New Approaches to Investigate Drug-Induced Hypersensitivity

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Perspective

New approaches to investigate drug-induced hypersensitivity Monday O. Ogese, Shaheda Ahmed, Ana Alfirevic, Catherine J. Betts, Anne Dickinson, Lee Faulkner, Neil S. French, Andrew Gibson, Gideon M. Hirschfield, Michael Ernst Kammüller, Xiaoli Meng, Stefan F. Martin, Philippe Musette, Alan Norris, Munir Pirmohamed, Brian Kevin Park, Anthony Wayne Purcell, Colin F. Spraggs, Jessica Whritenour, and Dean John Naisbitt Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00333 • Publication Date (Web): 02 Nov 2016 Downloaded from http://pubs.acs.org on November 4, 2016

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Chemical Research in Toxicology

New approaches to investigate drug-induced hypersensitivity Monday O. Ogese,1,2 Shaheda Ahmed,4 Ana Alferivic,2 Catherine J. Betts,1 Anne Dickinson,4 Lee Faulkner,2 Neil French,2 Andrew Gibson,2 Gideon M. Hirschfield,5 Michael Kammüller,6 Xiaoli Meng,2 Stefan F. Martin7, Philippe Musette,8 Alan Norris,2 Munir Pirmohamed,2,3 B. Kevin Park,2 Anthony W Purcell,9 Colin F. Spraggs,10 Jessica Whritenour,11 Dean J. Naisbitt,2*

1

Pathology Sciences, Drug Safety and Metabolism, AstraZeneca R&D, Darwin Building 310,

Cambridge Science Park, Milton Rd, Cambridge CB4 0WG, UK 2

MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology,

University of Liverpool, Ashton Street, Liverpool, L69 3GE, UK 3

The Wolfson Centre for Personalised Medicine, Department of Molecular and Clinical

Pharmacology, University of Liverpool, Ashton Street, Liverpool, L69 3GE, UK 4

Alcyomics Ltd c/o Haematological Sciences, Institute of Cellular Medicine, Newcastle

University Newcastle upon Tyne UK NE2 4HH, UK 5

Centre for Liver Research, NIHR Birmingham Liver Biomedical Research Unit, Institute of

Immunology and Immunotherapy, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK 6

Novartis Institutes for Biomedical Research, Klybeckstrasse 141, CH-4057, Basel,

Switzerland 7

Department of Dermatology and Venereology, Allergy Research Group, University of

Freiburg, Hauptstraße 7, 79104 Freiburg, Germany 8

Department of Dermatology and INSERM, University of Rouen, 905 Rouen, France

9

Infection and Immunity Program and Department of Biochemistry and Molecular Biology,

Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia

1

ACS Paragon Plus Environment


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GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY,

UK 11

Pfizer, Drug Safety Research and Development, Eastern Point Road, Groton, CT 06340,

USA

*Correspondence to: Dr. Dean J. Naisbitt, MRC Centre for Drug Safety Science, Department of Clinical and Molecular Pharmacology, Sherrington Building, the University of Liverpool, Ashton Street, Liverpool L69 3GE, United Kingdom Telephone: 0044 151 7945346 Fax: 0044 151 7945540 Email: [email protected]

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Abstract: The workshop on “New approaches to investigate drug-induced hypersensitivity” was held on June 5 2014 at the Foresight centre, University of Liverpool. The aims of the workshop were to (1) discuss our current understanding of the genetic, clinical and chemical basis of small molecule drug hypersensitivity (2) highlight the current status of assays that might be developed to predict potential drug immunogenicity, (3) identify the limitations, knowledge gaps and challenges that limit the use of these assays and utilise the knowledge gained from the workshop to develop a pathway to establish new and improved assays that better predict drug-induced hypersensitivity reactions during the early stages of drug development. This perspective article reviews the clinical and immunological bases of drug hypersensitivity and summarises various experts’ views on the different topics covered during the meeting.

KEYWORDS: drug hypersensitivity reactions, liver, skin, T-lymphocytes.

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Introduction Drug hypersensitivity reactions (DHRs) are rare, complex, mostly unpredictable and potentially fatal. These reactions are often immune-mediated, resulting in patient morbidity, ineffective drug therapy, drug attrition and/or drug withdrawal. Although they are rare, it is important to predict these reactions at the early stages of drug research and development. Early prediction of the potential of new drugs and/or their metabolite(s) to cause hypersensitivity will not only significantly reduce the rate of drug attrition in the pharmaceutical industry but will greatly enhance the therapeutic use of drugs in clinical settings. Multiple factors are critical to the development of DHRs; these include: the drug antigen, environmental factors and most importantly, patient-specific factors. These factors can be viewed as “the TRIANGLE of susceptibility to drug hypersensitivity”. Thus, Drug hypersensitivity = f [drug + patient genotype + patient phenotype]. A clear insight into the relative contributions of each of these factors is important in clinical diagnosis but also for the development of novel systems to predict the immunogenicity of a candidate drug. Characterization of the molecular pathophysiological mechanism(s) of drug hypersensitivity using a combination of in vitro assays and animal models is a critical step towards designing assays that will accurately predict which new drug will cause these reactions before they become widely used as therapeutics. Unexpected but potentially fatal DHRs are among the most important reasons why drugs have failed at advanced stages of research and development.1 These reactions constitute approximately 14% of adverse drug reactions (ADRs) with symptoms ranging from mild rash to more severe clinical manifestations such as Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN) and idiosyncratic drug-induced liver injury (DILI).2-5 Although the risk factors of DHR have been well studied, their relative contributions are still poorly understood. Complications are also inherent in capturing the contributions of co-morbidities and co-medications in a particular case of DHR. During the early stages of drug development, an enormous amount of information regarding the chemistry, efficacy, pharmacokinetics and pharmacodynamics of a candidate drug is available to researchers/scientists. Also available at this stage is an in-depth understanding of the disease pathogenesis, risk factors, co-morbidities, and diagnostic and prognostic biomarkers. In contrast, very little is known about the genetic (epigenetic and nucleotide polymorphism) and non-genetic peculiarities of the potential patient(s) for whom the given drug is being designed. The difficulty to fully predict DHRs during the preclinical stages of drug development may partially be related to the fact that safety studies are usually 5



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performed in young, healthy animals. Although high throughput assays exist to evaluate potentially toxic molecules based on in vitro covalent binding,6 these assays alone are inadequate to predict the potential of a candidate drug and/or its metabolite(s) to induce DHRs. Hence, there is a need to develop assays that can integrate the various risk factors and enhance predictability of drug hypersensitivity. Drug hypersensitivity can be defined as a serious ADR often with an immunological aetiology to an otherwise safe and effective therapeutic agent. Of note, the definition is used to describe reactions targeting skin and internal organs that manifest in a typically very small percentage of individuals exposed to a therapeutic drug. The frequency and severity of drug hypersensitivity are variable, increasing with disease and dose. Hence, it is important to understand the biology of the patient/immune system, the pathophysiology of the disease in question and the chemistry of the drug antigen. There are multiple mechanisms by which a drug may act as an antigen or immunogen to activate the immune system, and induce targeted tissue damage. The hapten, the pharmacological interaction with immune receptors and the altered self-peptide hypotheses go some way to explain the molecular pathomechanisms underlying DHRs. These running hypotheses define and characterise the different aspects of the drug antigen such as haptenicity, antigenicity, immunogenicity and hypersensitivity. Haptens are defined as low molecular weight chemicals with the propensity to bind covalently to protein. In the context of this review, the term antigen is used to describe a drug-related substance that interacts specifically with immunological receptors such as antibodies or T-cell receptors. Finally, immunogens are molecules capable of stimulating a cellular and/or humoral immune response. Also important is a clear understanding of the co-stimulatory signals (signal two) that augment the antigenic signal (signal one). Co-stimulatory signals can be either biological (infections) or mechanical (irritants and contact allergen), that provide danger signals (PAMPS-pathogen associated molecular patterns; DAMPS-damage associated molecular patterns, ROS-reactive oxygen species and ATP-adenosine triphosphate) capable of immune activation. Similar to the co-stimulatory molecules, the role of co-inhibitory checkpoint molecules such as PD-1 (Programmed Death 1 receptor),7,8 CTLA-4 (Cytotoxic TLymphocyte-Associated protein 4)9, LAG3 (Lymphocyte Activation Gene-3)10 and TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3)11 in the regulation of the immune system have been extensively researched. However, their importance in regulating drug antigen-specific T-cell responses in tolerant patients has not been studied. The evolution of pharmacogenetics has provided in-depth insights into individual susceptibility to various 6


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DHRs, and represents an important milestone towards stratified/precision medicine. Expression of specific HLA alleles is now known to be a major determinant of whether drug exposure will result in an inadvertent immune response and tissue injury. In fact, preprescription genotyping of patients for the risk allele HLA-B*57:01 prior to administration of abacavir has significantly reduced the incidence of hypersensitivity reactions.12,13 This concept has also been recommended for HLA-B*15:02 (Chinese) and HLA-A*31:01 (Caucasians) before carbamazepine prescription.14,15 However, prediction of DHRs in the clinic, based solely on HLA-genotype, remains very limited. This is because the majority of individuals who carry known HLA risk alleles do not develop immunological reactions when exposed to a culprit drug. We must therefore assume that immunological parameters, other than HLA genotype, may also contribute to the development of a drug-specific T-cell response. Since susceptibility to drug hypersensitivity is a function of the patient’s individual biology, the prediction of drug hypersensitivity will involve capturing the patient’s biology and variability during the early stages of drug development within pre-clinical test systems. This workshop, hosted by the CDSS (Centre for Drug Safety Science) in conjunction with the ABPI (Association of British Pharmaceutical Industry) and MHRA (The Medicines and Healthcare Products Regulatory Agency) provided the framework for speakers to discuss the current status of drug hypersensitivity research. Speakers and delegates were also encouraged to identify areas of deficiency and offer recommendations for future research strategies.

The key questions for consideration during and after the workshop included: 1. Which current in vitro endpoints are fit for the purpose of diagnosing drug hypersensitivity in humans? 2. Is there an animal model that can be used to determine the potential of a drug to produce hypersensitivity reactions in humans? If so, how can we validate such a model? 3. What predictive in vitro assays show the most promise at this time and what types of in vitro assays should be developed in the future? 4. Can assays discriminate between drugs which produce reactions at a higher incidence (>510%) or at a lower incidence ( 90% sequence homology and as such have peptide-binding repertoires which substantially overlap.32 Other HLA associations with DHRs include lumiracoxib with HLA-DRB1*15:01, and lapatinib with HLA-DRB1*07:01 which will be discussed later in this review. It is important to bear in mind that large linkage disequilibrium exists between genes in these loci, so an apparent association may in truth be related to another allele or haplotype associated with the detected allele.33,34 Since most individuals who express a HLA risk allele do not develop a reactions when exposed to a candidate drug, genetic, environmental or disease risk factors must impact on patient susceptibility. As drug metabolites are implicated in a number of DHRs, it is arguable that the expression of polymorphic drug metabolising enzymes may expose individuals within a population to varied quantities of antigenic moieties. Indeed, this may affect both phase I and II metabolism pathways, where an individual may be more susceptible to the formation of active products but also be less susceptible to their subsequent detoxification. Despite this, genetic variation in drug metabolism may rarely be a simple susceptibility to DHRs, but instead metabolic rate may be a factor for the rate of onset of a DHR. Impaired renal clearance and co-morbidity are other important factors to consider. Therefore, individuals are still exposed to potentially immunogenic metabolites independent of metabolic rate and thus it is unclear how metabolic variation translates to a predisposition to hypersensitivity. This may be explained by danger signalling, as certain individuals would be exposed to higher and thus more toxic concentrations of certain compounds, and would therefore be subject to enhanced danger signalling and an enhanced likelihood of T-cell activation. One of the most extensively studied drugs with regards to the role of drug metabolism in DHRs is the sulfonamide antibiotic sulfamethoxazole (SMX). SMX is susceptible to Nacetylation by both N-acetyl transferase-1 (NAT1) and NAT2 enzymes. This process reduces the in vivo availability of SMX and the downstream metabolite nitroso SMX (SMX-NO), both of which can stimulate T-cells from hypersensitive patients. Thus, exposure to SMXderived immunogenic antigens in a given individual is determined by the rates of metabolic activation and detoxification. As exposure of keratinocytes to SMX is associated with cell death, and the release of danger signals,35,36 a higher SMX concentration due to slower detoxification has the propensity to increase the likelihood of immune activation. Indeed, it has been found that patients with hypersensitivity more frequently express the NAT2 slow 12


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acetylator phenotype than do tolerant individuals. However, when Pirmohamed and colleagues addressed the role of acetylator genotype on predisposition to SMX hypersensitivity by investigating enzyme polymorphisms in HIV-positive patients with and without hypersensitivity, no significant associations were found.37 A number of studies have assessed the role of the microsomal NADH-dependent hydroxylamine reductase (NADHR)mediated detoxification of SMX-hydroxylamine, an intermediate in the metabolism of SMX to the protein-reactive species SMX-NO.38,39 Differential expression or activity may affect the ability of an individual to reduce SMX. Furthermore, promoting the formation of SMXhydroxylamine and SMX-NO by manipulating redox cycling processes has been proposed to partly mediate SMX-NO-mediated cytotoxicity. Of interest, the anti-oxidants glutathione and ascorbate aid the formation of SMX-hydroxylamine by promoting the reduction of SMXNO.40 However, similarly to NAT expression, genetic association analysis has ruled out a role for glutathione-S-transferase enzymes in the predisposition of an individual to these reactions.37 Interestingly, Wang et al demonstrated that HIV patients with rs761142 polymorphism in their glutamate cysteine ligase catalytic subunit (GCLC) had lower levels of GCLC mRNA expression and an increased incidence of SMX-induced hypersensitivity. However, in a more recent study by Reinhart et al investigating genetic risk factors associated with sulfonamide hypersensitivity in immunocompetent patients, the authors concluded that there was no genetic risk factor linked to the observed hypersensitivity reactions reported41. Therefore, while genetic association studies have not strongly supported a role for genetic variations in metabolising enzymes for predisposition to SMX hypersensitivity, metabolic variants may influence the balance of a response towards a parent drug- or metabolitespecific reaction. Presumably, drug metabolism is also influenced by co-administration of other drugs, diet exercise etc. As metabolism seldom represents a one-step process, it is important to consider the implications of metabolism and detoxification pathways downstream of the parent compound, particularly in cases where metabolites are capable of activating T-cells. Indeed, the interplay of complex metabolism pathways is thought to account for the lack of association between isoniazid hepatotoxicity and acetylator phenotype.42

Idiosyncratic DILI: clinical problems, genetics, lesson learnt and unanswered questions Idiosyncratic DILI presents with an array of clinical symptoms and can vary in severity from a mild increase in liver enzymes (alanine aminotransferase (ALT), bilirubin and alkaline phosphatase (ALP)) to acute liver failure and death. DILI is often difficult to distinguish from 13



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natural causes of liver injury such as viral hepatitis or autoimmune conditions.43 DILI often has a delayed onset (5 to >100 days) during continuous therapy and even may occur after cessation of therapy. Assessment is based on clinical and biochemical findings44 and accurate diagnosis with drug causality requires detailed case patient records reviewed by multiple expert hepatologists. Based on biochemical measures, three types of DILI can occur45 ‘hepatocellular’ caused by damage predominantly to hepatocytes, where serum ALT at the time of maximum elevation is greater than ALP, ‘cholestatic’ caused by disruption in biliary excretion of bilirubin, where serum bilirubin is elevated and ALP at the time of maximum elevation is greater than ALT and ‘mixed’, where ALT, ALP and bilirubin are elevated. A concomitant rise in ALT (>3x upper limit of normal range, ULN) and bilirubin (>2x ULN) is suggestive of severe liver injury where hepatocyte damage is coupled with disrupted biliary excretion, increased serum bilirubin and jaundice. These symptoms are collectively referred to as “Hy’s Law”. In cases analysed by Hy Zimmerman, Spanish and Swedish registries,46-48 concomitant ALT/bilirubin elevations are associated with a 10% mortality risk. Such cases of possible Hy’s Law require prompt and permanent discontinuation of the suspected drug to prevent further liver injury and possible mortality. DILI events typically resolve within 30 days following drug cessation, and in some situations adaptation during continuous treatment can occur, however the risk of DILI exacerbation following re-challenge means that drug reintroduction is contraindicated in cases where Hy’s Law criteria has been diagnosed. Since the lack of distinguishing diagnostic features and predictive or prognostic biomarkers for DILI makes it difficult to establish drug-induced DILI events, safety risk management in clinical trials must include regular monitoring of serum liver enzymes (ALT, ALP and bilirubin) levels, with defined thresholds for drug discontinuation and causality determination of potential DILI cases by hepatologist expert adjudication. Since DILI events can occur rapidly, at any time during drug treatment and may occur between monitoring visits, frequent serum liver enzyme monitoring in clinical trials is recommended for early safety evaluation and management, however this is not always achieved. Where HLA alleles are validated for DILI risk, a possible safety management approach may be to schedule risk allele carriers for more frequent liver chemistry monitoring than non-risk allele carriers. The delayed onset of liver injury (several days to several months) and development of more rapid and more severe symptoms following inadvertent re-challenge are indicative of an adaptive immune mechanism. Moreover, analysis of liver from patients with flucloxacillin- and sulfasalazineinduced liver injury showed an increase in T-cells secreting the cytolytic molecule granzyme B,49,50 which suggests that T-cells might play a direct role in the disease pathogenesis. In 14


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contrast to the skin reactions, the role of drug-specific T-cells in reactions targeting liver has not been extensively investigated. An early study detected T-cells in blood of certain DILI patients could be activated in vitro in the presence of the suspect drug(s),51 however, the phenotype and function of these cells were not investigated and additional studies have not been forthcoming. For this reason, the team in the CDSS has recently focussed on three drugs: flucloxacillin, co-amoxiclav and isoniazid, and in each case drug-responsive T-cells have been detected in patients with liver injury. Drug-specific T-cells were cloned and characterized in terms of cellular phenotype, HLA-restriction and mechanisms of cytotoxicity.31,52 The association between MHC class I alleles and a number of drug reactions has been well established.53 Notably, HLA-B*57:01 is associated with abacavir hypersensitivity syndrome54 and with pazopanib- and flucloxacillin-induced liver injury.55,56 However, it is now clear that abacavir and flucloxacillin bind to HLA*B57:01, and initiate immune responses in different ways. The molecular interaction between pazopanib and HLA-B*57:01 has not been characterised. The mechanism of immune response proposed for the pathogenesis of abacavir hypersensitivity involves a non-covalent interaction between the drug molecule and the Fpocket of the HLA-B*57:01 binding groove. The outcome is an alteration in the repertoire of self-peptides presented to T-cells, and subsequent activation of the immune system.32,57 Interestingly, the molecular mechanism of T-cell activation by flucloxacillin is dependent on a number of variables. Flucloxacillin is a β-lactam antibiotic that generates protein adducts spontaneously through nucleophilic attack by amino acids and ring opening.58 Wuillemin et al demonstrated that while T-cells generated from HLA-B*57:01-negative healthy donors were activated via a processing-dependent pathway by flucloxacillin-modified peptide (hapten hypothesis), T-cells generated from HLA-B*57:01-positive healthy donors responded to the drug antigen via a processing-independent, non-covalent interaction of flucloxacillin with HLA-B*57:01 allele (pharmacological interaction with immune receptors).59 The pathways of flucloxacillin-induced T-cell activation were also studied by Yaseen et al.60 Specifically, they compared the mechanism of T-cell activation using T-cells generated from both DILI patients and healthy volunteers. They demonstrated that in contrast to T-cell clones from healthy donors; those from patients with DILI were activated with flucloxacillin via the hapten mechanism. Moreover, the response to flucloxacillin-modified peptides was highly HLA allele-restricted. These data suggest that the pathway of T-cell activation by flucloxacillin may be dependent on multiple factors including, the expression of the HLAB*57:01 risk allele and disease. 15



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In contrast to the HLA class I-restricted reactions, as yet no findings have linked the expression of specific HLA class II risk alleles to the activation of drug-specific T-cells in drug involved in drug hypersensitivity. Thus, two speakers from the pharmaceutical industry were invited to discuss the potential role of MHC class II alleles in DILI. Michael Kammüller (Novartis) gave insight into the clinical problems and genetics of lumiracoxib-induced liver injury. Lumiracoxib is a selective COX-2 inhibitor indicated for osteoarthritis and acute pain. It has favourable PK properties, attaining a high concentration in the synovial fluid relative to the plasma. Furthermore, this drug had a better safety profile on the gastrointestinal tract when compared with the non-selective COX-1/2 inhibitors.61 However, signs of reversible transaminase elevations were observed during the development program,61 thus prompting a product labelling requiring monthly hepatic monitoring in chronic administration. In a large randomised controlled trial, the proportion of patients with transaminase concentrations >3x ULN differed significantly between lumiracoxib (2.6%) and non-steroidal anti-inflammatory drugs (0.6%), but changes were reversible on drug discontinuation.61 Cases of liver injury, liver failure and death associated with lumiracoxib were observed during post-marketing surveillance.62,63 Although the exact mechanism of lumiracoxib-induced liver injury is unclear, the genetics of susceptible individual appears to be an important risk factor, indicating an immune-mediated pathogenesis. An exploratory genome-wide association study of 41 cases (>5 x ULN ALT/AST) and 176 matched controls identified a significant association with the MHC class II region.34 HLA fine mapping identified a highly significant association with the haplotype (HLA-DRB1*15:01-HLADQB1*06:02-HLA-DRB5*01:01-HLA-DQA1*01:02, P = 6.8 x 10-25; allelic odds ratio = 5.0, 95% CI 3.6-7.0). Among patients with liver enzyme elevations, carriers of the HLADQA1*01:02 allele had more severe hepatotoxicity than did non-carriers. Restriction by human MHC class II alleles may explain the lack of predictability of preclinical toxicology studies for human hepatotoxicity. Moreover, the convergence of exogenous and endogenous risk factors, which likely determine frequency and severity of rare events such as DILI in humans, is not present in currently used preclinical animal models. Initial studies using naïve T-cells from healthy volunteers with this haplotype have failed to detect lumiracoxib-specific T-cells. However, it is important to note that not all individuals that express the abovementioned haplotype develop lumiracoxib-induced liver injury. Therefore, characterising the importance of this allele in the mechanism of lumiracoxib-induced liver-injury remains an unmet need. In view of the delayed onset of lumiracoxib-induced liver-injury (weeks to months after initiation of drug treatment), a more detailed characterisation of the various 16


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stages (initiation, progression and repair or adaptation) and events of DILI will be essential. This information may help in identifying risk factors, biomarkers and the molecular mechanisms involved in disease pathogenesis over and above the HLA class II associations. Both the chemistry of drug-antigen and patients’ immunogenic susceptibility factors need to be considered during clinical and mechanistic studies. The lack of liver biopsies, patients’ peripheral mononuclear cells (PBMCs), urine and DNA are obvious limitations to mechanistic studies. The ultimate challenge for drug safety sciences remains to identify drugs with the potential to cause HLA-associated DILI at the early stages of drug discovery without the risk of discarding potentially valuable new drugs. Colin Spraggs (GlaxoSmithKline) discussed the genetics of lapatinib-induced liver injury. Lapatinib (Tykerb/TyverbTM Novartis, formerly GlaxoSmithKline), is an inhibitor of human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR), receptor tyrosine kinases, which are signalling pathways for tumor growth. Lapatinib is approved for use within the US, EU and a number of other countries for the treatment of HER2-positive metastatic breast cancer, in combination with chemotherapy, such as trastuzumab,64 capecitabine or letrozole.65,66 The potential for liver toxicity was not identified in pre-clinical animal toxicology studies of lapatinib. However, during clinical trial evaluation, a small proportion of lapatinib-treated patients experienced isolated elevated serum alanine transaminase (ALT>5x upper limit of normal (ULN)) (3%) and/or concomitant elevated ALT (>3x ULN) with elevated serum bilirubin (>2x ULN), consistent with possible Hy’s Law cases (0.6%).62 These findings resulted in prominent ‘Black Box’ safety warnings for hepatotoxicity in Tykerb/Tyverb product labelling. Liver injury during lapatinib treatment exhibits a variable, but typically late onset elevation in ALT, which resolves upon lapatinib discontinuation. Symptoms may resume rapidly during re-challenge; a pattern consistent with a delayed immunological hypersensitivity reaction. Lapatinib is metabolised primarily by CYP3A4 and CYP3A5, with minor contribution from CYP2C8 and CYP2C19.67 The formation of protein-reactive quinone imine metabolites, possibly generating neoantigens that can activate the immune system has been demonstrated using a number of in vitro systems.68,69 Genome-wide screening of prospectively collected clinical trial samples performed to assess the significance of pharmacogenetic factors in lapatinib-induced liver injury implicated HLA-DQA1*02:01 and HLA-DRB1*07:01 (which are co-inherited) as risk alleles.70-73 Patients that expressed the HLA-risk alleles had higher ALT elevations and Hy’s Law risk. The association of MHC class II risk alleles suggests that the adaptive immune system may play a role the pathogenesis of lapatinib-induced liver injury in susceptible 17



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patients. To date however, no HLA binding studies have been carried out to investigate the interaction of either lapatinib or its reactive metabolites with these risk alleles. Moreover, lapatinib (metabolite)-specific T-cells have not been isolated from DILI patients or healthy volunteers post priming. Thus, similar to lumiracoxib, a knowledge gap exists in our understanding of the molecular basis of lapatinib-induced liver injury, most especially how the presence of HLA-DQA1*02:01 and HLA-DRB1*07:01 regulate immune responses resulting in DILI.

Immunological studies on patients with natural liver injury Gideon Hirshfield (University of Birmingham, UK) presented on the role of the immune system in naturally occurring hepatic disease. He and his colleagues are interested in liver diseases of varying aetiology, some of which are associated with HLA risk alleles, mostly HLA class II alleles. Their major research interest is primary biliary cirrhosis (PBC), which is associated with a number of HLA class II alleles in European and Japanese populations. However, the exact role of the implicated HLA alleles in the pathophysiology of PBC remains to be defined. Following a liver transplant, a biopsy is taken to isolate various cellular subsets such as liver infiltrating lymphocytes, hepatocytes and sinusoidal endothelial cells for further in-depth analysis. He described four cellular compartments implicated in DILI, namely: liver, bile duct, gut and immune compartments. Interestingly, although genome-wide association studies have strongly implicated certain HLA alleles, other genes such as IL-12, IRF5, CXCR5 and SOCS1 have been identified as important in the immunoregulation of PBC.74-76 Furthermore, analysis of the cytokine microenvironment of tissue infiltrates has implicated Th1 cells in early disease while Th17 cells predominate in the later stages of PBC.77 Such qualitative data provides important information for the clinical management of patients, most of who are diagnosed late in the disease process. Access to and analysis of clinical samples (liver tissues and whole blood) to investigate the molecular pathomechanisms of DILI is critical. There is a need for the rigorous phenotyping of T-cells from liver biopsies to assess the role of different populations of infiltrating T-cells in the different forms of drug-induced liver disease.

Moving Forward: Critical questions: (1) Why are specific organs (skin, liver, heart, brain etc.) targeted by the immune system? (2) What are the molecular mechanisms underlying HLA risk alleles? (3)

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Why do only some patients with risk alleles develop DHRs? (4) What are the critical events that cause immune activation in the tolerogenic environment of the liver?

LESSONS

FROM

PATIENTS

WITH

CUTANEOUS

HYPERSENSITIVITY

REACTIONS Skin reactions are traditionally listed as the most frequently observed reactions to drugs. Drug-induced skin injury affects about 2-3% of hospitalised patients.78,79 Morphologically, these reactions can be subdivided into maculopapular rash, bullous reactions (SJS/TEN), pustular reactions (AGEP) and hypersensitivity syndromes. Given the varying clinical presentations, it is unlikely that a single mechanism of T-cell recognition/activation is responsible for all the types of drug-induced cutaneous eruption. Drug-specific cytotoxic CD8+T-cells have been implicated in the death of keratinocytes, resulting in skin detachment observed in drug-induced SJS and TEN.80,81 A number of studies have implicated CD4+ Tcells in the pathogenesis of milder drug reactions.60,82,83 However, it is still not clear whether these CD4+ cells are activated to secrete the same effector molecules as cytotoxic CD8+ Tcells in patients. The different phenotypes of drug-induced cutaneous adverse reactions are summarised below.

Maculopapular exanthemas (MPE) and Fixed drug Eruptions (FDE) Maculopapular simply refers to the generalised morphology of the rash which is observed as macules or papules which are flat or raised skin lesions of up to 1cm in diameter respectively. This lends itself to the description of these reactions as exanthematous in reference to these eruptions. Other commonly used descriptions include the term morbilliform eruption alluding to similarities to the eruptions seen with measles infection. Indeed, MPE may be caused by certain diseases such as lymphoma and by viruses like cytomegalovirus and herpes, however it is most often induced by drugs.84 There is still some confusion surrounding the exact mechanism of drug-induced MPE but it may be that there are two; one being a Th2 response with subsequent eosinophil recruitment and the involvement of interleukin (IL)-4 and IL-5, the other a joint CD4/CD8 cytotoxic response involving various cell death pathways including Fas-FasL, perforin, and granzyme B.85 Other mild and generalised reactions may not affect the entire body, but just one area. For example, fixed drug eruptions (FDE) are characterised by recurring skin lesions, on the same area of the skin, in response to repeated exposure to an antigenic drug. FDE is often associated with accompanying nausea, vomiting,

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and fever, and lesions in the skin of patients with these reactions have infiltrating lymphocytes as well as neutrophils and eosinophils.86 Upon recognition of FDE, the drug is usually withdrawn and topical steroids administered.87 There are, however, a number of better defined hypersensitivity reactions, which are detailed below.

Acute Generalised Exanthematous Pustulosis (AGEP) AGEP is a rare cutaneous reaction which is estimated to affect a maximum of just 5 cases per million per year,88 and with 5% mortality. It is classified as more serious than MPE which is essentially non-lethal.89 This reaction is often observed less than 48 hours after initial drug administration but spontaneously resolves by around 2 weeks. Due to this rapid resolution, patients may be treated with corticosteroids but the vast majority only require supportive care.90 Morphologically, AGEP defines a rash formed of numerous pustules atop an inflamed, reddened section of skin and is strongly linked to simultaneous development of fever, as are to a lesser extent symptom such as blisters and facial oedema. In approximately one fifth of cases there is involvement of mucus membranes, particularly that of the mouth.90 A wide variety of drugs have been associated with AGEP including many anti-infective agents such as the sulfonamides containing medication co-trimoxazole, and much more common overthe-counter drugs such as acetaminophen.91,92 While both CD4 and CD8 T-cells are implicated in these reactions, more defining is the fact that these responsive T-cells are able to recruit and induce neutrophils through the production of IL-8. In fact, it is this large influx of neutrophils that forms the sterile pustular eruptions associated with AGEP.93

Drug reaction with eosinophilia and systemic symptoms (DRESS) DRESS, also referred to as drug hypersensitivity syndrome (DHS), is considered a severe cutaneous adverse reaction due to a mortality rate of 10%, double that of AGEP.89 The key distinguishing features of this reaction are, as its name implies, eosinophil involvement and immersion of this reaction within a number of systems other than the skin, which include the liver and hematologic system.94 DRESS has a reported incidence of 1 in 10,000 exposures and a delayed onset of 2-6 weeks post drug exposure. Associated symptoms are fever, eosinophilia, lymphadenopathy, severe skin eruption, and the inability of drug withdrawal to reduce symptoms.95,96 Other features are often present such as facial oedema, involvement of the liver, kidneys, lungs, and/or heart, with liver failure being the major cause of death for DRESS patients. Both CD4+ and CD8+ T-cells are thought to be involved which produce IL-5 prior to eosinophil recruitment.97 Recently, most interest in DRESS surrounds the potential 20


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role of viral reactivation in the induction of this reaction. Reactivation of cytomegalovirus, Epstein-Barr virus, and HHV6/7 has been linked with DRESS, which is thought to arise due to a hypogammaglobulinaemic environment induced by the culprit drug. For this scenario it is the viral reactivation that is then responsible for the development of DRESS which accounts for the lengthy time to onset due to the time required for viral expansion.96,98.However, it is also possible that it is the drug-specific activation of T-cells that reactivates the virus. Identifying which comes first, drug-specific T-cell activation or viral activation, is hindered by the fact that viral deoxyribonucleic acid (DNA) and virus-specific IgE, which are routinely used to assess viral activity, are only able to be measured many weeks after onset of a reaction.99 Oral corticosteroids and drug withdrawal are used to treat DRESS, as is the immunosuppressive agent cyclosporine. At least 50 drugs are known to cause DRESS.95 A key question that remains unanswered is: what is the mechanism of viral re-activation since this can be triggered by different drugs in DRESS patients? Philippe Musette (Rouen University Hospital, France) discussed recent findings of his group showing reactivation of multiple viruses (HHV6, CMV and EBV) in 80% of DRESS patients (n = 40) recruited in France. The reason why viral reactivation is not observed in the remaining 20% of patients is unclear. Musette also described the expansion of EBV-specific CD8+ T-cells in DRESS patients.100 T-cells reactive against viruses migrate to the skin and the liver, and cutaneous eruptions ensue upon re-exposure to “culprit drug”. An obvious knowledge gap is whether these viral-specific T-cells are capable of cytotoxicity in the same manner as other drugspecific T-cells that can be isolated from the same patients. The involvement of cytokines in DRESS was also discussed by Musette. IL-2, IL-10 and TNF-α have all been shown to be upregulated in patients with DRESS. These cytokines can be modulated and viral reactivation blocked; thus, providing a potential treatment option in the management of DRESS patients. Since cytokines and a pro-inflammatory microenvironment are important factors in the pathogenesis of DRESS, Musette emphasized the need to access PBMCs from patients and tolerant controls during clinical trials to allow prospective mechanistic studies. One other factor yet to be fully explored in the development of cutaneous reactions such as DRESS is the role of regulatory T-cells (Tregs). These CD25+ CD4+ T-cells, which account for 5-10% of all circulating CD4+ T-cells, function to primarily suppress effector T-cell responses.101 Musette concluded by hypothesizing that secretion of IL-10 and TNF-α, as a result of both drug and viral recognition, are important determinants of the severity of clinical symptoms in the pathogenesis of DRESS. Delegates agreed that further experiments are required to 21



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determine the relative importance of the drug and viruses in activating an inappropriate immune response in DRESS patients. Also, given the diversity of implicated viruses, new technologies need to be applied to further explore the contribution of viruses to the disease pathogenesis.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) The most serious cutaneous reactions, due to their high mortality rates, are SJS and TEN. These two reactions are in fact thought to be variants of the same reaction, differing in terms of severity, and affecting between 10-30% of the epidermis. SJS and TEN are categorised by the extent of skin detachment, with TEN being the more debilitating. SJS and TEN are caused by a myriad of drugs such as NSAIDs, antibiotics, anticonvulsants, sulfonamides, and allopurinol. SJS/TEN has a reaction onset time of between 1-3 weeks. Both of these reactions are associated with characteristic blistered lesions that are described as painful with a burning sensation.86,102 In fact, the treatment of SJS/TEN has many similarities to that of a severe burn, and indeed the specialist care of a burn unit is often required for SJS/TEN patients. Although considered a life threatening cutaneous reaction, if the drug is withdrawn and the reaction dampened, the skin of patients with SJS/TEN recovers far more quickly than that of burn patients, as the reaction is limited to the epidermis in SJS/TEN.103 SJS involves a skin reaction with 10% body coverage and 1-5% mortality, while TEN classification requires 30% body coverage and is associated with 30-50% mortality.104 The initial damage during SJS/TEN is actually keratinocyte apoptosis, with epidermal necrolysis occurring later in the reaction causing the characteristic skin detachment as the epidermis and dermis separate.102 SJS occurs more frequently than TEN with incidences of 1-2 cases and 0.4-1.2 cases per million person per year, respectively.105 In both SJS and TEN, lesions have been found to contain natural killer T-cells (NKT) and cytotoxic T-cells as the effector cells responsible for the associated keratinocyte death.106 The skin homing receptor CLA appears to be particularly important in recruitment of these T-cells to the skin as patient T-cells obtained from blister fluid have four-fold higher CLA expression than PBMCs. The T-cell response, which involves both CD4 and CD8 T-cells, leads to the secretion of IFN-γ, which through inducing secretion of tumour necrosis factor (TNF)-α, as well as expression of human leukocyte antigen (HLA)-DR and Fas on keratinocytes, leads to the death of epidermal cells.107 Indeed, blister fluid, as well as the epidermis, have been reported to contain cytokines such as IFN-γ, TNF-α, and FasL.102 The role of FasL is of particular interest as groups have been able to show effective Fas-FasL inhibition of skin detachment 22


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using both synthetic blocking antibodies as well as natural anti-Fas antibodies found in intravenous immunoglobulins (IVIG). Initial reports have highlighted IVIG therapy as highly effective, with reduced skin detachment in all ten patients tested.102 However while FasL represents a possible therapeutic approach, the major cytotoxic effector during SJS-TEN seems to be granulysin which therefore has great therapeutic and diagnostic potential. Specifically, granulysin concentrations in the blister fluid of patients have been shown to be 2 to 4-fold higher than concentrations of other cytolytic mediators including FasL, granzyme B and perforin. This high expression translates to a significant functional effect, as mice exposed to granulysin from the blister fluid of patients developed SJS-TEN-like features, without the addition of any other factors.108

Immunological studies on patients’ tissue To understand that aetiology of cutaneous hypersensitivity reactions it is important to define the role of individual T-cell subsets. Traditionally, it was considered that two distinct CD4 Tcell subsets could be generated from the naïve CD4 (Th0) population which were defined by distinct cytokine secretion patterns. Th1 cells are considered pro-inflammatory and defined by the ability to secrete IFN-γ and IL-2, while Th2 cells are anti-inflammatory and secrete cytokines such as IL-4 and IL-5. Th1 cell development is driven by the transcription factors T-bet and STAT4, subsequent to which they activate NK cells and macrophages, and expand cytotoxic CD8+ T-cells to protect against intracellular viruses and bacteria largely through the secretion of IFN-type cytokines. Th2 cells on the other hand are induced by the expression of GATA3 and STAT5 and act to control pathogenic infections such as from helminths, but also bacterial and viral infections, by promoting eosinophil- and IgE-mediated responses.109,110 However, in recent years, a number of new T-cell subsets have been identified. Lesser known Th subsets include T follicular helper cells (Tfh), which aid antibody class switching and the secretion of antibodies with high affinity,111-114 and Th9 cells, so called because of their profound IL-9 secretion, and reported to be involved in autoimmune and allergic disease development. However, two distinct subsets termed Th17 and Th22 cells have received the greatest attention with regards to hypersensitivity reactions as both appear to have important consequences in the skin, particularly Th22 cells which are enhanced in patients with psoriasis, atopic eczema, and allergic contact dermatitis.115 Both Th17 and Th22 cells have dual capabilities; they can induce an anti-inflammatory innate immune response in keratinocytes to protect skin, but they also have a pro-inflammatory function. Specifically, Th22 cells promote the secretion of chemokines and cytokines from keratinocytes in inflamed 23



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skin thus enhancing the immune response. However, they also maintain the integrity of the skin by inducing the proliferation of keratinocytes. In contrast, the pro-inflammatory effects of Th17 cells are more pronounced as they ultimately lead to keratinocyte death through apoptosis due to upregulation of specific adhesion molecules. While both Th17 and Th22 cells secrete IL-22, the more prominent pro-inflammatory effects mediated by Th17 cells are due to the combined effects of IL-17 and IFN-γ.116 Of immense importance are new mechanistic studies using cells from patients with cutaneous hypersensitivity to define the involvement (or not) of these new T-cell populations in the disease pathogenesis. Data derived from these studies will allow reclassification of hypersensitivity reactions by updating the system proposed by Pichler in 2003.4 The group led by Jean Francois Nicolas (Lyon, France) has demonstrated that there is a strong infiltration of amoxicillin-specific CD8+ T-cells into the epidermis during skin testing of patients with different forms of cutaneous hypersensitivity. These infiltrates can be detected before overt clinical manifestations of cutaneous adverse reactions, and they persist up to 48 hours after the “culprit drug” has been removed from the skin. Interestingly, the early CD8+T-cell infiltration is accompanied by IFN-γ secretion at 12 hours but IFN-γ expression does not persist at 48 hours. Furthermore, granzyme-B was shown to be involved in the predominant pathway of cell lysis. Aurore Rozieres (Lyon, France) and her colleagues hypothesise that the number of IFN-γ secreting CD8+ T-cells recruited into the skin during the initiation stage of cutaneous ADR is proportional to the severity of such reactions.

Innate immune response to chemicals and induction of drug hypersensitivity reactions In 1994, Matzinger and colleagues proposed a radical new concept for T-cell activation. Specifically she stated that a T-cell is tipped towards a responsive state if an antigen is presented to it in a “stressful” environment. This was termed the danger hypothesis and was rapidly applied to drug hypersensitivity.117,118 The idea is that if a drug is directly toxic to cells it will induce cell damage and potentially cell death. Antigen presenting cells consequently upregulate co-stimulatory molecule expression on their cell surface in response to stimuli from these dead cells which renders these cells ready for T-cell stimulation.119 Endogenous danger signals from dead cells are effectively the spilled contents of the cells and are known as DAMPs (damage associated molecular patterns), and include HMGB1 (high mobility group box-1 protein), S100 proteins, and heat shock proteins.119 No signalling 24


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occurs in the case of natural cell turnover where dying cells are effectively scavenged long before they could disintegrate, but signals are released in response to drug-induced necrotic cell death. Indeed, expression profiles of genes associated with oxidative stress changed shortly after administration of tielinic acid which is linked with hepatotoxicity,120 and dendritic cell expression of CD40 is enhanced by SMX-NO.121 One particular DAMP which has received considerable attention is uric acid, a product of homeostatic nucleic acid turnover. When an individual develops hyperuricemia, monosodium urate (MSU) crystals are deposited, particularly in the joints. These crystals are able to stimulate the NLRP3 inflammasome to produce the effector cytokine, IL-1β. The associated inflammatory response is known as gout. It appears that uric acid is a strong and therefore important danger signal as its depletion alone in mice can significantly dampen the immune response to induce cell death. In contrast, a similar effect was not detected in response to other inflammatory inducers such as sterile irritants or microbes indicating that certain DAMPs are selective for specific inflammatory responses.122,123 Dead cells are known to release intracellular stores of uric acid, which promotes an acute inflammatory response.122,124,125 Since most reactive drug metabolites induce direct toxicity, it is possible that uric acid released from tissue cells, such as keratinocytes, promotes inflammation that directs the nature of the adaptive response initiated against the drug antigen. In comparison, other DAMPs are involved in a wide array of inflammatory responses, such as HMGB1 which is found in response to both infectious and non-infectious stimuli. Interestingly, although danger signals are associated with inflammation, HMGB1 has also been observed to aid tissue repair and to restore damaged tissue.126 Other danger signals arising from exogenous sources such as bacterial LPS or viral RNA and are known as PAMPs (pathogen associated molecular patterns).127 Infection is a well-known danger signal for developing DHRs as exemplified by enhanced susceptibility to isoniazid hepatotoxicity with concomitant chronic hepatitis and HIV.128 These viral danger signals instigate myeloid dendritic cell production of CD1d mRNA leading to activation of NKT cells. Contact allergy is an inflammatory disease of the skin triggered by protein-reactive, low molecular weight organic chemicals and metals. The incidence in the general population has been estimated as 15-20%.129 The main effector cells in contact allergy are CD8+ T-cells with Th1 and Th17 cells and CD4+CD25+ICOS+ Treg and iNKT cells responsible for supporting the cytotoxic response and immune regulation, respectively. Contact allergens induce cellular stress and damage resulting in the production and release of danger signals critical for immune activation and skin inflammation. The activation of the innate immune 25



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cells upon compound exposure almost always precedes an inflammatory response and this process can be mimicked in vitro by culturing chemicals with dendritic cells at sub-toxic and toxic concentrations. Stefan Martin (University of Freiburg, Germany) and his colleagues have used the mouse model, MEST (Mouse Ear Swelling Test) to investigate the potential of chemicals to elicit contact hypersensitivity and the role of specific immune cells (effector Tcells, mast cells, Tregs, dendritic cells) in the reaction. The test consists of sensitization and elicitation phases. Sensitization can either be performed via skin painting or through intracutaneous injection of dendritic cells that have been modified in vitro. Measurement of ear swelling upon re-challenge or elicitation with the allergen of interest provides an assessment of the strength of the induced response. Using this model, the role of the different cellular components implicated in allergic contact dermatitis has been characterised. For example, neutrophil depletion before sensitization or before re-challenge has been shown to abrogate contact hypersensitivity.130 The innate immune response to pathogens is mediated by one or more Toll-like receptors (TLRs), reactive oxygen species (ROS) and NOD-like receptors. Interestingly, nickel and cobalt can bind directly to human but not mouse TLR4, and induce receptor dimerization with subsequent innate immune activation.131,132 Organic contact allergens like 2, 4, 6trinitrochlorobenezene (TNCB) induce hyaluronic acid (HA) degradation and ATP release from stressed and damaged cells resulting in an inflammatory response in the skin. Induction of skin inflammation by contact allergens is a highly regulated response, and involves TLR2/4, P2X7 and IL-1R receptors stimulated by HA, ATP and IL-1β respectively. Receptorligand interactions induce downstream signalling resulting in the activation of the innate immune system, leading to skin inflammation.133-135 Furthermore, generation of ROS by contact allergens is a well characterised pathway for the activation of the innate immune system. A major issue with the exploration of the immune system in DHRs is the limited number examples of established animal models that closely reflect the human disease. Until now, the nevirapine-induced skin rash observed in female Norway rats is the only model that has been shown to have close similarities with nevirapone-induced skin rash in humans.136,137 Moreover, in vitro models of drug or chemical immunogenicity that are being developed (see below) do not include the tissue-derived signals responsible for activation of dendritic cells. In recent years it has been shown that dysregulated co-inhibitory receptor signalling can propagate autoimmunity,138-144 and that blocking these pathways in cancer models can significantly enhance functional T-cell responses.145-147 Recently, a synergistic interaction of HLA-alleles and polymorphic variants of the co-inhibitory receptor CTLA4 has been shown 26


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to predispose individuals to the T-cell mediated disorder Grave’s Disease.148 Thus, it is conceivable that dysregulated immune regulation pathways may manipulate the threshold of T-cell activation to such an extent that they enhance a patient’s susceptibility to drug hypersensitivity. Indeed we have reported an inhibitory role for the co-inhibitory Programmed Death-1 (PD-1) pathway during the priming of naïve T-cells to drug-antigens.149 Although PD-1 is a critical immune checkpoint, complex interplay between multiple pathways means that it is crucial to elucidate the role of multiple co-signalling pathways to effectively analyse the role of inhibitory signalling during T-cell activation. In this respect, Uetrecht and colleagues have shown that inhibition of immune tolerance can unmask druginduced allergic hepatitis in animal models.150-152 Specifically, the amadiaquine-, isoniazidand nevirapine-induced liver injury is potentiated following blackade of CTLA-4 and PD1 signalling

THE CHEMISTRY OF DRUG MHC BINDING The hapten hypothesis and the pharmacological interactions with immune receptors (p-i), well-established mechanisms of T-cell activation, are briefly discussed below. Tony Purcell (Monash University, Australia) was invited to the workshop to elaborate on the altered selfpeptide repertoire concept, the most recently described mechanism of drug-specific immune activation, and to discuss the relevance of the individual pathways of drug-specific T-cell activation in the human disease. The hapten hypothesis provided an initial explanation for how small molecule drugs overcame their native small and thus non-immunogenic size to induce immune-mediated reactions. This hypothesis proposed that immune stimulation occurred through the binding of drug antigen to protein to form a hapten-protein complex. The hypothesis was first conceived after the identification of a relationship between sensitisation potential of chemicals and the extent of protein binding and reported in the landmark paper of Landsteiner and Jacobs in 1935.153 This complex, now large enough to be recognised by circulating dendritic cells, is taken and processed by antigen presenting cells. The peptide products of processing are subsequently presented on the cell surface bound to MHC molecules, which TCRs on passing T-cells can recognise.154 A slightly different mechanism is needed for drugs which lack chemical reactivity but are metabolised to protein reactive substances. Thus, the term prohapten hypothesis was conceived. It is identical to the hapten hypothesis but it is a metabolite that binds covalently to protein and not the parent drug.

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Recent advances in mass spectrometry-based proteomics have allowed for characterisation and quantification of hapten-protein complexes formed by drugs or metabolites in great detail. The chemical nature, quantity, and exact location of haptens that bind to proteins have been extensively studied in vitro and in patients. Human serum albumin and haemoglobin have been identified as targets for numerous drugs (metabolites) including β-lactam antibiotics,58,155-157 phase I reactive drug metabolites such as quinone imines (acetaminophen and lapatinib)158 and epoxides (carbamazepine),159 phase II metabolites such as acyl glucuronides (diclofenac)160 and sulphates (nevirapine),156 and covalent inhibitors (neratinib).161 Other proteins such as cytochrome P450 and glutathione S-transferase pi in the liver and keratin in the skin are also potential targets for haptenation. Hapten-protein complexes have been detected within cells as well as cell culture medium,162,163 in tissues (skin and liver),164-166 and in patients taking therapeutic drugs.58,157,158,160,167,168 Although much progress has been made in characterisation of hapten-protein complexes, for many drugs, the target proteins with respect to their location, the quantity, and in particular, their biological function in the immune system remain to be defined. Furthermore, the bona fide haptenated peptides that are presented on the surface of antigen presenting cells have not been characterized. Future research employing new proteomic technologies to identify these naturally processed immunogenic peptides will certainly provide affirmative evidence for the hapten hypothesis. It has also been shown that parent drugs can directly activate T-cells by a non-covalent, reversible interaction with the MHC and/or TCR. This theory, the “pharmacological Interaction” or PI concept, states that a drug may directly bind to an antigen receptor, whether this is the TCR, MHC, or both to stimulate a T-cell response independent of antigen uptake and processing by dendritic cells. Figure 1 illustrates the main models of T-cell activation. Drugs such as SMX are able to stimulate TCR via this direct interaction as assessed by positive T-cell stimulation in the presence of fixed antigen presenting cells (to abolish antigen processing but allowing MHC-antigen binding).169 Further proof lies in the recognition that stimulation of T-cells by some drugs occurs within seconds, which is too quick for a protein adduct to have been engulfed, processed, and externally displayed. Importantly, the PI concept has originated exclusively from in vitro assessment of T-cells isolated from human patients and it is now imperative to put these findings in the context of the iatrogenic disease that has a delayed onset. It is generally thought that hapten and pro-hapten associated drugs stimulate a novel response and as such naïve T-cells are implicated; however, memory T-cells may be preferentially 28


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activated by drugs associated with the PI-concept.154 It is likely that PI-dependent T-cell activation only occurs on T-cells with low thresholds for activation.169 While this rapid activation of T-cells may at first appear contradictory to the delayed nature of T-cell hypersensitivity reactions, it is important to bear in mind that even the first contact between dendritic cells and T-cells occurs 3-15 hours after administration in vivo. Moreover, the activation of naïve CD4+ T-cells in vivo has been reported to require a minimal stimulation period of 6 hours before subsequent clonal expansion, with a sustained CD4+ response being dependent on additional signals produced by dendritic cells which occur more than 24 hours after the initial exposure. Moreover, these events only represent the beginning of the T-cell response which requires time-consuming events such as gene activation and the production of cytokines. Indeed, these late signals appear to be important for distinct functions of the response that result in the development of clinically distinct DHRs.170-173 The altered-self peptide hypothesis was put forward in 2012 to explain the mechanism of abacavir hypersensitivity.32 About 5-7% of abacavir-exposed patients present with hypersensitivity characterised by fever, rash, malaise, nausea, vomiting and diarrhoea. GWAS demonstrated that 100% of patients that experienced abacavir hypersensitivity were positive for the MHC class I allele HLA-B*57:01.174 Tissue injury is thought to be CD8+ Tcell mediated since abacavir-specific CD8+ T-cells have been isolated from hypersensitive patients and generated from drug-naïve donors expressing HLA-B*57:01.175,176 T-cell activation involves abacavir-dependent alteration of the repertoire of self-peptide presented to T-cells in the context of HLA-B*57:01. Abacavir was found to interact specifically but noncovalently with the F-pocket of the antigen binding cleft of HLA-B*57:01. Purcell and his colleagues have been successful in isolating and identifying peptides bound to HLA-B*57:01 using the CIR cell line but also in delineating the structural basis of the abacavir hypersensitivity allele specificity. Their more recent research has looked at the kinetics of abacavir-induced changes in the self-peptide repertoire. Such elegant experiments have the potential to provide valuable information concerning specific peptides that activate T-cells responsible for abacavir hypersensitivity. Although drugs such as carbamazepine (HLA-B*15:02 and A*31:01) and allopurinol (HLAB*58:01) interact with proteins encoded by specific HLA alleles, the ability of these drugs to alter the immunopeptidome and trigger a hypersensitivity reaction has not been demonstrated conclusively. Interestingly, Metushi et al have shown that acyclovir interacts with HLAB*57:01 in a similar manner to abacavir but does not induce hypersensitivity reactions.177 They demonstrated that the changes in peptide affinity in the presence of acyclovir were 2-529



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fold compared to the 1000-fold change observed with abacavir. Could the induction of hypersensitivity be regulated by a defined threshold of altered-self peptides? The challenge and unmet need remains the development of high throughput assays capable of screening drugs and their metabolite(s) for the potential to alter the display of self-peptides by MHC molecules.

Moving Forward: Critical questions: (1) how do the cellular components and tissue-derived signals vary in different tissues as a result of drug/chemical-induced cell stress? (2) Do these differences explain why some drugs selectively target certain organs? (2) Are there organ-specific thresholds for signals required to drive inflammatory responses (3) what is the correlation between the nature of the immune effector mechanisms and the nature/intensity of the hypersensitivity reactions induced? Critical action points: (1) there is an urgent need to develop high throughput assays capable of predicting potential HLA-associated hypersensitivity during pre-clinical development of a new drug.

Figure 1: Models of TCR activation by drug or chemical antigens. (A) The hapten hypothesis states that a drug binds to protein to form a hapten and become recognisably immunogenic. The hapten is then internalised and processed by antigen presenting cells to form antigenic peptide fragments which are subsequently loaded onto

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MHC molecules (covalent binding) and presented at the cell surface to passing T-cells. (B) The PI concept states that chemically inert parent drugs or chemicals can interact (non-covalently) directly with the MHC-TCR without the need for protein binding or antigen processing. (C) The altered peptide concept states that a drug may bind to the MHC-peptide complex in such a way that altered self-peptides represent an antigenic signal. This may refer to binding of drug (i) to HLA outside the peptide binding groove, (ii) to HLA in the peptide binding groove, or (iii) directly to the self-presented peptide. Peptide A = normal self-peptide; peptide B = altered self-peptide.

TOWARDS THE DEVELOPMENT OF PREDICTIVE ASSAYS In vitro approaches to predict drug immunogenicity A meeting was held in Rome in 2009 to discuss T-cell recognition of chemicals, protein allergens and drugs with an overall aim of moving towards the development of in vitro assays. The status of the field and outcomes of the meeting were summarized in an article by Martin et al.178 The need to develop in vitro assays utilising human cells to predict immunogenicity, and to move away from animal-based models was based on ethical and moral obligations, and the subsequent legal enforcement of restricted animal use for drug testing and the assessment of cosmetic sensitisation potential (7th amendment of the EU cosmetics directive [March 2009]). The article by Martin et al highlights key developments allowing for the emergence of ‘state-of-the-art’ T-cell assays including the utilisation of magnetic beads and flow cytometry to isolate pure cell populations, the identification of further T-cell activation markers as readouts and the identification of optimised T-cell culture conditions. Such pure T-cell populations allow for the removal of immunosuppressive Tregs which would otherwise limit the detection of effector T-cell responses.178,179 Moreover, the authors highlighted the importance of advanced in vitro culture techniques for dendritic cells as the availability of well-characterized non-tolerogenic dendritic cells is important for the detection of primary T-cell responses to drugs and chemicals. The authors proposed that functional maturation of dendritic cells by LPS and TNF-α should be performed to surpass the need for specific tissue-derived danger signals received in vivo.178 Indeed, a number of groups have now described the priming of naïve T-cells sorted from human donors to drugs and chemicals, with mature dendritic cells. It is these assays which are now being utilised and developed to investigate the intrinsic immunogenicity of drugs and chemicals. Dean Naisbitt (The University of Liverpool) discussed the priming of human naïve T-cells to drugs using cells from a bank of 1000 HLA-typed healthy volunteers. The ready supply of PBMCs from healthy donors is critical to study the immune pathogenesis of the increasing 31



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number of HLA allele-restricted forms of hypersensitivity.180-183 Naisbitt discussed the applicability domain of existing T-cell assays. It was agreed that T-cell priming assays in their current form are useful to (1) study mechanisms when a problem has been identified in the clinic and (2) investigate structurally related possibly “second-in-line compounds.” However, they could not be used to list compounds in terms of immunogenic potency simply because the sensitivity and specificity of the assay had not been validated. The failure to solubilise certain compounds in a suitable form for the T-cell assay limits its usefulness. Furthermore, the absence of known synthetic drug metabolites and/or relevant carrier proteins for reactive metabolites restricts the interpretation of a negative result with a parent drug. Despite these limitations, in vitro T-cell priming assays such as that used in Liverpool180 have greatly enhanced our understanding of the origins of drug antigen-specific T-cell activation. Focussing on the model antigen, SMX, it has been possible to study (1) mechanisms of drugspecific T-cell priming, (2) the stress-signalling pathways involved in T-cell activation and (3) immune regulation. The oxidative metabolism of SMX has been fully-characterized, and its synthetic reactive metabolite SMX-NO is commercially available for functional assays.184 SMX and SMX-NO can stimulate primed naïve T-cells to proliferate and to secrete cytokines such as IFN-γ, IL-5 and IL-13. Moreover it is possible to clone drug- and drug metabolitespecific T-cells from the priming assay and what is observed is essentially no cross-reactivity (i.e., SMX-reactive T-cells are not activated with the nitroso metabolite and vice-versa). As discussed above, it has been hypothesized that drugs which activate T-cells via a PI mechanism might preferentially activate memory T-cells. Thus, the T-cell assay has been modified to utilize purified memory T-cells from healthy donors. Somewhat surprisingly, SMX and SMX-NO were able to stimulate memory CD4+ T-cells to proliferate and secrete cytokines. Although an array of co-stimulatory and co-inhibitory proteins are expressed on the surface of DCs, it is still unclear how the signalling of individual molecules act to regulate drug-specific T-cell priming. Naisbitt partly addressed this point by showing preliminary data using specific inhibitory antibodies alone or in combination8. The stepwise inhibition of immune regulation seemed to represent a promising technique to enhance the detection of a drug-specific immune response. Finally, immune regulation might then be fed back into the priming assay to determine whether responses in individual patients are differentially regulated. The T-cell priming assay has now been applied successfully to investigate HLA allele-restricted forms of hypersensitivity with some success. For 7 drugs highlighted in Figure 2 it has been possible to prime naïve T-cells or activate pre-existing

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memory T-cells from healthy volunteers who carry HLA risk alleles. In most cases, T-cells have been cloned from the priming assay and characterized in terms of (1) antigen specificity, (2) HLA restriction, (3) cellular phenotype and (4) mechanisms of cytotoxicity.

Figure 2 Definition of the causal alleles and immune responses associated with reactions targeting skin and liver Pharmacogenetics HLA association

Abacavir hypersensitivity

HLA-B*5701

OR = 132

Dapsone Skin reaction

HLA-B*1301

OR = 20

Carbamazepine Skin reactions

HLA-B*1502

Allopurinol Skin reactions

HLA-B*5801

OR = 580

Flucloxacillin DILI

HLA-B*5701

OR = 72

Amoxicillin/ clavulanic acid DILI

HLA-A*0201 HLA-DRB1*1501

OR = 2.3 OR = 2.8

Ticlopidine DILI

HLA-DRB1*3303

OR = 13.0

(Chinese) OR = 1000 HLA-A*3101 (Caucasians) OR = 30

Detection of T-cells Patient T-cells

Volunteer T-cells

Yes

Yes

No

HLA restriction

Phenotype

Cytotoxicity

class I

CD8 only

yes

Yes

?

?

?

Yes

Yes

class I

CD8 >CD4

yes

Yes

Yes

class I

CD8 >CD4

yes

Yes

Yes

class I

CD8 >CD4

yes

Yes

Yes

class II

CD4>CD8

yes

No

Yes

class I

CD8>CD4

yes

Integration of tissue cells into in vitro approaches to predict drug immunogenicity Shaheda Ahmed (Alcyomics Limited) discussed the technologies available to the pharmaceutical, biotechnology, cosmetic and chemical industries to assess immunogenicity of drug/metabolites and chemical compounds. The use of human in vitro models such as the SkimuneTM have the ability to identify the potential for monoclonal antibodies, protein therapeutic immunodulators and small chemical molecules to induce cutaneous hypersensitivity reactions. The test compound is incubated with dendritic cells, derived from CD14+ monocytes isolated from PBMCs of a healthy volunteer. Dendritic cell exposure to antigens induces T-cell maturation, proliferation and cytokine secretion.185 Skin samples obtained from the same volunteer are then incorporated into the assay followed by histological analysis. In vitro tissue damage is graded from I-IV as described by Lerner et al186 as a marker of immune sensitisation and cytotoxicity. Data obtained from this human in vitro system correlates well with the disease and can be used to predict clinical outcomes. These data also correlates well with T-cell proliferation and cytokine release assays, which can be used as sensitive screening tools for predicting potential reactions. Ahmed stressed that that this model also has the potential to predict rare reactions during clinical trials such as 33



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the cytokine storm associated with TGN1412 IN 2006.187 However, interestingly, the SkimuneTM Test shows a positive response for drugs which show adverse reactions to be associated with a specific HLA allele. An example of this is abacavir, which despite the association of hypersensitivity reactions to the MHC class I allele HLA-B*57:01, shows a positive response in the SkimuneTM tests. This goes against clinical evidence and studies with T-cells from HLA-B*57:01 positive and negative donors and suggests HLA is not imperative to the induction of skin injury.

Moving Forward: Critical questions: (1) Why is the skin the site of most hypersensitivity reactions when most drug metabolism does not occur in the skin? (2) What are the mechanisms of viral reactivation in DRESS since this can be triggered by different drugs? (3) Is viral reactivation in DRESS critical for tissue damage? (4) How does the nature of the immune microenvironment in the skin change over time? (5) Do investigations using immune cells in the blood accurately reflect what is happening in the skin?

In vivo approaches to predict drug immunogenicity Some of the challenges associated with the development of animal models of drug hypersensitivity include incomplete understanding of the mechanisms driving the reactions, the relative roles of different mechanisms and risk factors, and the rarity of observing these reactions during early toxicity studies. Jessica Whritenour (Pfizer) and her colleagues have developed a mouse allergy model based on lymph node proliferation.188,189

The assay

involves subcutaneous administration of test compounds once daily for 3 days followed by a 2 day rest and then assessment of lymph node cellularity by flow cytometry on day 6. A stimulation index [mean total cell number (treatment group)/mean total cell number (vehicle group)] ≥ 2.5 is considered a positive response. Using positive and negative control drugs (defined as such by the occurrence of hypersensitivity reactions in the clinic), the potential of a test compound to induce hypersensitivity reactions can be assessed. Positive control drugs were shown to increase the number of immune cells (T- and B-cells) in draining lymph nodes and induce changes in the expression of the homing receptors CD62L and CCR7 on T-cells. Low bulk requirement, high throughput relative to other mouse models and an absence of radioactive endpoints are advantages of the mouse allergy model. However, the obvious 34


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limitations are lack of insight on the immunological mechanism induced by the compounds and restriction of the model to subcutaneous injection. Furthermore, Whritenour highlighted that certain compounds had a tendency to produce false positive and false negative results. While this model may be used to help de-risk backup molecules (once an issue has been identified), the translation of a positive response in the model to human risk is unknown. Therefore this assay requires further refinement and validation to prevent pharmaceutical companies from discarding potential candidate drugs at an early stage of development.

Expert opinion and conclusion In vitro T-cell priming assays are now established and used by several groups to explore mechanisms of T-cell activation by drugs/chemicals. However, at the moment, such assays cannot be used by the pharmaceutical industry to screen, ab initio, the potential that individual compounds will cause DHRs when administered widely to humans. One of the critical action points discussed at the meeting highlighted the urgent need to develop high throughput assays capable of predicting potential HLA-associated DHRs. However, the demands of adapting an in vitro system used in a research setting into a screening assay for industry are considerable. Following this workshop, it is believed that a number of groups have made important advances in their assays to investigate drug hypersensitivity, which will potentially form part of the agenda for the next meeting. At the CDSS we have challenged ourselves to identify screening strategies where we can use an established biobank of PBMCs isolated from 1000 HLA-typed volunteers. The current form of the T-cell priming assay can only look at a single HLA risk allele, is labour intensive, uses different plate formats, has multiple readouts and takes 3-4 weeks to run. A screening assay would need to include multiple donors with and without several different HLA risk alleles and be simplified to run on a single plate format using a single readout. In a screening assay for HLA Class I risk alleles, we could potentially include 3 donors with B*13:01, 2 donors with A*33:03 and B*15:02 and a minimum of 5 donors for the remaining alleles (A*02:06, A*31:01, A*68:01, B*35:05, B*44:03, B*56:02, B*57:01, B*58:01 and C*04:01). Five donors without the HLA risk alleles would then be selected as negative controls and a unique donor would be used as a positive control. We are currently developing this screening assay and our initial efforts have concentrated on optimising assay conditions using SMX-NO as a test compound. Next the sensitivity of the assay will be explored using blocking antibodies against co-inhibitory molecules of T-cell

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activation such as CTLA4, PD-1 and Tim-3 before testing the screening assay with a set of test compounds known to cause hypersensitivity.

Obviously, there remains a need to foster collaboration between the pharmaceutical industry, academia and healthcare professionals to devise well thought out experiments with the potential to enhance our understanding of the molecular mechanisms underlying DHRs. This workshop provided an excellent opportunity for researchers with varying interests to assess the current status of methods to predict drug hypersensitivity in humans. Substantial advances in our understanding of mechanisms of the iatrogenic disease have been made in recent years; however, knowledge gaps still exist, most especially concerning the role of HLA class II risk alleles in drug-induced skin and liver injury. Addressing this and other important unmet needs mentioned in this report requires access to patient samples and a panel of experiments designed around the drug antigen, environmental, disease and patient-specific factors.

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AUTHOR INFORMATION Corresponding Author *Telephone: 0044 151 7945346. Fax: 0044 151 7945540. Email: [email protected] Funding MOO, LF, NF, AG, XM, AN, MP, BKP and DJN are supported by grants from the Medical Research Council (grant number MR/L006758/1) and the European Community under the Innovative Medicine Initiative project MIP-DILI [grant agreement number 115336]. MOO is an AstraZeneca postdoctoral research fellow. AWP is a Fellow of the Australian National Health and Medical Research Council. GMH is supported by the National Institute of Health Research Birmingham Liver Biomedical Research Unit.

This paper presents independent research supported by the

National Institute for Health Research (NIHR) Birmingham Liver Biomedical Research Unit (BRU). The views expressed are those of the author and not necessarily those of the NHS, the NIHR or the Department of Health.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors would like to thank the audience of the workshop who provided valuable comments and suggestions. Thanks are also extended to Audrey Nosbaum (Lyon, France) who provided slides to discuss at the workshop, but unfortunately could not attend. ABBREVIATIONS

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ADRs, adverse drug reactions; DHR, drug hypersensitivity reactions; DILI, drug-induced liver injury; SJS, Stevens-Johnson syndrome; TEN, toxic epidermal necrolysis.

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Author Biographies Dr Andrew Gibson, received his PhD from the Department of Molecular and Clinical Pharmacology at the University of Liverpool in 2016 for his work developing in vitro human T-cell assays to characterise drug-specific T-cell activation and further elucidating the role of co-inhibitory T-cell pathways during these responses. He is currently a postdoctoral researcher, based at the University of Liverpool, investigating the role of T-cells in hypersensitivity reactions to drug antigens. Dean J Naisbitt, is a Reader of Pharmacology at the MRC Centre for Drug Safety Science, The University of Liverpool (UK). His research combines aspects of genetics, cell biology and chemistry in order to study the fundamental principles of immunological drug reactions. Dr Naisbitt has been recognized with several honours and awards for research. Awards of note include the ISSX New Investigator Award (2009), the British Pharmacological Society Novartis prize for excellence in research (2012) and the American Chemical Society CRT Young Investigator Award in 2013. He serves on the Editorial Advisory Board of Chemical Research in Toxicology. Stefan F. Martin is Associate Professor for Immunobiology and head of the Allergy Research Group in the Department of Dermatology at the Medical Center – University of Freiburg. His work focusses on the understanding of mechanisms underlying adverse reactions to chemicals, particularly irritant and allergic contact dermatitis. Cellular and molecular immune as well as stress responses are investigated. The aim of the work is the detailed mechanistic understanding of cellular and molecular responses to chemicals, the improvement of treatment and diagnosis of contact dermatitis and the development of mechanism-based in vitro assays to replace animal testing for contact allergen identification. Dr Shaheda Ahmed completed her PhD in Clinical Medical Sciences at Newcastle University (UK) in 2006. She worked as a Research Associate within the field of bone marrow transplant biology before making a successful transition from academia to industry. Her work has involved development of a human in vitro skin explant model for testing adverse events of pharma, cosmetic and chemical substances as alternatives to the use of animal models. More recently she has developed 3D skin models and contributes to the growth and expansion of a start-up life sciences company where she currently holds the position of Senior Scientist. Dr Catherine Betts completed a PhD in parasitology at the University of York and joined the Department of Immunology at the University of Manchester as a Welcome 5yr post doc looking at the immune response in resistant and susceptible mice upon parasitic infection. In 2001 she moved to Syngenta Agrochemicals at the Central Toxicology Laboratory to investigate contact and respiratory sensitisation to chemicals and models of food allergy. Since 2007 she has run an immunotoxicology team within the Safety department of AstraZeneca investigating adverse immune drug reactions. Anne Dickinson is Professor of Marrow Transplant Biology in Haematological Sciences, Institute of Cellular Medicine and Director of the Newcastle Cellular Therapies Facility Newcastle University, UK. Professor Dickinson founded Alcyomics Ltd based on over 20 years experience with a human in vitro skin explant model for predicting graft-versus-host disease, a severe allo-immune complication of haematopoietic stem cell transplantation. This skin model ( Skimune™) is now used commercially to predict allergy and sensitisation to chemicals and therapeutic monoclonal antibodies and drugs. 51



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Dr Neil French is Senior Scientific Operations Manager for the MRC Centre for Drug Safety Science at the University of Liverpool. He has responsibility for all operational activities in the MRC Centre, including oversight of industry interactions and major grant applications. Previously, he worked for a biotechnology company (Renovo) in Manchester, developing the company’s bioinformatics resources and led a team of scientists with responsibility to prioritise lead candidates. Dr French has a PhD in Molecular Oncology from the Paterson Institute for Cancer Research at the University of Manchester and has 4 years postdoctoral research experience at the Karolinska Institute, Sweden. Dr Gideon Hirschfield is a clinician and academic scientist with a special interest in hepatology, including in particular autoimmune liver disease, as well as the conduct of clinical trials in liver disease generally. He is well published scientifically and clinically in his field having made important contributions to the understanding and clinical management of diseases such as primary biliary cholangitis/cirrhosis, primary sclerosing cholangitis and autoimmune hepatitis. His overarching goal is to continue the translation of scientific advances towards new rational therapies for patients with chronic inflammatory liver diseases. Dr Ana Alferivic is a Senior Lecturer at the MRC Centre for Drug Safety Science, The University of Liverpool. Her research interests include: systems stems pharmacology approaches to drug-induced hypersensitivity reactions, personalisation of treatment in cardiovascular disease through next generation sequencing in adverse drug reactions and identification of genetic markers for drug-induced hypersensitivity syndromes. Dr Michael Kammüller received his PhD in toxicology and immunology from the University of Utrecht (Netherlands) and was a visiting scientist at The Scripps Research Institute in La Jolla (USA). He is heading the immunology group in Translational Medicine – Preclinical Safety at the Novartis Institutes for Biomedical Research in Basel (Switzerland). His work focusses on investigating mechanisms of immune disregulation at molecular, cellular and in vivo levels to understand adverse effects of low molecular weight and biotherapeutic drugs on immune system homeostasis, in particular regarding drug-induced hypersensitivities, autoimmune disorders and more recently risk for infections. Dr Xiaoli Meng is a postdoctoral research fellow currently working in the MRC Centre of Drug Safety Science at the University of Liverpool. Xiaoli received her PhD in medicinal chemistry from the University of Liverpool, with a focus on the synthesis of reactive drug metabolites. Her research primarily focuses on understanding the mechanisms of adverse drug reactions by defining the relationship between metabolism and toxicity. She specializes in mass spectrometric characterization of covalent binding and immune-mediated adverse drug reactions. Philippe Musette MD, PhD is professor of Dermatology at Rouen University Hospital and is the director of a team focussed on immunodermatology and rheumatology at the INSERM. His research topics include the role of viral reactivation in severe cutaneous side effects and the immune mechanisms of auto-immune bullous diseases and he conducts innovative clinical trials in immunodermatology. Dr Alan Norris, a pharmacologist, is the Industry Programme Manager for the MRC Centre for Drug Safety Science with responsibility for managing interactions with industry partners. 52


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Prior to this he was Director for Strategic Alliances at Novartis Institutes for Biomedical Sciences with responsibility for all global business development activities for the company in the gastrointestinal and respiratory fields from pre-clinical stage to proof of concept in man. Dr Lee Faulkner is a post-doctoral researcher at the MRC Centre for Drug Safety Science, The University of Liverpool. Her current research interest is the development of T-cell assays to predict the immunogenicity of drugs and chemicals. Previously, she has worked on the role of T cells in toxic shock and nasopharyngeal carcinoma. Dr Monday Ogese graduated with bachelors in Pharmacy from the University of Jos, Nigeria in 2005. In 2009, he enrolled for his postgraduate studies at the University of Liverpool where he obtained a Masters of Research and PhD in Pharmacology. His PhD research focused on “antigenic determinants of drug hypersensitivity reactions”. He is currently a postdoctoral fellow with AstraZeneca, UK, and his research interest revolves around understanding the molecular pathomechanism of adverse drug reactions targeting the liver. Kevin Park is currently Professor of Pharmacology and Head of the Institute of Translational Medicine at the University of Liverpool. He is a Fellow of the Royal College of Physicians and a Fellow of the Academy of Medical Sciences. The over-riding theme of his work is bringing “molecule–to-man” and back again, to enable the prediction of adverse drug reactions based on the chemical structure of the drug and the identification of susceptible individuals. This work has been expanded by using pharmacogenomics and toxicogenomics to link findings in patients to the chemical structure of the drug. Professor Sir Munir Pirmohamed is currently David Weatherall Chair in Medicine at the University of Liverpool, and a Consultant Physician at the Royal Liverpool University Hospital. He is also the Associate Executive Pro Vice Chancellor for Clinical Research for the Faculty of Health and Life Sciences. He holds the only NHS Chair of Pharmacogenetics in the UK, and is Director of the MRC Centre for Drug Safety Sciences, Director of the Wolfson Centre for Personalised Medicine and Executive Director, Liverpool Health Partners. His research focuses on personalised medicine in order to optimise drug efficacy and minimise toxicity. Tony Purcell is a Professorial Fellow and Deputy Head Department of Biochemistry at Monash University. He is currently an executive committee member of the Australasian Proteomics Society and the Australasian Peptide Society and was recently elected to the Human Proteome Organisation Council. His laboratory focusses on how the diverse array of peptides presented to the immune system, the immunopeptidome, is influenced by infection, inflammation and the environment. He has made important contributions to understanding the role of antigen presentation, including the characterizing peptide epitopes involved in T cell recognition, in several autoimmune diseases, drug hypersensitivity, cancer and infectious diseases. Dr Colin Spraggs (Target Sciences, GlaxoSmithKline, UK) has broad experience in drug discovery and development as a pharmacologist, clinical development leader and geneticist. His recent work has utilised pharmacogenetics to identify and characterise the association of a specific HLA allele (DRB1*07:01) with liver injury caused by lapatinib and resulted in validated HLA biomarker data inclusion in the Tykerb/Tyverb product label. His current research utilises human genomic data to identify and select novel disease targets as potential medicines.

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Dr Jessica Whritenour is a Senior Principal Scientist in the Immunotoxicology group within the department of Drug Safety Research and Development at Pfizer Inc. She has a special interest in understanding mechanisms of drug hypersensitivity reactions, and in the development of predictive and diagnostic tools that can be used during drug development.

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New Approaches to Investigate Drug-Induced Hypersensitivity

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