated overnight in trypsin. 820

dx.doi.org/10.1021/tx400098z | Chem. Res. Toxicol. 2013, 26, 817−827

Chemical Research in Toxicology

Article

Figure 4. Using skin isolated from rats cotreated with NVP and topical 1-phenyl-1-hexanol using the schematic shown in Figure 3B, we performed epidermal immunoblot analysis. (A) Immunoblot of epidermis from rash areas versus vehicle areas from the epidermis of inhibitor-treated rats cotreated with NVP. (B) Immunoblot of epidermis from topical 1-phenyl-1-hexanol areas versus vehicle areas from the epidermis of inhibitor-treated rats compared with that of an untreated control and a NVP-treated control. Fifteen micrograms of protein per lane was loaded in each of the lanes in A and B. protein, skin (final concentration 1 mg/mL), Dulbecco’s PBS with MgCl2 and CaCl2, and drug. Following the 5 min of preincubation, PAPS was added to a final concentration of 0.3 mM, and the samples were vortexed. The final concentration of all drugs tested was 1 mM. Samples were taken at time 0 (before the start of the incubation), 30, and 60 min, and reactions were terminated by placing the samples on dry ice. Negative controls were the skin fractions incubated at 37 °C without the addition of any drug or PAPS (see Results for specific details). All samples were stored at −80 °C until use for immunoblotting experiments. Incubation of Rat Liver or Skin Cytosol or Human Liver Cytosol with 12-OH-NVP and 1-Phenyl-1-hexanol in the Presence and Absence of PAPS. To an Eppendorf tube containing Dulbecco’s PBS with MgCl2 and CaCl2 was added 12-OH-NVP dissolved in methanol (stock 50 mM) and 1-phenyl-1-hexanol to a final concentration of 1 mM each. Methanol was partially removed by nitrogen evaporation in order to limit the amount of solvent in the incubation to