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TRANSCRIPT
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
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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: [email protected]
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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: [email protected]
Funding
MOO, LF, NF, AG, XM, AN, MP, BKP and DJN are supported by grants from the Medical
Research Council (grant number MR/L006758/1) and the European Community under the
Innovative Medicine Initiative project MIP-DILI [grant agreement number 115336]. MOO is
an AstraZeneca postdoctoral research fellow.
AWP is a Fellow of the Australian National Health and Medical Research Council.
GMH is supported by the National Institute of Health Research Birmingham Liver
Biomedical Research Unit. This paper presents independent research supported by the
National Institute for Health Research (NIHR) Birmingham Liver Biomedical Research Unit
(BRU). The views expressed are those of the author and not necessarily those of the NHS, the
NIHR or the Department of Health.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors would like to thank the audience of the workshop who provided valuable
comments and suggestions. Thanks are also extended to Audrey Nosbaum (Lyon, France)
who provided slides to discuss at the workshop, but unfortunately could not attend.
ABBREVIATIONS
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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