Pichler WJ (ed): Drug Hypersensitivity. Basel, Karger, 2007, pp 115 128 Nevirapine Hypersensitivity a, b a, c a J.M. Shenton M. Popovic J.P. Uetrecht a Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ont., Canada; b Department of Immunotoxicology, Bristol-Myers Squibb Co., Syracuse, N.Y., USA; c Department of Investigative Toxicology, Novartis Pharma AG, Basel, Switzerland A b s t r a c t Nevirapine (Viramune TM ) can cause severe and life-threatening idiosyncratic skin rash. No clear mechanistic understanding exists; thus, it is impossible to predict which patients will suffer nevirapine-induced rash or to design a safer nevirapine analogue. Animal models of idiosyncratic drug reactions are rare, but animal models are arguably the key to mechanistic understanding. Recently, an animal model of nevirapine-induced rash was discovered and characterized; nevirapine causes rash in female Brown Norway rats with characteristics akin to nevirapine-induced rash in humans. The animal model has permitted considerable gains in the mechanistic understanding of nevirapineinduced rash, although it has yet to be determined if the animal model findings reflect the pathogenesis of nevirapine-induced rash in humans. Investigations with the animal model confirmed the essential role of the immune system. Ongoing research should determine the relative importance of parent drug versus reactive metabolite as the root cause; this is a fundamental and unanswered question in the field of idiosyncratic drug reactions. Importantly, the animal model of nevirapine-induced rash does not provide a predictive test of the ability of other drugs to cause idiosyncratic reactions, but may provide a sufficient mechanistic understanding of nevirapine-induced rash to allow the prevention of the disease in patients. Introduction Copyright 2007 S. Karger AG, Basel In 1996, Boehringer-Ingelheim received Food and Drug Administration (FDA) approval for nevirapine (Viramune TM ) indicated in combination with nucleoside analogues for the treatment of human immunodeficiency virus (HIV)-1 infections. Nevirapine was the first in its class, the non-nucleoside reverse transcriptase inhibitor (NNRTI), to reach the market. In clinical trials, some patients developed idiosyncratic skin rash and/or liver toxicity. Rashes occurred in 16% of patients and varied from mild and maculopapular to severe and life-threatening prompting permanent discontinuation of nevirapine treatment. The severe and life-threatening rashes, Stevens-Johnson syndrome (SJS) and SJS-toxic epidermal necrolysis (TEN)-transition syndrome were responsible for 0.3% of rashes in early clinical trials [1] and cases of TEN have since been reported [2]. More recent Boehringer-Ingelheim compiled data indicate that nevirapine-induced rash occurs in 8.9% of patients with 16% of these rashes being severe or life-threatening [3]. Liver toxicity is reported both in combination with and in the absence of rash. The reported incidence of liver toxicity is much lower than for rash; Boehringer-Ingelheim reports the incidence to be 2.8% [3]. Thus, many patients are affected by nevirapine-induced rash in the absence of liver toxicity. Several risk factors for developing nevirapine-induced rash and/or hepatotoxicity have been identified (table 1 ). These reactions can have dire consequences for those affected. Severe rashes such as TEN are
Table 1. Risk factors identified for the development of nevirapine-induced rash and/or liver toxicity Risk factor Details Ref. Sex Antiallergic drugs Third trimester in pregnant women Previous nevirapineinduced rash Nevirapine plasma concentrations CD4 count HLA genes Liver enzymes Severe rash and/or liver toxicity is/are more common in females with body mass index <18.5 Increased incidence of rash with prophylactic use of glucocorticoids or antihistamines Indications that nevirapine-induced hepatotoxicity may have a higher incidence in women who are given the drug late in pregnancy Earlier onset and an increased risk of severe rash on rechallenge with nevirapine if rash occurred on first exposure 23, 34 Findings with regard to the role of nevirapine plasma concentrations in patients that develop nevirapine reactions are contradictory: Higher nevirapine plasma levels predict rash and/or liver toxicity 27, 28 Nevirapine plasma levels are not predictors of rash and/or liver toxicity 29 Lower pretreatment CD4 counts in patients are protective: initiation of treatment is contraindicated in females with CD4 counts >250 cells/mm 3 and males with CD4 counts >400 cells/mm 3 Combinations of hepatitis, fever, or rash are associated with a HLA-DRB1*0101 haplotype; no associations were detected for isolated rash Increased liver enzymes may warrant permanent discontinuation of nevirapine and should prompt investigation into alternative treatment options depending on the degree of increase 24 25 26 3 30 31 not understood and supportive measures are usually the only available treatment. Patients can die or are left with permanent sequelae such as visual impairment or blindness [4]. Nevirapineinduced liver toxicity has also caused deaths or required life-changing interventions including liver transplantation [5]. Rash and liver toxicity also occur in HIV-negative patients, and nevirapine for post-exposure prophylaxis is discouraged [6]. Idiosyncratic reactions restrict the prescription of efficacious drugs to patients by clinicians, villainize the government regulators responsible for the drug approval, and frustrate the pharmaceutical manufacturers who have invested hundreds of millions of dollars in drug development. Despite significant research efforts, the mechanism(s) of idiosyncratic drug reactions remain(s) a black box. The ultimate goal is to be able to treat, predict, and/or prevent these reactions and other idiosyncratic reactions. To reach this goal, an understanding of the mechanisms of idiosyncratic drug reactions is imperative. Reactions such as nevirapine-induced rash have characteristics incriminating the immune system as a key mechanistic player. Thus, understanding the mechanism of nevirapine-induced rash will not only help in the handling of nevirapine-induced rashes, but may also significantly contribute to an understanding of how the immune system recognizes drugs [discussed in the chapter by Park et al., pp 55 65]. Elucidation of the mecha- 116 Shenton Popovic Uetrecht
Fig. 1. Basic characteristics of the nevirapine animal model. Female Brown Norway rats administered nevirapine (150 mg/kg/day) in the diet develop red ears and skin lesions between days 7 10 and 10 21, respectively. When nevirapine treatment is stopped the rats recuperate and the skin lesions resolve. If the animals are then rechallenged with nevirapine, red ears develop within approximately 8 h and skin lesions (albeit fewer than on first exposure) within 4 9 days. The lower number of skin lesions is misleading because the animals actually present with more severe clinical signs than on first exposure such as weight loss, hunched posture, and chromorhinorrhea. nism of nevirapine-induced rash may also help in the comprehension of other idiosyncratic drug reactions. Important contributions to the understanding of mechanisms are facilitated via animal models. Nevirapine-induced rash is one of the few reactions for which a reasonable animal model is available [7]. This chapter describes the discovery and characterization of the nevirapine animal model, the most important contributions the model has made toward understanding the mechanism of nevirapine-induced rash, and the importance of the model in ongoing mechanistic investigations. Discovery and Characterization of an Animal Model of Nevirapine-Induced Skin Rash The discovery of the animal model of nevirapineinduced rash ultimately stemmed from an observation in the late 1990s by a graduate student, George Lai, in the laboratory of Jack Uetrecht at the University of Toronto. George was administering nevirapine (150 mg/kg/day) to female Sprague-Dawley rats in order to investigate in vivo covalent binding of nevirapine to liver proteins. During this investigation he noted that 2 of 4 rats developed erythema, exhibited excessive scratching around the nose/mouth area, and lost body weight after several weeks of treatment. This study was replicated and expanded to result in a characterization of the response of various sexes and strains of rats (and a mouse strain) to nevirapine [8]. Female Brown Norway rats developed nevirapine-induced rash following a consistent time course ( fig. 1 ) [8] and with a high incidence (100%). These characteristics of the reaction were advantageous in that they facilitated mechanistic studies [7]. Interestingly, none of the rats tested developed liver toxicity; thus, the Brown Norway model represents the portion of patients developing rash in the absence of liver Nevirapine-Induced Skin Rash in Rats 117
Table 2. Similar characteristics of nevirapine-induced rash in humans and female Brown Norway rats Characteristic Humans Rats Rashes Nevirapine plasma levels Time to onset Vary from mild erythematous maculopapular rashes to blistering skin eruptions [32] 1 10 g/ml [3] 20 40 g/ml [7] Highest risk within first 6 weeks of treatment; rash generally develops 1 3 weeks after initiation of nevirapine treatment [1] Vary from mild to severe, no blistering skin reactions observed [8] Skin lesions occur after 2 3 weeks of nevirapine dosing [8] Dose response Incidence increases with dose [1] Incidence increases with dose [8] Female sex Increased susceptibility [23] Increased susceptibility (likely due to metabolic differences) [7] Escalating dose regimen Rechallenge Skin histology T-cell dependence 200 mg/day for 2 weeks prior to starting full dose 400 mg/day decreases incidence of rash by 50% [1] Immediate onset and increased severity in some patients (especially after a severe rash) [26] Little data mild perivascular lymphocytic inflammatory infiltrate [33] Reactions may be dependent on CD4+ T cells rash with other constitutional findings is more commonly seen in HIV-negative and HIV-positive patients with higher CD4+ T-cell counts [6] 40 or 75 mg/kg/day for 2 weeks prior to starting full dose 150 mg/kg/day prevents rash [7] Decreased time to onset and increased severity [8] Predominantly lymphocytic infiltrate including CD4+ T and CD8+ T cells, but also macrophages [8] Reaction is dependent on T cells; CD8+ T cells do not appear to mediate the reaction, evidence points to CD4+ T cells [7] toxicity. Nevirapine-induced rash in rats shares many characteristics with nevirapine-induced rash in humans ( table 2 ), suggesting a common mechanistic pathway. This is the most important attribute of an animal model; indeed, the nevirapine-induced rash animal model meets many of the requirements of a viable animal model of an idiosyncratic drug reaction [for review, see 7 ]. The nevirapine-induced rash model is arguably the most practical animal model of an idiosyncratic drug reaction currently available for mechanistic studies. Contributions of the Animal Model toward an Understanding of the Mechanism of Nevirapine-Induced Rash CD4+ T Cells Play a Key Role in the Development of Nevirapine-Induced Rash The immune system is believed to play an important role in the development of many idiosyncratic drug reactions including rash. Many of the characteristics of nevirapine-induced rash in humans and rats implicate the immune system in the mechanism of this reaction. For instance, T cells and macrophages were present in the skin of rats with rash, and rats developed more severe 118 Shenton Popovic Uetrecht
Fig. 2. Splenocytes, total T cells, or CD4+ T cells, but not CD8+ T cells, adoptively transfer susceptibility to nevirapine-induced rash. Total T, CD4+ T, or CD8+ T cells were purified from splenocytes obtained on day 9 of second exposure and injected intravenously into naive recipients. The naive rats responded to nevirapine similarly to previously exposed (sensitized) rats after receiving total splenocytes, total T, or CD4+ T cells, and like previously naive rats after receiving CD8+ T cells. (systemic) symptoms and with a faster onset on second exposure (rechallenge) to nevirapine ( fig. 1 ). Furthermore, the spleens of the rats rechallenged with nevirapine were significantly larger than those of control animals, and sensitivity to the rash could be adoptively transferred with total splenocytes from these rats to naive rats, that is, the naive rats responded to nevirapine similarly to previously exposed (sensitized) rats ( fig. 2 ) [8]. For further studies it would have been useful to elucidate a biomarker of the reaction. Although serum total immunoglobulin E (IgE) concentration appeared promising because it was significantly increased on day 7 of primary nevirapine exposure, no increase in IgE was detectable on rechallenge [9]. Thus, a subjective endpoint was decided upon for further mechanistic studies. Skin rash was not used as the endpoint, because on rechallenge, the rats developed far fewer skin lesions than on primary exposure despite more severe systemic symptoms and a more intense inflammatory infiltrate in the skin (tail, footpad, ear, and torso (ventral and dorsal) skin were all affected). However, female Brown Norway rats developed red ears prior to the onset of skin lesions on both primary and secondary exposure to nevirapine day 7 of primary exposure and within approximately 8 h after rechallenge ( fig. 1 ). Thus, red ears was chosen as albeit a crude endpoint. To determine which splenocyte population was responsible for transferring susceptibility to nevirapine-induced rash, splenic total, CD4+, or CD8+ T cells were purified from rats with severe nevirapine-induced rash (second exposure) and subsequently adoptively transferred into naive recipients [9]. It was evident that total T cells, or purified CD4+ T cells in some cases, could transfer susceptibility (naive rats developed red ears in approximately 8 h), whereas CD8+ T cells could not ( fig. 2 ). Rats partially depleted of CD4+ T cells, or depleted of CD8+ T cells, via intravenous injection with depleting monoclonal antibodies prior to nevirapine treatment, provided corroborating evidence. The development of nevirapineinduced rash was delayed in rats partially depleted of CD4+ T cells, but depletion of CD8+ T cells appeared to have no effect or actually worsen the reaction [9]. The observations that depletion of CD8+ T cells appeared to worsen the reaction, that partial depletion of CD4+ T cells did not completely prevent nevirapine-induced rash, and that CD4+ T cells could not consistently transfer sensitivity to nevirapine-induced rash to naive recipients led us to hypothesize that regulatory T Nevirapine-Induced Skin Rash in Rats 119
cells (CD4+ and/or CD8+) are also involved in the mechanism of nevirapine-induced rash. To this end, the Uetrecht laboratory has preliminary data demonstrating that CD8+ T cells in rats exposed to nevirapine upregulate FoxP3, a transcription factor expressed by regulatory T cells. An important future experiment would be to illustrate whether depletion of regulatory T cells from a purified population of CD4+ T cells prior to adoptive transfer would generate a consistent response, mimicking prior sensitization to nevirapine, in the naive recipient rats. Importantly, CD4+ T cells have also been implicated in the mechanism of nevirapine-induced skin rash in humans. In fact, Boehringer-Ingelheim has added an additional warning to the product label for nevirapine that states that nevirapine should not be initiated in women with CD4 cell counts 1 250 cells/mm 3 and men with CD4 counts 1 400 cells/mm 3 unless the benefit outweighs the risk [3]. Nevirapine-specific T cells have also been identified ex vivo in an HIV-positive patient with nevirapine-induced hepatitis [10]. The evidence that T cells are involved in human nevirapine reactions lends further credence to the validity of the animal model and the animal model allows one to delve more deeply into the role CD4+ T cells play in the reaction than would be possible in human studies. Inflammatory Infiltrates and Changes in the Expression of Cell Surface Molecules in the Skin over the Course of Nevirapine-Induced Rash Microscopic and immunohistochemical evaluation of skin from rats with rash on first exposure (measured after 21 days) and second exposure (measured after 9 days) illustrated significant inflammatory cell infiltrates in the skin. On second exposure the infiltrate was denser than on primary exposure [8]. T cells and macrophages were the predominant cell types, and cells were observed in the dermis, epidermis, and at the dermal-epidermal junction. Similar expression patterns of T-cell receptor, CD4, and CD8 were observed, and thus it is believed that the inflammatory infiltrate contained both CD4+ and CD8+ T cells. Lesions of satellite-cell necrosis, i.e., apoptotic cells (likely keratinocytes) adjacent to mononuclear cells, were observed in the epidermis. This lesion may represent immune-mediated keratinocyte cell death [8]. The infiltrates were visible in all the skin samples analyzed, i.e., ear, tail, footpad, thorax (dorsal and ventral) skin; however, ear skin provided the clearest picture. A time course investigation using immunohistochemical evaluation looked at histological changes in ear skin in the days preceding rash [11]. Skin sections were evaluated on days 7, 14, or 21 of first exposure to nevirapine and prior to second exposure or 1 or 9 days after second exposure. On first exposure, CD4+ and CD8+ mononuclear cells were not visible in the skin on day 7 despite the characteristic ear redness that occurs on day 7 of nevirapine exposure. By day 14, skin lesions were erupting and dermal CD4+ and CD8+ cells were visible. On second exposure, a similar picture was observed, that is, CD4+ or CD8+ mononuclear infiltrates did not occur concurrently with red ears (onset occurs within 8 h of rechallenge), but were present by day 9 as reported previously [8]. However, macrophages were present in the dermis at the onset of the ear redness illustrating that macrophages enter the skin before T cells and suggesting an important role for macrophages in the early stages of the immune response; indeed, the dermal macrophage infiltrate grew denser over time. Macrophages were mostly observed throughout the dermis, around the hair follicles, and surrounding sebaceous and apocrine glands. Some macrophages were also detected in the skin muscle layer. Questions yet to be answered include what causes the macrophage infiltration into the skin and if and how the infiltrating macrophages contribute to the pathology. Other early changes, observed in the skin beginning early after the initiation of nevirapine 120 Shenton Popovic Uetrecht
Fig. 3. Potential pathway leading to nevirapine-induced rash. (1) Nevirapine is metabolized in the liver by cytochrome P 450 enzymes to several hydroxylated me tabolites including 12-hydroxynevirapine (2). (3) 12-Hydroxynevirapine would likely circulate through the blood to the skin. In the skin, 12-hydroxynevirapine may be sulfated (4) in skin-resident cells such as fibroblasts [21]. The sulfate is a good leaving group and may dissociate from the molecule leaving a reactive quinone methide (5). The reactive quinone methide may covalently bind to nearby cellular proteins (6). The covalent binding may disrupt cellular homeostasis and result in the production of danger signals [22], such as release of cytokines, into the surrounding milieu. This may result in the recruitment of macrophages and the upregulation of MHC-I, MHC-II, and ICAM-1 on local cells such as Langerhans cells (skin-resident antigen-presenting cells) resulting in activation and mobilization of Langerhans cells (7). Nevirapine-specific CD4+ T cells may be activated in the draining lymph nodes by Langerhans cells presenting quinone methide-modified peptides and then home to the skin (8). Adhesion molecules such as ICAM-1 facilitate infiltration of T cells into the skin and antigenpresenting cell-t cell interactions (9). Nevirapine-specific T cells may result in direct attack on quinone-methidemodified skin cells and/or initiate downstream events that result in destruction of skin cells with the final result being an outbreak of nevirapine-induced rash (10). Nevirapine-Induced Skin Rash in Rats 121