Identification of Danger Signals in Nevirapine-Induced Skin Rash

Subscriber access provided by FLORIDA INTL UNIV

Article

Identification of ‘Danger’ Signals in Nevirapine-Induced Skin Rash Xiaochu Zhang, Amy M Sharma, and Jack Uetrecht Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/tx400232s • Publication Date (Web): 15 Aug 2013 Downloaded from http://pubs.acs.org on August 20, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Chemical Research in Toxicology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

Identification of ‘Danger’ Signals in Nevirapine-Induced Skin Rash

Xiaochu Zhang†, Amy M. Sharma†, Jack Uetrecht†*

† Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 3M2

*Correspondence: Jack Uetrecht Leslie Dan Faculty of Pharmacy University of Toronto 144 College St. Toronto ON, M5S 3M2 Canada Phone: 416-978-8939 Fax: 416-978-8511 [email protected]

TITLE RUNNING HEAD: Danger Signals in Nevirapine-Induced Skin Rash

KEYWORDS: adverse drug reactions, immune-mediated, reactive metabolite, skin rash, danger signals, bioactivation, cell stress

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC GRAPHIC

2 ACS Paragon Plus Environment

Page 2 of 24

Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABSTRACT Nevirapine (NVP) can cause serious skin rashes and hepatotoxicity. It also causes an immunemediated skin rash in rats but not hepatotoxicity. This rash is caused by a metabolite of NVP; specifically, NVP is oxidized in the liver to a benzylic alcohol (12-OH-NVP), which travels to the skin where it forms a reactive benzylic sulfate. This could both act as a hapten and induce a danger signal. In contrast, most of the covalent binding in the liver involves oxidation of the methyl group leading to a reactive quinone methide. In this study we examined the effects of NVP and 12-OH-NVP on gene expression in the liver and skin. Both NVP and 12-OH-NVP induced changes in the liver, but the list of genes was different, presumably reflecting different bioactivation pathways. In contrast, many more genes were up-regulated in the skin by 12-OHNVP than by NVP, which is consistent with the fact that 12-OH-NVP is an obligate intermediate in formation of the reactive sulfate in the skin. Genes up-regulated by 12-OH-NVP in the skin included TRIM63 (18 fold increase), S100a7a (7 fold increase), IL22-RA2 (4 fold increase), and DAPK1 (3 fold increase). TRIM63 acts as an ubiquitin ligase, which is consistent with protein damage leading to an increase in protein turnover. In addition, TRIM proteins are involved in inflammasome activation, and it appears that inflammasome activation is an essential step in the induction of NVP-induced skin rash. S100A7 is considered a danger signal and its upregulation supports the danger hypothesis. Upregulation of IL-22 RA2 gene marks an immune response. DAPK1 is involved with inflammasome assembly through binding directly to NLRP3, a NODlike receptor expressed in keratinocytes. These results provide important clues to how NVP causes induction of an immune response, in this case leading to a skin rash.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION Nevirapine (NVP; TOC graphic) is a non-nucleoside reverse transcriptase inhibitor used to treat HIV-1 infections. However, its use is associated with a relatively high incidence of serious idiosyncratic drug reactions, especially skin rashes and hepatotoxicity.1 NVP-induced skin and liver reactions are idiosyncratic drug reactions (IDRs) that do not occur in most of the patients who take the drug. We developed an animal model of NVP-induced skin rash in female Brown Norway rats.2 The rash is clearly immune-mediated in rats3 and has characteristics very similar to the milder form of rash that occurs in humans.4,5 Specifically, in both rats and humans, there is a delay between starting the drug and the onset of rash of a few weeks, a low CD4 T cell count is partially protective, onset is more rapid on rechallenge, and most important, T cells from both humans and rats produced interferon-γ on incubation with NVP.3,4,6 We have shown that covalent binding in the skin is dependent on oxidation of NVP to a benzylic alcohol in the liver followed by sulfation of the alcohol to a reactive benzylic sulfate in the skin, and inhibition of sulfation with a topical sulfotransferase inhibitor prevented covalent binding and the rash where it was applied.7,8 In contrast to the skin, most of the covalent binding in the liver is due to direct oxidation of NVP to a reactive quinone methide, which may be responsible for the hepatotoxicity observed in humans.9 However, because we were not able to reproduce the liver injury in animals9 we were unable to rigorously test this hypothesis. Reactive metabolites can induce an immune response by acting as haptens to modify proteins.10 It has been proposed that “foreign” protein alone is not sufficient to induce an immune response, and reactive metabolites must also cause some cell damage leading to upregulation of costimulatory molecules on antigen presenting cells; without costimulation the result is immune tolerance.11 This is a version of the danger hypothesis.10,11 If this hypothesis is


Page 4 of 24

Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


correct, the ability of a drug to induce danger signals could represent a biomarker of the risk that a drug candidate would be associated with a relatively high incidence of idiosyncratic drug reactions. It would be very difficult to test this hypothesis in humans and there are very few valid animal models of IDRs. The NVP model represents a unique opportunity to test the danger hypothesis in IDRs. Although there are some candidates that have been proposed as danger signals, there are a wide variety of changes that might act as a danger signal;12 therefore, we chose changes in gene expression as determined by a microarray analysis to sample a large number of changes that might represent danger signals.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

MATERIALS AND METHODS Materials. Nevirapine 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). 12-OH-NVP was synthesized as described previously.5 Methylcellulose was purchased from Sigma, (Oakville, ON). Rat gene expression microarrays (230 2.0) were obtained from (Affymetrix, Santa Clara, CA). RNase-free DNase kit, RNAlater RNA stabilization reagent and RNeasy mini kit were purchased from Qiagen (Mississauga, ON). The interleukin 22 receptor alpha2 ELISA kit was purchased from Antibodies-online (Atlanta, GA). All solvents used were HPLC grade. Animal Care. Female Brown Norway rats (150-175 g) were obtained from Charles River (Montreal, QC) and housed in pairs with a 12:12 h light/dark cycle with free access to water and Agribrands pellet lab chow (Leis Pet Distributing, Inc., Wellesley, ON). Animals were acclimatized for 1 week before experiments were initiated. At the end of the experiment, rats were killed by CO2 asphyxiation followed by cervical dislocation. All of the animal studies were conducted in accordance with the guidelines of the Canadian Council on Animal Care. Microarray Study of Liver, Skin, Whole Ear, and Ear Skin. For the microarray study of rat liver, NVP, or 12-OH-NVP was administered as a suspension in 0.5% methylcellulose via gavage at equimolar dosages of 150 or 159 mg/kg/day, respectively. Six or 12 h after treatment, the rats were killed, and liver samples were taken. The size of sample was about 0.5 x 0.3 cm2, and the site of liver sampling was consistent for each animal. The liver samples were put in RNAlater RNA stabilization reagent for later RNA extraction. For microarray studies of rat skin, dosing of NVP or 12-OH-NVP and rat number for each drug were similar as that in rat liver microarray study. Samples of about 0.5 x 0.3 cm2, taken


Page 6 of 24

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


from rat back and cleared of hair and fat, and then they were put in RNAlater reagent for later RNA extraction. For the microarray study of rat whole ear or peeled ear skin, NVP or 12-OH-NVP was administered as a suspension in 0.5% methylcellulose i.p. at a dose of 150 or 159 mg/kg/day, respectively. Control rats were not administered the drug. The number of rats for each drug and time points are summarized in Table 5. Tissue samples of about 0.5 x 0.3 cm2 were taken from whole ear or peeled skin from the ear, cleared of hair and fat, and put in RNAlater RNA stabilization reagent for later RNA extraction. RNA extraction was performed with RNeasy mini kits and RNase-free DNase kits according to the manufacturer’s instructions. Extracted RNA samples were sent to Affymetrix microarray center at the Hospital for Sick Children, Toronto for microarray analysis. Data analysis was performed with Partek Genomics Suite software to identify RNA expression changes by comparing treatment groups with control groups. One-way or 2-way ANOVA was used for statistical analysis. The statistical significance of changes in gene expression was determined by the False Discovery Rate (FDR

Identification of Danger Signals in Nevirapine-Induced Skin Rash

Recommend Documents