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*

 

1Pathology Sciences, Drug Safety and Metabolism, AstraZeneca R&D, Darwin Building 310,

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

2MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology,

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

3The Wolfson Centre for Personalised Medicine, Department of Molecular and Clinical

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

4Alcyomics Ltd c/o Haematological Sciences, Institute of Cellular Medicine, Newcastle

University Newcastle upon Tyne UK NE2 4HH, UK

5Centre for Liver Research, NIHR Birmingham Liver Biomedical Research Unit, Institute of

Immunology and Immunotherapy, University of Birmingham, Edgbaston, Birmingham, B15

2TT, UK

6Novartis Institutes for Biomedical Research, Klybeckstrasse 141, CH-4057, Basel,

Switzerland

7Department of Dermatology and Venereology, Allergy Research Group, University of

Freiburg, Hauptstraße 7, 79104 Freiburg, Germany

8Department of Dermatology and INSERM, University of Rouen, 905 Rouen, France

9Infection and Immunity Program and Department of Biochemistry and Molecular Biology,

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

1

10GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY,

UK

11Pfizer, 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: dnes@liv.ac.uk

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TOC graphic

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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

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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), CTLA-4 (Cytotoxic T-

Lymphocyte-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

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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, pre-

prescription genotyping of patients for the risk allele HLA-B*57:01 prior to administration of

abacavir has significantly reduced the incidence of hypersensitivity reactions. This concept

has also been recommended for HLA-B*15:02 (Chinese) and HLA-A*31:01 (Caucasians)

before carbamazepine prescription. 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 (>5-

10%) or at a lower incidence (<0.01%)?

5. Is the evaluation of drug-antigen formation a useful approach? If yes, what type of

approach should be used? If no, why is this approach not useful?

6. Do drugs with the potential to elicit hypersensitivity reactions have particular chemical or

pharmacological properties in common?

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7. Can in vitro models be used to identify specific HLA associations where clinical genetic

studies are not able?

THE VIEWPOINT FROM INDUSTRY

Hypersensitivity issues faced by industry during drug development

Jessica Whritenour (Pfizer) opened the meeting by providing an industry perspective on the

impact of DHRs in drug development. She highlighted potential issues faced by

pharmaceutical companies during the different stages of drug development and the impacts of

these challenges. Although the potential issues related to DHRs could be different depending

on the development phase, the lack of predictive and diagnostic tools to aid in the

identification and management of the risk is a common gap at all stages of drug development.

For example, during nonclinical development, predictive tools are not available for

prospective risk assessment to identify molecules that, based on chemical structure, may have

the potential to cause DHRs. If a DHR is observed during toxicity studies (rare), tools are not

routinely available to investigate the mechanism for the observed reaction in the toxicity

species or to screen/de-risk back-up compounds to try and identify a compound without a

potential DHR liability. As a compound moves into early clinical development, a potential

issue that may be encountered is the occurrence of mild skin rash in a relatively high

percentage of patients. In this case, understanding the mechanism for the reactions (allergic

or non-allergic), whether the reactions will increase in severity with longer duration of

treatment, and whether the reactions will be observed in phase II/III studies at doses that did

not produce reactions in phase I studies are important considerations in risk management.

During phase III/commercialisation, a potential issue that currently is impossible to predict is

the rare but severe reaction without an identifiable risk factor. When such an issue is

encountered, diagnosis and confirmation that the reaction is related to the study drug are key

considerations for managing the risk and progressing with the development of the drug.

Unfortunately, there are limited (if any) tools to address the majority of these

questions/concerns highlighted above.

The potential issues discussed above underscore unmet needs in this area of predictive and

diagnostic drug safety science. Hence, there is an absolute need to establish novel tools to

help identify and manage risks. An important issue surrounding predictive testing is the

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suitability of a particular assay to address a given question. For example, does the assay

developed to assess the formation of reactive intermediates accurately predict the

development of drug hypersensitivity? Do we need to incorporate HLA alleles into in vitro/in

vivo predictive assays? What is the level of validation (i.e., the specificity and selectivity) of

an assay that needs to be established, with known positive and negative controls?

DEFINING THE CLINICAL PROBLEM

Phenotypic assessment of drug hypersensitivity and HLA alleles as genetic risk factors

Munir Pirmohamed (The University of Liverpool; director of the CDSS) gave an insight into

the various definitions associated with DHRs. He stressed the importance of consistent

terminology and of using the European Network on Drug Allergy’s (ENDA) definition of

drug hypersensitivity. ENDA defines drug hypersensitivity as objectively reproducible

symptoms or signs initiated by exposure to a drug at a dose normally tolerated by non-

hypersensitive persons. Drug allergy on the other hand refers to immunologically mediated

DHRs. Hence non-allergic hypersensitivity reactions are not necessarily immune-mediated.

DHRs can be broadly classified into two sub-groups: (1) immediate hypersensitivity or Type

I reactions that are IgE-mediated. Not much is known about any genetic predisposition to

these reactions. (2) Delayed hypersensitivity reactions that are mainly Type IV reactions

according to Coombs and Gell but may sometimes include Type III phenotypes.16 These

reactions are T-cell-mediated and have variable clinical manifestations. The epidemiology of

drug hypersensitivity is complicated by the number of drugs that cause reactions and the

difficulties encountered in accurate diagnosis. Thus, a systematic approach is needed to

identify how often reactions to specific drugs occur in a given population.

The risk factors for DHRs include: high mass dose, route of administration, sex, viral

infections and genetic factors. To date, approximately 25 HLA-associated ADRs have been

identified (Table 1), most of them reaching genome wide significance. The discovery of

strong associations between DHRs and particular HLA alleles further implicates MHC-

restricted T-cell responses in disease aetiology as the primary role of the proteins encoded by

HLA is to effectively present antigenic peptides to passing T-cells. The successful interaction

between HLA molecule, peptide, and corresponding T-cell receptor (TCR) is referred to as

the ‘immunological synapse’ and acts as signal one for the successful activation of T-cells. 17

Exactly how HLA-peptide binding mediates TCR triggering, and thus stimulation of a T-cell

signalling cascade, currently remains unresolved despite the proposal of multiple models

categorised as aggregation, segregation, and conformational alteration.18 The TCR interaction

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is sensitive and stimulation requires high avidity to antigenic HLA-(drug) peptide complexes,

which are often present at very low concentrations and thus potentially difficult to detect

amongst the more abundantly presented self-peptides. Such high affinity binding overcomes

an activation threshold, while self-peptides provide a suboptimal response and trigger a

negative feedback loop whereby the tyrosine phosphatase SHP-1 is recruited to inactivate

further T-cell signalling.18-20

Table 1: HLA-associated drug hypersensitivity and target organs affected.

A. HLA-associated hypersensitivity targeting the skin

Causative drug Risk HLA allele

Carbamazepine A*31:01

Lamotrigine A*68:01

Cold medicines A*02:06

DapsoneTrichloroethylene

B*13:01

CarbamazepinePhenytoin

B*15:02

Cold medicines B*44:03

Nevirapine B*35:05

Phenytoin B*56:02

Abacavir B*57:01

Allopurinol B*58:01

Nevirapine C*04:01

B. HLA-associated hypersensitivity targeting the liver

Causative drug Risk HLA allele

Ticlopidine A*33:03

Flucloxacillin B*57:01

Pazopanib B*57:01

XimelagatranLapatinib

DRB1*07:01

LumiracoxibCo-amoxiclav

DRB1*15:01

Lumiracoxib DQA1*01:02

Lapatinib DQA1*02:01

XimelagatranClometacin

DQB1*02:01

Co-amoxiclavLumiracoxib

DQB1*06:02

Ticlopidine DQB1*06:04

C. HLA-associated hypersensitivity targeting other organ

Causative drug Risk HLA allele

Statins DRB1*11:01

Aspirin DRB1*13:02

Clozapine DQB1*05:02

Aspirin DQB1*06:09

To account for the endless array of encountered non-self-antigens, the HLA locus is the most

polymorphic locus in the human body leading to a broad range of genetic diversity. HLA

alleles are subdivided into two major gene classes, defined by the type of antigen that each

presents, major histocompatibility complex (MHC) class I and II. MHC class I, encoded by

HLA class I alleles, is expressed on all nucleated cells and presents intracellular antigenic

peptides loaded in the endoplasmic reticulum, and mostly made up of the peptide remnants of

proteasome-mediated protein degradation, to CD8+T-cells. In contrast, MHC class II

expression is mainly restricted to professional antigen presenting cells. As antigen-loading

occurs within the endocytic pathway whereby antigens are derived from internalised proteins,

MHC class II molecules present peptides derived from extracellular proteins to CD4+T-cells. 21-23 These seemingly distinct pathways overlap in a process termed cross-presentation

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whereby HLA class I may present extracellular antigens to allow the development of a

cytotoxic T-cell response to exogenous antigens as part of the priming pathway for the

eradication of tumours and viruses. Similarly, endogenous antigens may be presented on

HLA class II when they traverse the secretory pathways of the cell or are produced under

specific circumstances such as autophagy. When discussing the mechanisms by which drugs

activate T-cells, one must consider these immunological processes in the context of

therapeutic drug exposure. It is highly likely different pathways of drug antigen-specific T-

cell activation occur in vitro when cells are sometimes exposed to millimolar drug

concentrations.

Both MHC class I and II are further subdivided with HLA class I consisting of HLA-A,

HLA-B, and HLA-C genes as well as the less abundant non-classical HLA-E, HLA-F, and

HLA-G, whilst HLA class II is made up of proteins deriving from HLA-DR, HLA-DP, and

HLA-DQ genes. A third set termed MHC class III represent 57-60 non-classical HLA genes

of which many do not play a direct role in antigen presentation.

The potential for inter-individual genetic variation in the development of hypersensitivity was

first indicated clinically after a pair of monozygotic twins developed carbamazepine-induced

hypersensitivity, postulating some familial genetic predisposition.27 Since then, the highly

polymorphic HLA locus has been associated with DHRs through the identification of

numerous different HLA allele associations with T-cell responses to different drugs, in both

the skin and liver. The best documented HLA association with DHRs is that of the reverse

transcriptase inhibitor abacavir (ABC) and HLA-B*57:01 first reported in 2002,28 which

remains the paradigm for the application of HLA genetic testing to clinical drug therapy in

order to reduce the incidence of hypersensitivity. Currently, the only other drug for which

administration requires the patient to be HLA-typed is carbamazepine (CBZ), a commonly

prescribed antiepileptic medication which causes SJS/TEN associated with HLA-B*15:02.

This genetic test is highly clinically informative, as it was found to have a 100% sensitivity,

and a specificity of 97%.29

Despite this, most HLA associations with DHRs have much lower prognostic values and thus

the expression of a specific HLA-allele is often not reason enough to withhold a potentially

lifesaving drug. For instance, HLA-B*57:01, the allele associated with ABC-hypersensitivity,

is also associated with flucloxacillin-induced DILI. However, in comparison to ABC, just 1

in 500-1000 HLA-B*57:01+ individuals who receive flucloxacillin will develop an adverse

reaction.30 Moreover, the binding of flucloxacillin-derived antigen to MHC molecules is

much less stringent. Specifically, binding of flucloxacillin to closely related alleles of the B17

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serological family provides antigenic determinants that activate T-cells. This does not occur

with abacavir.31 Members of the B17 family including HLA-B*57:01, HLA-B*58:01, HLA-

B*58:02, HLA-B*57:02, and HLA-B*57:03, share > 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.

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 N-

acetylation 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 SMX-

derived 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, 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

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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. Differential expression or activity may affect the

ability of an individual to reduce SMX. Furthermore, promoting the formation of SMX-

hydroxylamine 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 SMX-

NO.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 metabolite-

specific 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

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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 sulfasalazine-

induced liver injury showed an increase in T-cells secreting the cytolytic molecule granzyme

B, which suggests that T-cells might play a direct role in the disease pathogenesis. In contrast

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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.

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. 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 F-

pocket 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.

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 HLA-B*57:01 risk

allele and disease.

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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. 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-HLA-

DQB1*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 HLA-

DQA1*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 above-

mentioned 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

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. 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.

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 patients. To date

17

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)

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?

18

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. 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. A number of studies have implicated CD4+ T-cells in the pathogenesis

of milder drug reactions. However, it is still not clear whether these CD4+ cells are activated

to secrete the same effector molecules as cytotoxic CD8+ T-cells 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,

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

19

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 over-

the-counter drugs such as acetaminophen. 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. 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

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

20

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..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 drug-

specific 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

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

21

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. 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 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,

22

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 T-

cell 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.

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

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

23

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. 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 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.

24

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. Dead cells are known to release intracellular stores of uric acid, which promotes

an acute inflammatory response. 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 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

25

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 T-

cells, 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. Organic contact allergens like 2, 4, 6-

trinitrochlorobenezene (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. Receptor-

ligand 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.

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

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

26

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 drug-

induced allergic hepatitis in animal models.150-152 Specifically, the amadiaquine-, isoniazid-

and 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 self-

peptide 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 pro-

hapten 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.

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

27

have been identified as targets for numerous drugs (metabolites) including β-lactam

antibiotics, 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, in tissues (skin and liver), 164-166 and in

patients taking therapeutic drugs. 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

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

28

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+ T-

cell mediated since abacavir-specific CD8+ T-cells have been isolated from hypersensitive

patients and generated from drug-naïve donors expressing HLA-B*57:01. 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 non-covalently

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 (HLA-

B*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 HLA-

B*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 2-5-

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.

29

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

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

30

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. 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

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.”

31

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 drug-

specific 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-, IL-5 and IL-13. Moreover it is possible to clone drug- and drug metabolite-

specific 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

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.

32

Figure 2 Definition of the causal alleles and immune responses associated with reactions

targeting skin and liver

Abacavirhypersensitivity

DapsoneSkin reaction

CarbamazepineSkin reactions

AllopurinolSkin reactions

FlucloxacillinDILI

Amoxicillin/clavulanic acid DILI

TiclopidineDILI

Detection of T-cells

Yes Yes class I CD8 only yes

Patient Volunteer T-cells T-cells HLA restriction Phenotype Cytotoxicity

No 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

Pharmacogenetics

HLA-B*5701 OR = 132

HLA-B*1301 OR = 20

HLA-B*1502 (Chinese) OR = 1000HLA-A*3101 (Caucasians) OR = 30

HLA-B*5801 OR = 580

HLA-B*5701 OR = 72

HLA-A*0201 OR = 2.3HLA-DRB1*1501 OR = 2.8

HLA-DRB1*3303 OR = 13.0

HLA association

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

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

33

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. 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

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

34

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

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

35

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.

36

AUTHOR INFORMATION

Corresponding Author

*Telephone: 0044 151 7945346. Fax: 0044 151 7945540. Email: dnes@liv.ac.uk

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

37

ADRs, adverse drug reactions; DHR, drug hypersensitivity reactions; DILI, drug-induced

liver injury; SJS, Stevens-Johnson syndrome; TEN, toxic epidermal necrolysis.

38

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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.

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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.

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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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