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Chem. Res. Toxicol. 2003, 16, 1078-1089
Characterization of a Potential Animal Model of an Idiosyncratic Drug Reaction: Nevirapine-Induced Skin Rash in the Rat Jacintha M. Shenton,† Munehiro Teranishi,†,‡ Mones S. Abu-Asab,§ Julie A. Yager,| and Jack P. Uetrecht*,†,⊥ Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada, Ultrastructural Pathology, National Institutes of Health, Bethesda, Maryland, United States, Department of Pathobiology, Ontario Veterinary College, Guelph University, Guelph, Ontario, Canada, and Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada Received March 31, 2003
Idiosyncratic drug reactions are difficult to study in humans due to their unpredictability. Unfortunately, this characteristic also hinders the development of animal models needed for mechanistic studies. Nevirapine, used to treat human immunodeficiency virus (HIV) infections, results in a severe idiosyncratic skin rash in some patients. We found that nevirapine can also cause a significant rash in some strains of rats. At a dose of 150 mg/kg/day, the incidence in female Sprague-Dawley rats was 6/28 (21%), in female Brown Norway rats 32/32 (100%), and in female Lewis rats 0/6 (0%) while no male Sprague-Dawley or Brown Norway rats developed a rash. Female SJL mice 0/7 also did not develop nevirapine-induced skin lesions. The first sign of a reaction in Brown Norway rats was red ears at days 7-10 followed by a rash with scabbing mainly on the back; this was a shorter time to onset than in SpragueDawley rats. Light microscopy of the skin revealed a primarily mononuclear inflammatory infiltrate and lesions typical of self-trauma. Immunohistochemistry results suggest that the infiltrate was composed of CD4 and CD8 T cells as well as macrophages. A lower dose of either 40 or 75 mg/kg/day did not lead to a rash and, in fact, 2 weeks of the lower doses induced tolerance to the 150 mg/kg/day dose in female Brown Norway rats. A dose of 100 mg/kg/day resulted in rash in 2/4 (50%) of female Brown Norway rats. Rechallenge of Brown Norway rats that had been allowed to recuperate after a nevirapine-induced rash led to red ears in less than 24 h followed by hair loss and occasional skin lesions. Although the skin rash was less evident on rechallenge, microscopically, the cellular infiltrate was more prominent, especially surrounding the hair follicles. Moreover, there were lesions of interface dermatitis with apoptosis and satellitosis, indicative of a cell-mediated immune attack on the epidermis. While systemic signs of illness did not accompany the rash on primary exposure, on rechallenge, the animals appeared generally unwell and this forced sacrifice after 2 weeks or less of treatment. Importantly, splenocytes isolated from rechallenged animals were able to transfer susceptibility to nevirapine-induced skin rash to naı¨ve female Brown Norway recipients, which was illustrated by a faster time to onset of rash in the recipients. The characteristics of this adverse reaction are similar to that seen in humans; that is, it is idiosyncratic in that it only occurs in some strains of animals, is delayed in onset, is more common in females, is dosedependent, and appears to be immune-mediated. Therefore, it may represent a good animal model for the study of idiosyncratic drug reactions.
Introduction Idiosyncratic drug reactions are responsible for significant morbidity and mortality as well as significantly increasing the uncertainty of drug development (1). The term idiosyncratic drug reaction will be used here to designate adverse drug reactions that do not occur in most patients at any dose and do not involve the known * To whom correspondence should be addressed. Tel: 416-978-0185. E-mail:
[email protected]. † Faculty of Pharmacy, University of Toronto. ‡ Present address: Sankyo Co., Ltd. Medicinal Safety Research Labs, Fukuroi, Shizuoka, Japan. § Ultrastructural Pathology, National Institutes of Health. | Department of Pathobiology, Ontario Veterinary College, Guelph University. ⊥ Faculty of Medicine, University of Toronto.
pharmacological properties of the drug (2). Classic examples include clozapine-induced agranulocytosis and cotrimoxazole-induced TEN.1 Although the mechanism of most of these reactions is unknown, it is generally believed that they are immunemediated; the aberrant immune response is thought to arise as a result of covalent binding of drugs to proteins in the affected tissues (3). Mechanistic studies of idio1 Abbreviations: TEN, toxic epidermal necrolysis; NNRTI, nonnucleoside reverse transcriptase inhibitor; HIV, human immunodeficiency virus; SJS, Stevens-Johnson syndrome; SD, SpragueDawley; BN, Brown Norway; LEW, Lewis; EM, electron microscopy; CBC, complete blood count; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, γ-glutamyltranspeptidase; LDH, lactic acid dehydrogenase; BUN, blood urea nitrogen; SER, smooth endoplasmic reticulum; ABC, avidin-biotin complex.
10.1021/tx034064+ CCC: $25.00 © 2003 American Chemical Society Published on Web 08/07/2003
Animal Model of Nevirapine-Induced Skin Rash
Figure 1. Nevirapine.
syncratic drug reactions are very difficult. Ideally, prospective human clinical trials would be used to study these reactions; however, their unpredictable nature and low absolute incidence make this virtually impossible. Their complex nature makes it very unlikely that in vitro experiments would represent what happens in vivo. Animal models have the potential to greatly facilitate mechanistic studies; however, although animals have idiosyncratic drug reactions, they are just as unpredictable in animals as they are in people. Therefore, animal models are usually found by accident, and reasonable animal models of idiosyncratic drug reactions are very rare. It is likely that there are significant variations in the mechanisms of different idiosyncratic reactions so it is important to have several animal models. Animal models that have been discovered include a rat model of penicillamine-induced autoimmunity. This is probably the only reasonable model known in which a similar idiosyncratic drug reaction occurs in both rodents and humans (4). Other potential animal models of idiosyncratic drug reactions include halothane-induced hepatitis in the guinea pig (5), sulfadiazine-induced allergy in the dog (6), and propylthiouracil-induced autoimmunity in the cat (7). Although the latter two models have merit, nonrodent models pose several problems. The guinea pig model of halothane hepatitis is an attractive option, but the resulting hepatic damage is transient (8). It is imperative that other animal models of idiosyncratic drug reactions are found so that a spectrum of mechanisms can be studied and the differences as well as the common mechanistic features of idiosyncratic reactions can be determined. Recently, we have discovered another potential animal model of an idiosyncratic drug reaction: nevirapine (Viramune)-induced skin rash in the female rat. Nevirapine (Figure 1), is a NNRTI used in the treatment of HIV infections. It has been published that in controlled clinical trials skin rash occurred in 16% of patients; of these reactions, approximately 33% were severe or lifethreatening and resulted in the majority of permanent discontinuations of nevirapine treatment (9). However, the most recent product insert for nevirapine, reflecting data compiled by Boehringer-Ingelheim from many patients over time, indicates that nevirapine attributable skin rash occurs in 8.6% of patients with 20% of these rashes being severe or life-threatening.2 SJS or SJS/TEN transition syndrome were responsible for 0.3% of the rashes (9). Although TEN was not reported in clinical trials, there have since been case reports of TEN occurring in patients undergoing nevirapine therapy (10). In addition, in clinical trials, nevirapine-induced hepatotoxicity occurred in approximately 1% of HIV patients (9). Furthermore, following the release of nevirapine on 2 Boehringer-Ingelheim Pharmaceuticals, Inc., personal communication.
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the market and its subsequent more widespread use, it became apparent that the incidence of hepatotoxicity is higher than originally thought (11). Presently, Boehringer-Ingelheim reports that the incidence of nevirapineinduced hepatotoxicity is in fact 2.8%.2 Nevirapine has also caused severe liver toxicity and/or skin rash in persons taking nevirapine for postexposure (such as needle stick injury) prophylaxis (12, 13); therefore, these reactions are not limited to persons with an HIV infection. In studies to explore the possibility that nevirapine forms a reactive metabolite, we found that some female SD rats developed a skin rash; however, no signs of hepatotoxicity were observed. The following work was performed to further characterize this rash to determine whether it could serve as a model of the nevirapineinduced rash that occurs in some humans.
Materials and Methods Materials. Nevirapine was kindly supplied by BoehringerIngelheim Pharmaceuticals Inc. (Ridgefield, CT). PBS (10 mM, pH 7.4) dry powder pouches were obtained from Sigma (Oakville, ON) and dissolved in water as per package directions. RPMI 1640 medium and PBS (10 mM, pH 7.4, minus calcium and magnesium) used for adoptive transfer studies were prepared by the University of Toronto Media Services. Hydrogen peroxide was purchased from EM Science (Gibbstown, NJ). Tissue-Tek OCT compound embedding medium for frozen tissue specimens was purchased from Sakura Finetek USA, Inc. (Torrance, CA). Animal Care. Rats (150-175 g) and mice (29-35 days) were obtained from Charles River (Montreal, QC) and housed in pairs in standard cages with free access to water and Agribrands powdered lab chow (Leis Pet Distributing Inc., Wellsley, ON). After a 1 week acclimation period, during which food intake was monitored, the animals were either continued on the powdered lab chow diet (control) or switched to a diet containing nevirapine. Animals were monitored for development of skin rash, food intake, and body weight. At the termination of the experiment or after development of severe skin lesions, rats were killed by ip injection (0.1-0.3 mL/kg) of an anaesthetic mixture consisting of a 5:3 ratio by volume of ketamine (100 mg/ mL) to xylazine (20 mg/mL) and, in some cases, subsequent exsanguination from the abdominal aorta. Mice were killed by CO2 asphyxiation followed by cervical dislocation after blood samples were collected via the saphenous vein. Liver and spleen weights were recorded. Grading of Skin Lesions. The severity of skin lesions was graded as follows: no skin lesions (none), less than five lesions (mild), 5-20 lesions and/or a confluent patch of lesional skin (moderate), or greater than 20 lesions and/or multiple confluent patches of lesional skin (severe). Affected areas were reported as lesions when the diameter was 15 mm. It was necessary to move aside the animal’s fur to monitor lesion development, especially in the early stages of the reaction; thus, it was difficult to quantify the lesions. Strain, Sex, and Dose Dependence of the Rash. Inbred female (n ) 32) and male (n ) 9) BN, inbred female (n ) 6) LEW, and outbred female (n ) 28) and male (n ) 8) SD rats as well as inbred female SJL mice (n ) 7) were administered nevirapine for up to 8 weeks. Nevirapine was mixed with the powdered lab chow such that the animals would receive 150 mg nevirapine/kg/day. To determine the dose-response relationship between nevirapine administration and development of skin lesions, female BN rats were treated with various doses of nevirapine. Nevirapine was mixed with the powdered lab chow such that rats would receive 40 (n ) 4), 75 (n ) 8), 100 (n ) 4), or 150 mg/ kg/day (n ) 32; as outlined above) for up to 8 weeks. Female BN rats were also treated with nevirapine at either 40 (n ) 4)
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Table 1. Primary Antibodies Used in Immunohistochemistry Studies antibodya
dilution
catalog no./supplier
specificity
anti-rat CD2 anti-rat Rβ TCR anti-rat CD8a anti-rat CD8b anti-rat CD4 anti-rat ED1
1:10 000 1:1000 1:1000 1:10 1:1000 1:100
CL034AP/Cedarlane (Hornby, ON) CL057AP/Cedarlane CL004AP/Cedarlane 554971/Pharmingen (San Diego, CA) CL003AP/Cedarlane MCA341R/Serotec (Raleigh, NC)
T cells, NK cells, dendritic cells, subset of macrophages Rβ T cells CD8 T cells, NK cells, activated CD4 T cells CD8 T cells CD4 T cells, macrophages, dendritic cells macrophages
a
All primary antibodies are of the mouse IgG1 isotype with the exception of CL034AP, which is mouse IgG2a.
or 75 mg/kg/day (n ) 4) for 2 weeks and then switched to the regular high dose (150 mg/kg/day) for up to 8 weeks. Histology and EM Studies. At sacrifice, liver and skin samples for histology were obtained from selected animals. Liver and skin samples were fixed and stored in 10% buffered formalin (VWR, Mississauga, ON) until processed. Tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Liver tissue for EM was cut into pieces (∼1 mm3) and fixed in cold 2.5% glutaraldehyde (Sigma) in PBS. Samples were stored at room temperature in fixative and analyzed at the National Institutes of Health (Bethesda, MD). Immunohistochemistry. At sacrifice, skin (∼10 mm × 2 mm) for immunohistochemistry was immersed in a Tissue-Tek cryomold (Miles Inc., Elkhart, IN) containing Tissue Tek OCT compound embedding medium and then snap-frozen in liquid nitrogen. Frozen blocks were then stored at -80 °C until use. For immunohistochemical staining, frozen sections were airdried (10 min), fixed by immersion in cold acetone (10 min), and then rehydrated in PBS. After they were rehydrated, the slides were submerged in 0.3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidases. After a washing step, nonspecific sites were blocked by applying 5% horse serum (Vector, Burlington, ON) to each section for 30 min. This was followed by blocking of endogenous avidin and biotin using the Vector avidin/biotin blocking kit. At this point, the primary antibodies were diluted and applied to the sections (Table 1). Sections were incubated with primary antibodies overnight at 4 °C. After a washing step, biotinylated secondary antibody (horse anti-mouse IgG, rat adsorbed; Vector) was applied to the sections at a dilution of 1:250 in 2% horse serum. Sections were incubated with secondary antibody for 30 min followed by a washing step. Then, an avidin:biotinyated horseradish peroxidase complex (Vectastain Elite ABC kit, Vector) was applied for 30 min. After a final washing step, Vector NovaRED was added as a substrate for the peroxidase following the package instructions. Sections were then counterstained with Mayer’s hematoxylin (Sigma), dehydrated by sequential immersion in increasing concentrations of ethanol, cleared in xylenes, and then mounted using Permount mounting medium (Fisher, Markham, ON). Washes were carried out using three changes of PBS (5 min each). Dilutions were prepared using PBS. All incubations were carried out at room temperature in a moist chamber unless otherwise stated. Blood Chemistry and Hematology. At sacrifice, serum and/or whole blood from selected animals (Table 2) were sent to a commercial lab (Vita-Tech Veterinary Laboratory Services, Markham, ON) for analysis. The tests were carried out as follows: CBC; liver function, including ALT, AST, bilirubin, albumin, globulin, alkaline phosphatase, GGT, total protein, and bile acids; and LDH and BUN. Rechallenge (Secondary Exposure) Studies. Both female BN rats (n ) 9) with moderate to severe skin lesions and female SD rats (n ) 4) with mild skin lesions were removed from drug (placed on regular rodent chow) and allowed to recuperate. After a 3 week recuperation period in which the animals were monitored for weight gain and resolution of rash, the rats were again placed on a diet containing nevirapine at a dose of 150 mg nevirapine/kg/day. Animals were monitored for reoccurrence of skin lesions, food intake, and body weight. Adoptive Transfer Studies. Female BN rats (n ) 3) with rash on day 9 of secondary exposure were used as splenocyte donors. The donor rats were treated with nevirapine for 3 weeks,
Table 2. Number of Animals of Each Sex and Strain Selected for Blood Tests sex and straina female BN male BN female SD male SD female LEW female SJL
treatment
Nb
nevirapine none nevirapine none nevirapine none nevirapine none nevirapine none nevirapine none
6 3 3 3 3 3 2 2 2 1 2 2
a BN, SD, and LEW are all rat strains whereas SJL is a mouse strain. b Female BN treated CBC n ) 5; male BN treated CBC n ) 2; female SJL treated ALT, AST, alkaline phosphatase, total protein (n ) 1), and bile acids only; and female SJL control ALT, AST, albumin, globulin, alkaline phosphatase, total protein, and bile acids only.
followed by a 4 week no treatment period, and finally rechallenge. Recipient rats (n ) 3) were untreated female BN rats. Spleens were removed from the donor rats, and single-cell suspensions were prepared under sterile conditions. Whole spleens were placed in Petri dishes containing RPMI 1640 medium. Spleens were crushed to obtain a single-cell suspension using the butt end of a 10 cm3 syringe. This cell solution was passed through a 70 µm Falcon cell strainer (Becton Dickinson, Franklin Lakes, NJ) and subsequently centrifuged at 340g for 5 min. The cells were washed one time in PBS. Finally, the cells were pooled and resuspended in an appropriate volume of PBS for iv injection (
