How Reactive Metabolites Induce an Immune Response That

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How Reactive Metabolites Induce an Immune Response that Sometimes Leads to an Idiosyncratic Drug Reaction Tiffany Elizabeth Cho, and Jack Uetrecht Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00357 • Publication Date (Web): 24 Oct 2016 Downloaded from http://pubs.acs.org on October 25, 2016

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Chemical Research in Toxicology

How Reactive Metabolites Induce an Immune Response that Sometimes Leads to an Idiosyncratic Drug Reaction

Tiffany Cho and Jack Uetrecht*

Faculty of Pharmacy, University of Toronto, Toronto, Canada M5S 3M2

Corresponding Author: Jack Uetrecht E-mail address [email protected] Telephone # 416 9788939

Key words: idiosyncratic drug reactions, liver injury, danger, immune tolerance, inflammasomes, mitochondria, bile salt exporter protein, genetic polymorphisms, heterologous immunity

ACS Paragon Plus Environment


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ABSTRACT Little is known with certainty about the mechanisms of idiosyncratic drug reactions (IDRs); however, there is substantive evidence that reactive metabolites are involved in most, but not all, IDRs. In addition, evidence also suggests that most IDRs are immune mediated. That raises the question of how reactive metabolites induce an immune response that can lead to an IDR. The dominant hypotheses are the hapten and danger hypotheses. These are complementary hypotheses: a reactive metabolite can act as a hapten to produce neoantigens, and it can also cause cell damage leading to the release of danger-associated molecular pattern molecules that activate antigen presenting cells. Both are required for an immune response. In addition, drugs may induce an immune response through inflammasome activation. We have found examples in which the ability to activate inflammasomes differentiated drugs that cause IDRs from similar drugs that do not. There are other hypotheses that do not involve an immune mechanism such as mitochondrial injury and bile salt export pump (BSEP) inhibition. With some possible exception, these hypotheses are unlikely to be able to completely explain IDRs. However, some types of mitochondrial injury or BSEP inhibition could produce danger signals. The major mechanism that protects us from IDRs appears to be immune tolerance. Consistent with this hypothesis, we used checkpoint inhibition to develop the first animal model of idiosyncratic drug-induced liver injury that has the same characteristics as the idiosyncratic injury in humans. This was accomplished by treating Pd-1-/- mice with anti-CTLA-4 antibodies and amodiaquine. The combination of the Pd-1-/- mouse and anti-CTLA-4 also unmasks the ability of other drugs such as isoniazid to cause delayed type liver injury. This model should allow rigorous testing of mechanistic hypotheses that was impossible in the past.


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BACKGROUND

At the time of the inauguration of Chemical Research in Toxicology (CRT), significant progress had been made in establishing a role for reactive metabolites in the mechanism of many adverse drug reactions. Pioneering work performed at the National Institutes of Health more than a decade earlier under the guidance of B.B. Brodie and then Jim Gillette produced conclusive evidence for the involvement of a reactive metabolite in the mechanism of acetaminopheninduced liver injury.1 There were also studies of bioactivation of other drugs that caused liver injury such as halothane2 and isoniazid.3 The liver injury caused by acetaminophen was easily reproducible in mice, and this led to literally thousands of studies into the mechanism of acetaminophen-induced liver injury. The development of atmospheric pressure ionization mass spectrometry just prior to the inauguration of CRT greatly facilitated the study of drug metabolism, and studies showed that most drugs that caused a relatively high incidence of liver injury also formed reactive metabolites;4 however, unlike acetaminophen, treatment of animals with drugs that cause idiosyncratic liver injury in humans did not lead to the characteristic liver injury observed in humans. Many of the features of idiosyncratic drug reactions (IDRs) suggested an immune mechanism;5 however, without valid animal models, rigorous studies to test immune mechanisms were virtually impossible. In fact, even though there was a large amount of circumstantial evidence to suggest most IDRs are caused by reactive metabolites, without a valid animal model, even the involvement of a reactive metabolite is not certain. Furthermore, even for the IDRs caused by drugs that readily form reactive metabolites such as sulfamethoxazole, which is a primary aromatic amine - a notorious structural alert - it was proposed that these IDRs did not involve a reactive metabolite, but rather a reversible interaction


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between the parent drug and the major histocompatibility complex – T cell receptor (MHC – TCR). This is the so-called pharmacological interaction (p-i) hypothesis.6 In addition, many drugs form several reactive metabolites, and there was no method to determine which reactive metabolite, if any, was responsible for a given IDR. For example, the relevance of acyl glucuronides to IDRs is controversial because most drugs such as diclofenac that form reactive acyl glucuronides also form oxidative reactive metabolites. Although progress has been made in the last 30 years in the understanding of IDRs, our mechanistic understanding remains superficial. Until recently, there continued to be a lack of valid animal models to test mechanistic hypotheses. Given the lack of definitive evidence for the mechanism of IDRs, it is important to obtain clues from the clinical characteristics of IDRs, and it is essential that any hypothesis be consistent with these characteristics.

IDR CLINICAL CHARACTERISTICS

There is no perfect definition of an IDR. Idiosyncratic means “specific to an individual”, and in general, IDRs are unpredictable adverse reactions that, with the exception of immune modulators, probably do not involve the therapeutic effect of the drug. With very rare exceptions, there is a delay between starting a drug and the onset of the IDR on first exposure, but classically there is a much more rapid onset of the IDR if a patient is rechallenged with a drug that has previously caused an IDR in a specific patient. IDRs can affect any organ, but the liver, skin, and blood cells are the most common targets. If a drug causes a severe IDR, it virtually always causes a higher incidence of mild IDRs of the same type, and these mild IDRs often resolve despite continued treatment with the drug.5 The same drug often causes several


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types of IDRs. For example, amodiaquine can cause liver injury in one patient and agranulocytosis in another, while nevirapine can cause a serious skin rash in one patient and liver injury in another. This presumably reflects where the reactive metabolite is formed. In the case of amodiaquine, it is oxidized to a reactive iminoquinone in both the liver and by myeloperoxidase in neutrophils. While in the case of nevirapine, it forms a reactive benzylic sulfate in the skin and a reactive quinone methide in the liver. These characteristics and those described in the following sections provide mechanistic clues.

INVOLVEMENT OF REACTIVE METABOLITES

As mentioned, there is a large amount of circumstantial evidence that most IDRs are caused by reactive metabolites rather than the parent drug unless the drug has intrinsic chemical reactivity.7,

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However, with very few exceptions, there is no direct evidence that IDRs are

caused by reactive metabolites. The fact that the liver is a frequent target of IDRs is presumably because of the high capacity of the liver to metabolize drugs, including the formation of reactive metabolites. One exception is penicillin-induced anaphylaxis. Anaphylaxis is mediated by IgE antibodies, and in the case of penicillin, these IgE antibodies recognize penicillin-modified proteins.9 Therefore, it is clear that the chemical reactivity and covalent binding of penicillin is involved in the mechanism of penicillin-induced anaphylaxis. More recently, it was shown in a rat model that a nevirapine-induced skin rash is caused by a chemically reactive benzylic sulfate formed in the skin because application of a topical sulfotransferase inhibitor prevented covalent binding of the drug and the manifestation of a skin rash where it was applied.10 This also provides very strong evidence for the involvement of a specific reactive metabolite in the


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mechanism of this rash. It has been shown that there is a correlation between the amount of covalent binding to hepatocytes multiplied by the daily dose of the drug and the risk that a drug will cause idiosyncratic liver injury; this is referred to as the total body burden of a reactive metabolite.4 However, there are drugs such as ethacrynic acid that are chemically reactive and can covalently bind to proteins, but virtually never cause IDRs. In addition, there are drugs such as allopurinol and pyrazinamide that cause serious IDRs but do not appear to form a reactive metabolite. Therefore, the formation of a reactive metabolite is considered a liability in a drug candidate, but the relationship between the formation of a reactive metabolite and IDR risk is far from perfect.

INVOLVEMENT OF THE IMMUNE SYSTEM IN THE MECHANISM OF IDRS

The clinical characteristics of IDRs are most easily explained by an immune mechanism. However, as with the involvement of reactive metabolites, definitive evidence for an immune mechanism is lacking with the exception of a few IDRs. As mentioned, one type of an IDR that is clearly immune mediated is ß-lactam-induced anaphylaxis, which is mediated by IgE antibodies that recognize ß-lactam-modified proteins. Another type of IDR that is clearly immune mediated is autoimmune reactions.11 Autoimmune IDRs can take the form of generalized autoimmunity that resembles idiopathic systemic lupus erythematosus, or they can involve a single target such as minocycline-induced autoimmune hepatitis or α-methyl dopainduced autoimmune hemolytic anemia. Although definitive evidence for an immune mechanism is absent for most IDRs, even the idiosyncratic nature of IDRs is most easily explained by an immune mechanism. It is common for other immune mediated reactions such as allergies to be


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idiosyncratic and difficult to reproduce in animals. As mentioned, with very few exceptions, there is a lag between first exposure to a drug and the onset of an IDR. This is typical of an immune response, at least in part because it requires some time for the specific adaptive immune cells involved to proliferate to sufficient numbers to cause a clinical response. As mentioned, IDRs classically occur rapidly on rechallenge, another characteristic of an immune response. However, there are many exceptions in which rechallenge leads to the same adverse reaction, but it takes just as long as on the first exposure, or it does not recur at all. For example, rechallenge of patients with a history of clozapine-induced agranulocytosis usually, but not always, results in recurrence, and the time to onset is usually shorter than on first exposure; however, it still usually takes more than a month of treatment before the onset of agranulocytosis.12 In the case of heparin-induced thrombocytopenia, which is mediated by antibodies against the heparin-platelet factor 4 complex, rechallenge does not usually result in recurrence.13 Some IDRs are accompanied by fever and eosinophilia, characteristics that are typical of some types of immune responses, but the absence of such features is by no means evidence against an immune mechanism. Anti-drug antibodies have been detected in the IDRs caused by some drugs such as halothane14 and isoniazid,15 but this has not been tested for the IDRs caused by most drugs because the detection of such antibodies requires knowledge of how the drug binds to proteins and the synthesis of the appropriate drug-modified proteins used for the assay. The presence of such antibodies is also not proof that the IDR is immune mediated because such antibodies could be a response to the drug-mediated injury rather than the cause of the injury. The histology of the target organ of an IDR such as the skin or liver most commonly includes a mononuclear immune cell infiltrate, 16 often including CD8+ T cells.17 This also suggests an immune mechanism, but again it is possible that these immune cells are a response to the injury rather than a cause of the


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injury. In contrast, the presence of a significant number of neutrophils, which are often the “first responders” to injury, is uncommon in the histology of most IDRs with the exception of acute generalized

exanthematous

pustulosis.18

IDRs

have

been

reported

to

respond

to

immunosuppressive drugs, but because in most cases IDRs resolve when the drug is stopped, there is a lack of randomized trials to demonstrate the efficacy of such treatments. In many cases, lymphocytes from patients with a recent history of an IDR proliferate when exposed to the drug implicated in the IDR; this is referred to as the lymphocyte transformation test. The fraction of cases with a positive lymphocyte transformation test is highest for drugs such as ß-lactams that have intrinsic chemical reactivity.19 This is presumably because in most cases it is a chemically reactive species, usually a reactive metabolite rather than the parent drug, that initiates the immune response. It is surprising how often the lymphocyte transformation test is positive because the enzymes required for reactive metabolite formation are usually lacking in the assay. Positive results in the absence of reactive metabolite formation appear to be because even when the immune response is initiated by a reactive metabolite, when a strong immune response is produced, the response spreads to include the parent drug. Another observation that strongly suggests an immune mechanism is that a specific human leukocyte antigen (HLA) genotype is often a major risk factor for a specific IDR.20 It is unknown how common this is because it requires DNA samples from patients with a history of a specific IDR as well as samples from matched controls, and depending on how common the implicated HLA is in the control population, it may require many samples to demonstrate a clear relationship. Associations with other immune-associated genes have also been noted.20 Taken together these observations provide strong evidence that most IDRs are immune mediated; however, the data are far from complete, and it is possible that there are exceptions.


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MECHANISTIC HYPOTHESES TO LINK REACTIVE METABOLITE FORMATION TO AN IMMUNE RESPONSE

If, as suggested above, most IDRs are caused by reactive metabolites and are immune mediated, the question becomes how do reactive metabolites induce an immune response that in some patients leads to an IDR. The first immunogenic model proposed by Burnet21 was known as the self-nonself model. It posited that each lymphocyte recognizes foreign substances through many copies of a single surface receptor that is specific for a non-self entity. This, in turn, is able to initiate an immune response whereas the immune system responds to self with tolerance.22, 23 Since then, the original models have undergone several modifications to explain other observations.24 The two major hypotheses that can be used to link reactive metabolites to an immune response are the hapten and danger hypotheses (Figure 1). These two hypotheses are not mutually exclusive, and both may be required to explain the mechanism of IDRs. In addition, there are other pathways that may be involved in the induction of a drug-induced immune response as described below.

Hapten Hypothesis. The word “hapten” originates from the Greek word meaning “to fasten” and is used to describe small, low molecular weight molecules that bind to proteins making the proteins “foreign” with the potential to initiate an immune response.25 The idea of haptens and the hapten hypothesis was first proposed by Landsteiner and Jacobs26 in 1935 when it was revealed in classical studies that an immune response could not be induced by small molecules unless they were chemically reactive and irreversibly bound to endogenous protein; thus,


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creating a hapten:self-peptide complex. Modification of the proteins occurs when chemically reactive drugs or reactive drug metabolites covalently bind to them. These “foreign” proteins could be taken up by antigen presenting cells (APCs), processed, and the drug-modified peptides subsequently presented to T cells; in some cases, leading to an immune response (Figure 1).27, 28 The reason that drugs do not usually directly lead to an immune response is presumably because MHC and T cell receptors have evolved to recognize peptides as the major product of processing antigens, and the binding affinity of most drugs to these molecules is simply too low. Again, penicillin-induced anaphylaxis can be used as a classic example of a hapten.29 Penicillin is a βlactam antibiotic, the structure of which is chemically reactive because of ring strain. A small fraction of an administered dose of penicillin covalently binds to proteins. This often leads to the production of antibodies against penicillin-modified proteins, but in most cases the dominant antibody is IgG, and the titre is low. Only occasionally is the dominant antibody IgE with a titre sufficient to lead to mast cell degranulation and an anaphylactic reaction. Given that this IDR involves covalent binding of penicillin to proteins and is mediated by IgE antibodies that recognize penicillin-modified proteins, it represents a clear example that confirms the hapten hypothesis, although it does not mean that the hapten hypothesis is relevant for all IDRs.27, 29 Similarly, halothane and tienilic acid form reactive metabolites that covalently bind to hepatic proteins. Halothane- and tienilic acid-induced idiosyncratic hepatitis is associated with antibodies against drug-modified proteins.14, 30 The specificity of these antibodies is different in different patients, and some of the antibodies are autoantibodies such that they recognize native proteins. Although this demonstrates that these drugs can induce an immune response that coincides with the liver injury, unlike penicillin-induced anaphylaxis, there is no evidence that these antibodies mediate the liver injury. In fact, from the histology of idiosyncratic drug-


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induced liver injury, it is most likely that the injury is mediated by T cells.16 Most immune responses involve a complex combination of immune cells and antibodies so it would not be surprising if halothane induces a mixed immune response involving both antibodies and T cells. It has not been possible to test whether anti-drug antibodies are involved in the idiosyncratic liver injury caused by halothane because, despite many attempts, none were able to reproduce liver injury in animals with the same characteristics as the human halothane-induced IDR. The one exception is a recent study that found a marked increase in the immune response and liver injury caused by halothane in mice when myeloid-derived suppressor cells (MDSCs) were depleted with an antibody, and even in this case, the injury resolved. In this model, CD4+ T cells appeared to be responsible for most of the liver injury.31

Danger Hypothesis. Although covalent binding of drugs or their reactive metabolites clearly has the potential to produce “foreign” proteins, injection of such proteins rarely results in a significant immune response without the addition of an adjuvant. Janeway referred to adjuvants as the immunologist’s “dirty little secret”.32 Induction of a significant immune response requires activation of APCs leading to the upregulation of costimulatory molecules such as CD80, CD86, and CD40. Matzinger proposed that the immune system ignores antigens unless they represent a danger to the organism.33 This is referred to as the danger hypothesis, and it involves damage to cells resulting in the release of danger-associated molecular pattern (DAMP) molecules that lead to the activation of APCs. DAMPs are analogous to pathogen-associated molecular patterns (PAMPs) associated with pathogens, which are involved in the induction of an immune response to protect us from pathogens and are often used in adjuvants.34 The hapten and danger hypotheses are complementary. The covalent binding of drugs to proteins produce novel antigens


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that can be recognized by specific T cells; this delivers to T cells what is referred to as signal 1. DAMPs upregulate costimulatory factors on APCs that are required for T cell activation, and these costimulatory factors deliver what is referred to signal 2. Both signals are required for activation of T cells and an active immune response (Figure 1). If a reactive metabolite covalently binds to proteins, such as albumin, it is unlikely to lead to cell damage, but some covalent binding can involve critical molecules and can lead to cell injury. This could lead to the release of DAMPs and generation of signal 2. This provides an attractive hypothesis of how reactive metabolites can lead to IDRs, and why not all drugs that form reactive metabolites are associated with a significant risk of causing IDRs. Specifically, if the reactive metabolite does not also cause cell damage with the release of DAMPs it is unlikely to lead to an IDR. This also has the potential to lead to the development of biomarkers that would predict the risk of a drug candidate in causing IDRs; if it leads to the release of DAMPs, it is more likely to cause IDRs. This leads to two questions: what molecules can act as DAMPs, and how do DAMPs activate APCs? Although not close to a complete list, molecules that can act as DAMPs include extracellular high mobility group box protein 1 (HMGB1), ATP, heat shock proteins (HSPs), S100 proteins, and mitochondria-derived DAMPs.35 HMGB1 is an important and complex DAMP.36 It is a nuclear protein that contains several lysine groups, which bind to the negative charges on DNA. During necrosis, HMGB1 is released and can act as a powerful DAMP. With inflammasome activation, in particular the NACHT, LRR, and PYD domains-containing protein 3 (NALP3) inflammasome described below, HMGB1 can also be hyperacetylated, which blocks its binding to DNA, and leads to secretion by macrophages and other immune cells.37 In principle this is a positive feedback system because HMGB1 is reported to prime inflammasomes by binding to toll-like receptor (TLR) 2 and 4. Another receptor for HMGB1 is


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the receptor for advanced glycation end products (RAGE) leading to the activation of nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB). There are several oxidation states of HMGB1.38 There are 3 sulfhydryl groups located on the cysteines at C23, C45, and C106. The sulfhydryl groups at C23 and C45 can form a disulphide, the formation of which is reversible. The sulfhydryl group at C106 can be irreversibly oxidized to a sulfonic acid. The fully reduced HMGB1 forms a complex with chemokine (C-X-C motif) ligand (CXCL) 12 and acts as a chemokine with increased affinity for C-X-C chemokine receptor (CXCR) 4. The disulphide form is reported to activate TLR4. Oxidation of the C106 leads to an inactive or even tolerogenic form. Therefore, HMGB1 can have multiple effects, depending on the redox environment. There are other posttranslational modifications of HMGB1 such as methylation, phosphorylation, and glycation, which affect binding to DNA and facilitate the transfer of HMGB1 from the nucleus to the cytoplasm. Such structural modifications also modulate its immunological effects. The extracellular concentration of ATP is generally low; however, it is released during necrosis, and there are also pathways for active release of ATP-containing lysosomes from inflammatory cells such as neutrophils. ATP binds to P2X purinergenic receptors, which are involved in inflammation and wound healing.35 ATP is also a classic activator of the NALP3 inflammasome, but phosphatases markedly limit its duration of action. Products released from mitochondria such as mitochondrial DNA and N-formyl peptides can also act as DAMPs.39 In general, necrosis leads to the release of inflammatory forms of HMGB1 and other DAMPs, and apoptosis leads to inactive or even tolerogenic forms of DAMPs. However, there are other forms of cell death such as pyroptosis that are inflammatory, and stressed cells may also release DAMPs in the absence of cell death.36 These are only a few of the many molecules that can act


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as DAMPs. In addition, their activities can be quite different depending on posttranslational modifications and context; as well multiple triggers lead to their release. These factors make it a very complex system. Overall, the danger hypothesis complements the hapten hypothesis with the inclusion of signal 2.

Inflammasome Activation. The term inflammasome comes from the word “inflammation” and the suffix from the Greek word “soma” meaning body.40 As members of the NOD-like receptor (NLR) family, the inflammasomes act as sensors for DAMPS or PAMPS. Structurally, inflammasomes are high-molecular weight, multiprotein complexes that reside in the cytoplasm. There are many inflammasomes; the NALP3 inflammasome will be discussed as a prototypical example (Figure 2). It contains a sensor protein, an adaptor protein, and the zymogen – procaspase-1.40,

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The NALP3 inflammasome is expressed in monocytes, lymphocytes, and

granulocytes.42 Activation of the NALP3 inflammasome is a two-step process. It is assembled in response to a large range of stimuli, such as the binding of lipopolysaccharide (LPS) to TLR4 to increase the transcription of NALP3 via NF-κB signaling.43 In the second step, activation of the NALP3 inflammasome leads to the conversion of procaspase-1 to its active form mediated by adaptor proteins.44 Caspase-1 cleaves precursors of the interleukin (IL)-1 cytokine family members, most notably IL-1β and IL-18, which promote the activation and induction of pyrotosis – a highly pyrogenic form of cell death. These cytokines function in response to infection and injury, but they can also cause acute and chronic inflammation.41, 45, 46 Although both IL-1 and IL-18 are of a proinflammatory nature, each one has a specific and individual function. As a pyrogenic cytokine, IL-1β can be involved in skin rashes; inflammatory pain hypersensitivity; upregulation of prostanoid synthesis; joint and bone disease; tumour


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angiogenesis; and neurodegeneration. In contrast, IL-18 is known for its ability to stimulate interferon (IFN)-γ production and T cell polarization.47 However, both are important for T cell activation and can provide a link between innate and adaptive immunity.48,

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Both reactive

oxygen species that are often produced by reactive metabolites50 and the misfolded protein response (discussed below) have the potential to lead to inflammasome activation. Inflammasome activation plays a large role in the mediation of the inflammatory response, and this may extend into IDRs. For example, animals that are deficient in components of the inflammasome are resistant to contact sensitizers, which are caused by covalent binding of reactive xenobiotics.51 In a similar manner, the skin rash caused by covalent binding of a reactive benzylic sulfate metabolite of nevirapine formed in the skin may involve inflammasome activation.10 Overall, inflammasomes may be important in detecting DAMPs that are produced by drugs, which consequently lead to a proinflammatory response and the induction of an IDR. The ability of a drug to cause inflammasome activation may be a biomarker of IDR risk.51 For example, in comparing telaprevir, which has a black box warning because of severe skin rashes, and boseprevir, which does not, only the telaprevir caused significant inflammasome activation. Likewise, in comparing dimethyl fumarate and ethacrynic acid, both of which are reactive Michael acceptors, only dimethyl fumarate is associated with contact sensitization and immune modulation, and only dimethyl fumarate activated inflammasomes. We first assumed that only chemically reactive drugs would activate inflammasomes in the model system of THP-1 cells because of their limited drug metabolizing capacity. However, troglitazone activates doublestranded RNA-activated protein kinase (PKR), which can lead to inflammasome activation.52 In preliminary experiments, we also found that troglitazone, but not pioglitazone, activated inflammasomes in THP-1 cells (unpublished observation). Therefore, reactive metabolite


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formation may not be a requirement for inflammasome activation.

Misfolded Protein Response/ER Stress. Reactive metabolites are formed primarily in the liver. Most of the enzymes involved, principally cytochromes P450, are localized in the smooth endoplasmic reticulum (ER). The rough ER functions to synthesize, modify, and deliver properly folded proteins along the secretory pathway to appropriate target sites. Proteins that are folded into their correct conformation, including the addition of post-translational modifications, are allowed to travel into the Golgi apparatus.53 Quality control is implemented through ER-specific mechanisms that monitor the integrity of folding and assembly of the proteins. If improper or incomplete folding occurs, there are safeguards to identify and remove these misguided proteins to prevent their release from the ER into the secretory pathway. This is done to reduce the potential harm of protein aggregation. The misfolded or unfolded protein response (UPR) is a series of signaling events that react to the presence of folding-incompetent, aggregate proteins that cause ER stress through the accumulation of misfolded proteins (Figure 3).53,

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It is an

adaptive mechanism to protect cells against the defects in protein folding in the ER.55 Recognition of a higher free-energy state than a peptide in its normal conformation is an indication to the system that something has gone awry. This may occur if the ER protein-folding system reaches an overcapacity and is unable to halt excess peptide synthesis or diminish the amount of misfolded proteins in the ER. Viral infections can be a cause of this response.53, 54 Adaptive mechanisms are then activated to restore the folding capacity of the ER to match its needs and alleviate the misfolded protein burden. This includes the upregulation of molecular chaperones and foldases; downregulation of protein synthesis at the transcriptional and translational levels; expansion of the ER membrane and the synthesis of additional protein-


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folding machinery; and increased clearance of the misfolded protein in the ER through upregulated degradation pathways. If these adaptive mechanisms cannot mitigate ER stress, the cell is programmed for apoptosis.53, 55 Three transmembrane proteins help transduce the UPR signal across the ER membrane to potential downstream effects to regulate transcription. There are at least three stress-response mechanisms or branches that act to mediate the UPR to re-establish homeostasis.56,

57

This

includes the ER-resident type I transmembrane protein kinases, inositol-requiring enzyme 1 (IRE1) and protein kinase RNA-like ER kinase (PERK), whereas the type II transmembrane protein includes activating transcription factor 6 (ATF6). Immunoglobin binding protein (BiP), a HSP70 family member that is regulated by the UPR, is associated with IRE1 and PERK luminal domains in an inactive state. Upon ER stress, BiP is competitively displaced from IRE1 and PERK by the excess of misfolded proteins in the ER lumen, resulting in the oligomerization and transphosphorylation of IRE1 and PERK, which activate signal transduction pathways (Figure 3).53, 54 As such, BiP represses IRE1 and PERK signalling under physiological states, but this repression is relieved by detection of misfolded proteins to release BiP from the kinases.58 The activated signalling pathways regulate chaperone induction, translational repression, expand ER space in response to ER stress, induce antioxidant response, and upregulate genes involved in immune response. In addition, PERK phosphorylates the alpha subunit of the eukaryotic initiation factor 2 (eIF2α) to inhibit protein translation and expression. ATF6 is regulated similarly by BiP as PERK or IRE1, where it translocates to the Golgi complex after release of BiP from its ER luminal domain. The major difference is that BiP does not regulate the activity of oligomerization domains in ATF6.53, 54


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Covalent binding of drugs to proteins may lead to misfolding of the modified proteins, and this could lead to an immune response. The ER stress-responsive transcription factor that is hepatocyte specific, known as the cyclic AMP-responsive element-binding protein 3-like protein 3 (CREBH), can increase the expression of genes involved in innate immune system activation and inflammation.59 Defective UPR pathways in non-immune cells can also overwhelm mechanisms of immune tolerance, which have been shown to lead to the development of experimental autoimmune encephalomyelitis, atherosclerosis, and colitis in mice. Furthermore, ER stress and the UPR are implicated in activating NF-κB via phosphorylation of its inhibitor protein, I kappa B (IκB), by IκB kinase (IKK).60 Phosphorylated IκB is ubiquitinated and degraded by the proteasome, and NF-κB is free to enter the nucleus, bind DNA, and undergo transcription of its target gene battery. This tight regulation of NF-κB transcriptional activity is critical to avoid the emergence of a disease state.61 The misfolded protein response can lead to inflammasome activation;62 therefore, the inflammasome activation and misfolded protein response may be directly linked. An important question is whether the degree of protein adduction caused by reactive drug metabolites is sufficient to lead to the UPR. Although it is easy to detect drug-protein adducts with the appropriate antibody, it is difficult to identify the adducted proteins because the degree of adduction is actually quite low. Therefore, the adducted proteins are lost in a sea of native proteins even with drugs such as isoniazid in which the degree of covalent binding is relatively high.63 There may be other mechanisms by which a reactive metabolite could induce ER stress such as undergoing redox cycling with the generation of reactive oxygen species.

HYPOTHESES NOT REQUIRING A REACTIVE METABOLITE


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Pharmacological Interaction (p-i) Hypothesis. As mentioned, T cells from patients with a history of an IDR often proliferate in response to the incriminated drug. This is referred to as the lymphocyte transformation test.64 There are related tests that use the release of cytokines as an assay instead of cell proliferation. In these systems, it is very unlikely that a reactive metabolite is formed, and in most cases the parent drug was not chemically reactive. This suggests that covalent binding might not be required for the induction of an immune mediated IDR. Pichler65 proposed the p-i concept, which posits that an unreactive drug can directly stimulate T cells without haptenation through a non-covalent “pharmacological interaction” with MHC molecules or TCRs. This, in turn, could initiate an immune response leading to an IDR.66, 67 Consistent with this hypothesis, Schnyder et al.67 found that sulfamethoxazole was not chemically reactive but could be presented in a MHC-restricted fashion without the need for drug processing. Furthermore, Zanni et al.68 noted two different and distinct pathways of drug presentation for T cell activation by sulfamethoxazole in the absence of metabolism. This response was also noted in cells from lidocaine-sensitized patients.69 Another study found that drug-specific T cell clones from patients with a history of lamotrigine-induced skin rash were activated in a HLA-DR- and HLA-DQ-dependent manner by lamotrigine in the absence of metabolism or antigen processing. Likewise, carbamazepine was found to be presented on MHC class II molecules expressed by antigen-presenting cells, which led to T cell receptor activation.70, 71 In addition to the lack of need for a reactive metabolite, if the drug directly activates T cells through the TCR, involvement of the innate immune system would not be required, and there would be no need for signal 2.72 Using this hypothesis, the factor that was proposed to


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make IDRs idiosyncratic is the chance binding of the drug to one of many TCRs, which have been estimated to number 1011 for each individual. It is ironic that the first drug that was considered an example of a drug working through the p-i mechanism was sulfamethoxazole because sulfamethoxazole is a primary aromatic amine. Primary aromatic amines are considered a structural alert because they readily form reactive metabolites, and essentially all drugs that have a primary aromatic amine functional group cause IDRs, presumably because they form reactive metabolites. Even though the TCR-p-i hypothesis does not require signal 2 produced by DAMPs, it is possible that the reactive metabolites of aromatic amines do cause cell damage, which may facilitate an immune response. A larger issue is that the p-i hypothesis is based on an unstated assumption. That assumption is what the T cells respond to in a lymphocyte transformation test is what initiated the immune response. That is a plausible assumption, but it is false. We found that the reactive benzylic sulfate metabolite of nevirapine, which covalently binds in the epidermis, is responsible for nevirapine-induced skin rash.10 Yet T cells from animals that had developed the rash responded to nevirapine in the absence of reactive metabolite formation.73 Although this is an animal model, the rash in rats has characteristics very similar to the rash in humans, and if a reactive metabolite can induce an immune response in mice and yet the lymphocyte transformation test is positive to the parent drug, this may also be true in humans. Another interesting example was reported by Warrington.74 The study found that lymphocytes from patients who had mild liver injury caused by isoniazid proliferated on exposure to isoniazid-modified hepatic proteins, but not to isoniazid itself. In contrast, lymphocytes from patients with severe isoniazid-induced liver injury also proliferated in response to isoniazid. It is very likely that isoniazid-induced liver injury is caused by a reactive metabolite of the drug.75 This may be an example of epitope spreading in which


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when there is a strong immune response, it triggers a response to related molecules including the parent drug. In vitro experiments such as those to support the p-i hypothesis, most of which use cloned T cells that have been selected for by their proliferation to the parent drug, are prone to artefacts. Specifically, the selection for T cells that respond to the parent drug is analogous to finding anti-drug antibodies in patients with an IDR. Specifically, the presence of such antibodies does not indicate that the injury was mediated by antibodies, the dominant mechanism of the injury could be cell-mediated. Unlike the hapten hypothesis for which there are at least a few examples that clearly involve this mechanism, it is very difficult to perform experiments that would prove the p-i hypothesis, especially in the absence of a valid animal model. The p-i model may be involved in some IDRs. If it is, given that it involves the direct activation of T cells, it is most likely to be involved in IDRs characterized by a generalized activation of the immune system. This can include drug reactions with eosinophilia and systemic symptoms (DRESS) rather than an IDR that targets only one organ such as idiosyncratic drug-induced liver injury (IDILI), idiosyncratic drug-induced agranulocytosis, or even most skin rashes. It is interesting that allopurinol does not appear to form a reactive metabolite, which suggests that it might involve the p-i hypothesis, and yet unlike abacavir, patch tests are virtually always negative with allopurinol-induced IDRs.

Altered Presentation of Endogenous Peptides. T cells potentially encounter a large number of endogenous self-peptide/MHC ligands during development for which positive and negative selection occurs in the thymus.76, 77 If the MHC is altered it leads to the presentation of peptides to which negative selection of T cells has not occurred. This appears to be the mechanism of abacavir-induced hypersensitivity reactions. Abacavir binds very tightly, but non-covalently, to


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the F-pocket of the antigen-binding groove of HLA-B*57:01, which alters the repertoire of endogenous peptide ligands that are presented by this MHC.66 The T cells that recognize these peptides have not previously undergone negative selection, and this can lead to an alloreactive T cell response analogous to the graft versus host reaction. More that 50% of individuals who carry this gene and are treated with abacavir develop a severe IDR,78 which is much higher than other HLA-associated IDRs. This is a “new” and very interesting mechanism, and it would seem that other drugs would also be likely to operate by the same mechanism, and yet to date, abacavir appears to be unique. Although carbamazepine has also been reported to bind noncovalently to HLA-B*15:0279, which is the HLA associated with the highest risk of carbamazepine-induced toxic epidermal necrolysis, the ability of APCs treated with carbamazepine to activate T cells is much less than that of abacavir, and most patients who carry the HLA-B*15:02 gene do not develop toxic epidermal necrolysis. Although abacavir-induced hypersensitivity reactions may not involve a reactive metabolite, and abacavir clearly binds tightly but non-covalently to HLA-B*57:01, it also forms a Michael receptor-type reactive metabolite mediated by alcohol dehydrogenase.80 This reactive metabolite can be formed by APCs81 and cross-link proteins.82 Therefore, a reactive metabolite may also be involved in the mechanism of abacavir-induced IDRs. It may be the combination of non-covalent binding to HLA-B*57:01 to alter the pattern of endogenous peptides that are presented to T cells and the formation of a reactive metabolite that activates APCs that leads to such a high incidence of IDRs in susceptible patients.

Altering Immune Balance. Many new drugs are being developed to modulate the immune response, either to suppress the response to treat immune mediated reactions or to increase the


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response for the treatment of cancer. It is not surprising that agents that stimulate immune responses would lead to immune mediated IDRs. For example, monoclonal antibodies such as anti-programmed cell death protein 1 (PD-1), anti-programmed cell death 1 ligand 1 (PD-L1), and anti-cytotoxic T lymphocyte associated protein 4 (CTLA-4) that impair immune tolerance, or anti-CD137 that activates T cells can lead to autoimmune IDRs.83 They can also increase the risk of IDRs to other co-administered drugs.84 What is more surprising is that the agents, mostly monoclonal antibodies that block cytokines such as tumor necrosis factor α (TNFα), developed to suppress the immune system to treat diseases such as multiple sclerosis, inflammatory bowel disease, or rheumatoid arthritis can also lead to autoimmune reactions85 such as autoimmune hepatitis.86 This is another example of the complexity and unpredictability of the immune system.

Mitochondrial Injury. Another hypothesis for the mechanism of idiosyncratic drug reactions is mitochondrial injury.87 This is a plausible hypothesis given the importance of the mitochondria in the function of cells, in particular their role in determining the mode of cell death (Figure 4). In addition, several of the DAMPs come from mitochondria, and mitochondrial toxicity could promote an immune response. A classic example of mitochondrial toxicity is the tragic adverse reaction caused by fialuridine, which was being developed for the treatment of hepatitis B.88 It inhibited mitochondrial DNA synthesis leading to multisystem injury including the liver. It caused death in 5 out of 15 subjects and led to liver failure with steatohepatitis and lactic acidosis in others. This was not predicted by animal studies, but it was not idiosyncratic in humans. Other antiviral drugs such as the nucleoside reverse transcriptase inhibitors used to treat human immunodeficiency virus (HIV) infections also inhibit mitochondrial DNA synthesis and can lead


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to myopathy, neuropathy, and liver injury with steatohepatitis, but these reactions are not typical of IDILI. The liver injury associated with these drugs is usually characterized by steatosis and lactic acidosis, which are characteristics of mitochondrial injury.89 It does not appear that these agents markedly increase the risk of IDILI with co-administered drugs. Linezolid inhibits mitochondrial protein synthesis, and this is apparently the mechanism by which it can cause several adverse reactions when it is used for an extended period of time.90 The major adverse reactions that result are bone marrow suppression and neuropathy, but it can also cause liver injury with lactic acidosis.91 This is again not a typical idiosyncratic reaction in that it probably occurs in most patients with sufficient exposure to the drug. Linezolid also does not appear to increase the risk of IDILI with co-administered drugs. The one IDILI for which there is very strong evidence for involvement of the mitochondria is valproate-induced liver injury. It is not typical of other IDILI in that it is much more common in infants, but is similar to other IDILI in terms of other characteristics.92 It is branched at the position where the ß-oxidation of fatty acid oxidation occurs, and it inhibits fatty acid metabolism. Valproate-induced liver injury is often associated with steatosis and lactic acidosis, which provides strong evidence for the involvement of mitochondria. In addition, the major known risk factor for valproate-induced liver injury is a genetic mutation in mitochondrial DNA polymerase-γ.93 This represents very strong evidence for the involvement of mitochondria in the mechanism of valproate-induced liver injury. It is not clear whether the immune system is also involved in valproate-induced liver injury; however, valproic acid is associated with other IDRs such as toxic epidermal necrolysis that are likely immune mediated.94 It has also been suggested that reactive metabolites are involved,95 but the evidence is far from conclusive. It has also been proposed that IDILI can be caused by inhibition of the mitochondrial


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electron transport chain and opening of the mitochondrial pore transition.87 Certainly, the classic acute toxicity of acetaminophen involves reactive metabolites that target liver mitochondria and result in opening of the mitochondrial permeability transition pore,96 but acetaminophen-induced liver injury is also not idiosyncratic. In an in vitro study by Lee and Boelsterli97, the use of efavirenz and isoniazid were used to inhibit complex I and II of the electron transport chain, respectively. As a combinational therapy that is commonly used to treat HIV/tuberculosis coinfected patients, both drugs have been implicated to target the electron transport chain and have been associated with liver injury. It was hypothesized that the concurrent use of these drugs would synergistically increase the risk of IDILI compared to each drug alone because normally, complex I can compensate for the loss of complex II and shuttle electrons through the electron transport chain to bypass the inhibited complex. However, the acute in vitro toxicity in this model is very different from IDILI, which has a delayed onset, and there is no clinical evidence for such synergism that we are aware of. In a similar in vitro study, it was also found that the inhibition of complex I by rotenone increased the toxicity of isoniazid.98 In contrast to the in vitro studies, drugs such as metformin and phenformin that inhibit the mitochondrial electron transport chain and cause lactic acidosis99 virtually never cause IDILI in humans. It is possible that some other factor, in addition to the inhibition of the electron transport chain, is required to cause liver injury, but there is no evidence that metformin, a very common drug, increases the risk of IDILI with coadministered drugs. The classic agent that uncouples oxidative phosphorylation is dinitrophenol, which has been used for weight loss. Although dinitrophenol has caused many fatalities, it did not cause liver injury typical of IDILI.100 In short, with the exception of valproate-induced liver injury, the characteristics of IDRs are very difficult to explain on the basis of mitochondrial injury alone. If IDILI does not have hallmarks of


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mitochondrial injury such as microvesicular steatosis and/or lactic acidosis, it is questionable whether mitochondrial injury, especially inhibition of the electron transport chain, plays a significant role. However, the mechanism by which a drug inhibits the electron transport chain can affect the production of reactive oxygen species, and it is possible that a drug could cause the release of reactive oxygen species or other mitochondrial DAMPs that activate inflammasomes without causing lactic acidosis or steatosis. Several mechanisms have been proposed by which mitochondria may activate inflammasomes, but the evidence is inconclusive.101 It would markedly change the process of drug development if there were high throughput assays that would accurately predict the risk that a drug candidate would cause IDILI and other types of IDRs. In attempts to address this need, in vitro assays such as quantifying mitochondrial respiration have been developed to screen drug candidates for their risk of causing idiosyncratic drug-induced liver injury. However, in vitro assays often utilize concentrations that are 100-fold greater than the Cmax of the drugs, and their predictive value is questionable. The availability of animal models to test the mitochondrial injury hypotheses would be very useful. Boesterli developed an in vivo model of troglitazone-induced liver injury in a mitochondrial superoxide dismutase heterozygote mouse, but others have not been able to reproduce it.102

Bile Salt Export Pump Inhibition. The bile salt export pump (BSEP), or ABCB11, is the predominant, ATP-dependent efflux transporter responsible for transporting conjugated bile salts, monovalent bile acids, and cholate derivatives from hepatocytes into the bile (Figure 5). BSEP belongs to the ATP-binding cassette superfamily of transporters. It is expressed in the liver canalicular/apical membrane along with other biliary transporters, such as multidrug resistance protein 3 (MDR3).103-105


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Bile salts are toxic, and a genetic deficiency of BSEP leads to severe cholestatic liver injury, and in the most severe forms, liver failure.106 There are drugs that can inhibit BSEP, and it is plausible that this could cause liver injury. In fact, some of these drugs such as bosentan, tolcapone, nefazodone, and troglitazone are associated with IDILI.107, 108 An in vitro sandwichcultured hepatocyte assay has been developed to study BSEP inhibition, and this assay has been used to test drugs. An animal model to confirm that BSEP inhibition leads to IDILI would be very helpful to test the hypothesis; however, unlike humans, there appear to be other significant bile salt transporters in rodents, and the composition of rodent bile salts is less toxic than that of human bile salts. 107 It is always important to make links to humans. In the liver injury caused by a genetic deficiency in BSEP in humans, in the more severe forms in which BSEP is virtually absent, the progression to liver failure takes years, and although the alkaline phosphatase levels are elevated as in other forms of cholestatic liver injury, the gamma-glutamyl transpeptidase levels are normal.106 It is not clear whether the same is true in IDILI caused by drugs such as bosentan, and bosentan causes primarily hepatocellular IDILI rather than cholestatic IDILI. Although BSEP inhibitors do not appear to cause liver injury similar to the injury that occurs with a genetic deficiency in BSEP, it is quite plausible that inhibition of BSEP leads to mild liver injury, and this helps to promote an immune response to the drug that in some cases can lead to IDILI. Another possibility is that the physical properties of a drug that lead to BSEP inhibition also lead to a higher concentration of the drug in bile, and this could be associated with an increased risk of cholestatic liver injury. Along with testing for mitochondrial injury, drug candidates have also been tested for their ability to inhibit BSEP. It is claimed that the combination of assays help to predict IDILI risk, but certainly these tests are far from perfect.103 In particular, in the paper


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cited, progesterone and rosiglitazone were characterized as having the “most DILI concern” and several of the drugs associated with the highest risk of IDILI such as isoniazid, pyrazinamide, propylthiouracil, nevirapine, and tolcapone, were not tested. Although the emphasis has been on BSEP, inhibition of other transporters such as multidrug resistance-associated protein (MRP) 3/4 and MDR3.109

IMMUNE TOLERANCE The immune response is capable of causing a large amount of tissue damage; therefore, there has to be mechanisms to limit the immune response. The liver, in particular, is exposed to a large number of bacterial products and reactive metabolites formed during food metabolism. If the liver mounted a strong immune response to such stimuli it would lead to significant damage. It is therefore not surprising that the dominant immune response in the liver is immune tolerance.110, 111 If a drug causes serious IDRs, it always causes a higher incidence of mild IDRs of the same type, and these IDRs often resolve despite continued treatment with the drug. In the case of liver injury, this is called Temple’s corollary.112 Other than the severity, the general characteristics of the mild IDRs such as time to onset are usually the same as for the serious IDRs. In the case of lumiracoxib, the HLA association for the mild liver injury is the same as for the serious liver injury.113 This suggests that the mechanisms of the mild injury and the serious injury are the same. If the mild injury resolves despite continued treatment with the drug and the IDR is immune mediated, it implies that the resolution involves immune tolerance. We found that patients with mild isoniazid-induced injury that resolved despite continued treatment was


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associated not only with an increase in inflammatory Th17 cells, but also with T cells that produced IL-10,114 a cytokine classically associated with immune tolerance. That further suggests that serious IDRs represent a failure of immune tolerance (Figure 6). A promising area of cancer chemotherapy involves the use of checkpoint inhibitors, which impair immune tolerance and allow the immune system to kill tumour cells that often express antigens not present on normal cells.115 We tried for many years to develop animal models of IDRs by stimulating the immune system, but there are multiple mechanisms to down regulate the response to agents such as lipopolysaccharide.116 In contrast to the effects of trying to activate the immune system, checkpoint inhibition led to an animal model of IDILI very similar to IDILI in humans. Specifically, treatment of Pd-1-/- mice with amodiaquine produced greater liver injury than in wild-type animals, but it still resolved despite continued treatment with the drug. However, if we added an antibody to CTLA-4, the injury did not resolve with continued treatment, there was decreased liver function, and the histology demonstrated piecemeal necrosis similar to IDILI in humans.117 Using this model, we also saw liver injury with nevirapine and isoniazid that we did not see in wild type animals, although it was not as severe as with amodiaquine.118 It was also able to differentiate the potential of troglitazone and pioglitazone to cause liver injury (unpublished observation). Therefore, blocking immune tolerance appears to unmask the potential of drugs to cause liver injury; however, it is unlikely to work with agents that have a strong HLA dependency. As mentioned, checkpoint inhibitors also have the potential to increase the risk of IDRs caused by co-administered drugs in humans. In addition to checkpoint inhibition involving PD-1 and CTLA-4, which are expressed on T cells, there are many other molecules and cells involved in immune tolerance. In the liver, antigen presentation by Kupffer cells or liver sinusoidal endothelial cells promotes immune


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tolerance. T regulatory cells and MDSCs are other cell types important for immune tolerance.119 Cytokines such as IL-10 and transforming growth factor (TGF)-ß are, in general, associated with immune tolerance (Figure 6). Furthermore, the induction of immune tolerance is essential to prevent extensive immune damage, and therefore there are many redundant systems involving many other cells and molecules in addition to those mentioned. In the Pd-1-/-/anti-CTLA-4 model described above, immune tolerance still prevailed and prevented liver failure with upregulation of other tolerizing factors such as T regulatory cells. Another complexity is that a type of cell or molecule can have pleotropic effects depending on the context of the immune response; therefore, any strict categorization is likely to be incorrect in some cases.

PATIENT RISK FACTORS

In the previous sections, we have provided mechanistic hypotheses that may make it possible to predict which drug candidates are likely to be associated with IDRs. However, given that most patients do not have an IDR to a specific drug, if we could predict who is at high risk for such a reaction, it would be possible to use the drug safely in the rest of the population. Several hypotheses have been proposed, and multiple risk factors coming together in a specific patient may be required for that individual to have a specific IDR to a particular drug. Hostrelated risk factors such as disease state, age, gender, drug interactions, diet, and the microbiome may contribute to IDR risk, and different factors may be important for different drugs. For example, co-administration of valproate to patients taking lamotrigine markedly increases the risk of lamotrigine-induced skin rash,120 presumably because valproate inhibits glucuronidation of lamotrigine and significantly impairs its clearance.121 However, valproate does not increase


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the risk of IDRs to most other drugs. Another interesting example is that the gut microbiome appears to significantly affect the risk of ipilimumab-induced colitis.122

Genetic Factors. As mentioned earlier, there is a clear association between specific HLA alleles and specific IDRs. HLAs are the human alleles that encode for MHC receptors: class I (HLA-A, -B, and -C) are present on all nucleated cells and bind to CD8+ T cells, while class II (HLA-DP, DQ, and -DR) are present on APCs and bind to CD4+ T cells. Both HLA classes have allelic variants that have been implicated in leading to a higher incidence of specific IDRs.20 The strongest association that has been found is the link between HLA-B*57:01 and abacavirinduced hypersensitivity reactions as discussed earlier.78 Other notable associations include allopurinol-induced hypersensitivity and HLA-B*58:01; carbamazepine-induced Stevens Johnson Syndrome (SJS) / toxic epidermal necrolysis (TEN) and HLA-B*15:02; and flucloxacillin-induced cholestatic IDILI and HLA-B*57:01.123 Several other weaker associations have also been found. Nevirapine-induced skin rashes are associated with HLA-B*35:05124, 125, amoxicillin-clavulanate-induced IDILI is associated with a DRB1*15:01-DRB5*01:01DQB1*06:02 haplotype126,

127

, ticlopidine-induced IDILI is associated with HLA-A*33:03128,

ximelagatran-induced IDILI is associated with HLA-B*5701129, and lapatinib-induced IDILI is associated with HLA-DQA1*02:01.130 To elaborate on the carbamazepine example, the association between HLA-B*15:02 and carbamazepine-induced TEN is observed in the Han Chinese and South Asian populations while this allelic frequency is low in Caucasians and Japanese.131 It is interesting that individuals with this genotype are not at an increased risk of DRESS even though these two IDRs share several characteristics.132 In contrast, HLA-A*31:01 and HLA-B*15:11 are risk factors for


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carbamazepine-induced IDRs, mostly DRESS and maculopapular exanthema, in European and Japanese populations.125, 133 The strong association between specific HLA genes and specific IDRs provide strong evidence for an immune mechanism for the IDRs involved. However, even this evidence is not absolute because there could be linkage with some other unrelated gene that is the true IDR risk factor. Consistent with the postulated immune mechanism of most IDRs, polymorphisms in immune-related genes such as IL-10 have been described.20 TCRs also show a high degree of variation from one patient to another. However, the structure of TCRs is not inherited in the same manner as HLA alleles because the genes coding for the TCRs are produced by random recombination, and therefore, are different even in identical twins. There is evidence that carbamazepine-induced TEN is also associated with TCR sequences, specifically, VB-11-ISGSY and VB-11-GLAGVDN.134 Except for the association between a mutation in the mitochondrial DNA polymerase-γ and valproate-induced IDILI that was already mentioned, all of the strong associations between specific genes and IDR risk found to date have been HLA genes. Given the proposed role of reactive metabolites in the mechanism of many IDRs, it might be expected that polymorphisms in genes associated with drug metabolism and detoxification enzymes would represent IDR risk factors. Troglitazone-induced IDILI has been linked to a combined glutathione-S-transferase (GST) GSTT1-GSTM1 double null mutation.135 The gene associated with decreased Nacetyltransferase-2 activity appears to be a weak risk factor for isoniazid-induced IDLI; however, no strong associations between polymorphisms in drug-metabolizing enzymes and the risk of IDRs have been found.


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It is unclear at the present time what fraction of IDRs have a significant HLA association. The reactive metabolites of most drugs, such as isoniazid, bind to many, probably thousands, of different proteins, so there should be some combination of HLA and TCR that would recognize one of the many modified peptides that are produced during the processing of the altered proteins. Unfortunately, with the exception of abacavir-induced hypersensitivity reactions and carbamazepine-induced TEN in Han Chinese and some other South Asian populations, at the present time, none of the other HLA associations are sufficiently strong to be routinely used to determine who should not receive a specific drug.

Disease States and Inflammation. Given the need for signal 2 in the induction of an immune response, if IDRs are immune mediated, it would be expected that inflammatory disease states that activate APCs would markedly increase the risk of an IDR. A classic example to support this hypothesis is the marked increase in ampicillin- and amoxicillin-induced skin rash in patients who also have mononucleosis.136 The presence of an HIV infection clearly increases the risk of sulfonamide-induced IDRs.137 There is also evidence that patients with cystic fibrosis have a higher risk of antibiotic-induced IDRs.138 There may be an increased risk of IDILI in patients with some types of viral hepatitis,139 but this association is more controversial. However, in general, inflammatory conditions do not appear to be major risk factors for most IDRs. For example, given the inflammatory nature of ulcerative colitis and Crohn’s disease and the fact that they make the gut much more permeable to bacteria and bacterial products, it would be expected that patients with ulcerative colitis and Crohn’s disease would be at a markedly increased risk of IDILI. However, if there is any increased risk of IDILI in these patients, it must be small because it has never been documented. Likewise, non-alcoholic steatohepatitis is an inflammatory


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condition of the liver, and it would be expected to increase the risk of IDILI. There is one study that suggests that patients with non-alcoholic steatohepatitis may have a small increase in risk,140 but it was not a controlled study, and it is not clear that this is a significant risk factor for IDILI. If there is any increase in risk, it must be small. Hyman Zimmerman famously observed years ago that pre-existing liver disease did not increase the risk of IDILI.92 There very well may be exceptions to this rule, but they are not common. In general, somehow the immune system appears to be able to determine the source of inflammation and respond appropriately. In addition, there are several mechanisms to down-regulate the immune response in the presence of chronic inflammation to prevent the immune response from causing excessive cell damage. In an attempt to develop an animal model of IDILI, Roth co-treated animals with LPS and drugs that cause IDILI in humans.141 The first drug he studied was ranitidine, and he found that the combination of LPS and ranitidine caused liver injury while either LPS or ranitidine alone did not. However, this liver injury was acute rather than delayed in onset, and the injury was different in every important respect from IDILI in humans. Therefore, it is almost certain that the mechanism is different. The injury resembled LPS-induced liver injury when LPS is given at a higher dose. In addition, ranitidine is a safe over-the-counter drug that almost never causes serious liver injury; therefore, if this model were used to screen drug candidates it would likely lead to elimination of safe drugs from development. We have tried to use LPS and other agonists of toll-like receptors such as poly-inosine/cytosine, which activates APCs through TLR3, to develop animal models similar to IDILI in humans, but without success.

Heterologous Immunity. To date, abacavir is unique; if a patient carries the HLA-B*57:01 gene and receives abacavir, they have more than a 50% chance of developing a severe hypersensitivity


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reaction. In contrast, although flucloxacillin-induced IDILI happens to also be associated with HLA-B*57:01, if a patient carries this gene and receives flucloxacillin, less than 0.2% will develop IDILI.129 Many, if not most, IDRs probably do not have strong HLA associations. Other factors must be involved in most IDRs. The TCR repertoire may be very important, but given the large number of proteins modified by reactive metabolites, it seems unlikely that this is sufficient to explain the idiosyncratic nature of IDRs. It is possible that multiple risk factors are involved, each one only contributing a small amount to the overall risk. If most IDRs are immune mediated, differences in the immune system are likely to be a key risk factor. It is clear that the immune response is shaped by everything that it has been exposed to. Immune responses to each new pathogen changes the distribution, frequency, and responses of the pool of memory T cells generated from previous responses. Although the number of TCRs is almost limitless, the number of lymphocytes that express these TCRs is not. Every time a new pathogen is encountered and produces memory T cells, other T cells have to be eliminated to make room for them because the total number of T cells remains constant. Yet the immune system has to respond to almost any pathogen. This can be accomplished because two unrelated antigens can bind to the same TCR because of the large number of interactions between the TCR and antigen that are possible. A relatively new hypothesis, heterologous immunity, may apply to IDRs.142 Heterologous immunity describes the phenomenon in which antigen-specific memory T cells that were originally generated during a previous infection can become activated to a novel and unrelated infection.143 As mentioned, an antigen can bind to TCRs in multiple ways; therefore, unrelated peptides can bind to the same TCR. This flexibility in which the same TCR can respond to unrelated antigens comes at a cost. If a pathogen has produced a strong immune response that


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primes the immune system to react strongly to a subsequent exposure, and a drug-modified peptide binds to the same TCR, it could overcome immune tolerance and lead to an IDR. One issue with this hypothesis is that it might be expected to lead to a rapid immune response rather than the delayed onset typical of IDRs. However, as mentioned earlier, when patients with a history of clozapine-induced agranulocytosis are rechallenged, the agranulocytosis recurs faster than on first exposure, but it still usually takes more than a month before the onset of agranulocytosis.12 The number of memory T cells is very small and may require a significant time to proliferate to numbers sufficient to be clinically evident. The tempo of the immune response may be a function of the nature and severity of the insult. If a pathogen produces a large amount of PAMPs and DAMPs, the response may be much faster than when a drug produces relatively mild cell damage. The major problem with this hypothesis is that it is difficult to test, and unless a way can be found to test this hypothesis, it may not be very useful. At the present time, with the exception of a few very strong HLA associations, it does not appear to be possible to predict which patients are at high risk of a specific IDR.

CONCLUSIONS AND FUTURE DIRECTIONS

IDRs are complicated, and little about their mechanisms is known with certainty. As with most such complex and incompletely understood problems, there are many hypotheses. The major hypotheses were outlined in this review with an analysis of each, but the data are incomplete, and it is difficult to be totally objective. It is also quite possible that there are mechanisms that have yet to be discovered. It is essential to first be guided by clinical data and characteristics. Although the data are far from conclusive, there is compelling evidence that most IDRs are


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immune mediated and many are caused by reactive metabolites of the drug rather than the parent drug itself. That is what led to the question posed by this review: how can reactive metabolites induce an immune response that sometimes leads to an IDR? There are likely to be exceptions to the premise of this review, and it is important that methods be found to rigorously test hypotheses. The farther removed data are from in vivo human data the more likely they are to be irrelevant to human IDRs. However, it is impossible to perform controlled experiments in humans; therefore, the development of valid animal models that can be linked back to data from humans is imperative. In vitro experiments can be very useful, but only if they can be linked back to in vivo and ultimately, human data. The progress in understanding IDRs since the inauguration of CRT has been slow. However, there have been several recent developments in immunology and methodology that are likely to significantly facilitate its progress in the future. A major development was a better understanding of immune tolerance and agents such as the checkpoint inhibitors that can be used to impair tolerance. Such agents appear to unmask the potential of drugs to cause IDRs. However, more drugs need to be tested, and it is unlikely to succeed with all drugs; for example, if there is a strong HLA dependence such as with abacavir, it is less likely that the drug will cause an IDR in this model. However, if this type of immune modulation leads to valid animal models of IDRs, it will make it possible to rigorously test hypotheses in a manner that has never been possible before. It may also make it possible to test drug candidates for their potential to cause IDRs. Although it is unlikely to be accurate for drugs such as abacavir, if there are few false positives, such models could still be very useful. It is quite plausible that many, if not most, patients have an immune response to drugs that can cause IDRs, but this immune response is clinically silent and resolves with immune


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tolerance. This is obvious with clozapine: most patients treated with clozapine have an immune response with an increase in inflammatory cytokines such as IL-6, neutrophilia, and often fever.144 This immune response resolves over a period of weeks, presumably with immune tolerance. It is likely that this immune response is related to the mechanism by which clozapine can cause agranulocytosis and other IDRs, but that remains to be tested. It also remains to be tested whether many other drugs induce a clinically silent immune response that is related to the mechanism by which they cause IDRs. Such immune responses may be different for different drugs and different in different individuals, and the study of such immune responses in both animals and humans could provide very important mechanistic clues. There have also been advances in the study of DAMPs, such as HMGB1, and inflammasome activation. It appears that inflammasome activation is an important mechanism of immune activation, and studies of inflammasome activation hold promise for a better mechanistic understanding of IDRs and possibly a biomarker of drug candidate risk. There is a strong incentive to develop high throughput in vitro assays to screen drug candidates, but in the absence of a better mechanistic understanding, such assays are likely to provide misleading results. It is important to test drugs for which we know the outcome in humans before using assays on drug candidates, most of which will never be used in a sufficient number of patients to know whether the predictions were correct. However, the basic mechanistic studies that are now possible are likely to lead to better biomarkers of IDR risk and improved safety of new drugs. The study of IDRs has become quite interesting.

1-7, 9-51, 53-100, 102-108, 110-118, 120-143


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Funding Source: This work was funded by grants from the Canadian Institutes of Health Research

Notes: The authors declare no competing financial interest.

Abbreviations: APC, antigen presenting cell; ASC, apoptosis-associated speck-like protein containing a caspase-recruitment domain; ATF6, activating transcription factor 6; BiP, immunoglobin binding protein; BSEP, bile salt exporter pump; CARD; caspase recruitment domain; CREBH, cyclic AMP-responsive element-binding protein 3-like protein 3; CRT, Chemical Research in Toxicology; CTLA-4, cytotoxic T lymphocyte associated protein 4; DAMP, danger-associated molecular pattern; CXCL, chemokine (C-X-C motif) ligand; CXCR, C-X-C chemokine receptor; DRESS, drug reaction with eosinophilia and systemic symptoms; eIF2α, alpha subunit of the eukaryotic initiation factor 2; ER, endoplasmic reticulum; FAO, fatty acid oxidation; GST, glutathione-S-transferase; HIV, human immunodeficiency virus; HLA, human leukocyte antigen; HMGB1, high mobility group box protein 1; HSP, heat shock proteins; IDILI, idiosyncratic drug-induced liver injury; IDR, idiosyncratic drug reaction; IFN, interferon; IκB, I kappa B; IKK, IκB kinase; IL, interleukin; IRE1, inositol-requiring enzyme 1; LAG3, lymphocyte-activation protein 3; LPS, lipopolysaccharide; LRR; leucine rich repeats; MDR3, multidrug resistance protein 3; MDSC; myeloid-derived suppressor cell; MHC, major histocompatibility complex; MRP, multidrug resistance-associated protein; NACHT, nucleotide binding domain; NALP3, NACHT, LRR, and PYD domains-containing protein 3; NF-κB, nuclear factor kappa-light chain-enhancer of activated B cells; NLR, NOD-like receptor; PAMP,


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pathogen-associated molecular pattern; PD-1, programmed cell death protein 1; PD-L1, programmed cell death 1 ligand 1; PERK, protein kinase RNA-like ER kinase; p-i, pharmacological interaction; PKR, double-stranded RNA-activated protein kinase; PYD, pyrin domain; RAGE, receptor for advanced glycation end products; SJS, Stevens-Johnson Syndrome; TCR, T cell receptor; TEN, toxic epidermal necrolysis; TGF, transforming growth factor; Th17, T helper cell 17; TLR, toll-like receptor; TNFα, tumor necrosis factor alpha; UPR, unfolded protein response


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Author Biographies Tiffany Cho received her Honours Bachelor of Science from McMaster University (Hamilton, Ontario) in Biology and Pharmacology, which was later followed by a Master of Science from the University of Toronto (Toronto, Ontario) in Pharmacology and Toxicology for the study of the aryl hydrocarbon receptor and the effects mediated by the polycyclic aromatic hydrocarbon, 3-methylcholanthrene. Her current work with Dr. Jack Uetrecht as a graduate student in Pharmaceutical Sciences at the University of Toronto focuses on the effect of mitochondrial dysfunction and how this may play a role in the development of idiosyncratic drug-induced liver injury. Jack Uetrecht is a professor of pharmacy and medicine at the University of Toronto. He held the Canada Research Chair in Adverse Drug Reactions from 2001 to 2015, the maximum term for a Canada Research Chair. He received his Ph.D. in Chemistry from Cornell University, M.D. from Ohio State University, medical residency at the Kansas University Medical Center, and clinical pharmacology fellowship at Vanderbilt University. His research is focused on the mechanisms of idiosyncratic drug reactions involving the liver, skin, and bone marrow. The emphasis is on the role of the immune system using animal models and samples from patients.


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Figure 1. Hapten and Danger Hypotheses. In the hapten hypothesis (left), chemically reactive drugs or reactive metabolites bind irreversibly to endogenous proteins to produce a drugmodified proteins. This ensemble is known as a hapten:self-peptide complex and may act as an antigen when processed and presented by antigen-presenting cells (APCs) in a major histocompatibility complex (MHC) II-restricted manner.25, 66 In contrast, the danger hypothesis 113 posits that the presence of activation signals on APCs is required for a cell- and antibodymediated immune response. Signal 1 consists of recognition of the processed antigen, which is presented in the context of MHC molecules on APCs, by T cells through the T cell receptor (TCR). Stressed or damaged cells produce danger signals (DAMPs) that lead to the activation of APCs and upregulation of costimulatory interactions such as B7 on APCs that bind to CD28 on T cells – this is signal 2. The coupling of T cell recognition of an antigen on APCs (signal 1) and expressed costimulation (signal 2) upregulated by the presence of DAMPs lead to T cell activation and differentiation into specific T cell subsets. In contrast, if danger signals are absent, signal 2 is not initiated and the result is immune tolerance.24, 145 The converse is also true; if there is signal 2 but no signal 1, i.e. no modified peptides, there also would be no specific immune response. Therefore, the immune system is not concerned only with the recognition of non-self; it is also concerned with a dangerous entity that can cause damage to the host.


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Figure 2. Inflammasome Activation. Inflammasomes are multiprotein complexes that are present in the cytoplasm of monocytes, lymphocytes, and granulocytes. These proteins act as sensors for danger signals and activate caspase-1 for the cleavage and maturation of inflammatory interleukin (IL)-1 cytokine family members. NACHT, LRR, and PYD domainscontaining protein 3 (NALP3) is the prototypical inflammasome. Upon recognition of DAMPs or PAMPs by toll-like receptors (TLRs), NF-κB upregulates the expression of various inflammasome-related components. The assembly of the inflammasome complex and its activation triggers the self-cleavage of procaspase-1 to active caspase-1, which cleaves the inactive pro-forms of the IL-1 cytokines to their active forms and leads to their release. Inflammasomes also regulate the release of high mobility group box 1 (HMGB1), a protein danger-associated molecular pattern (DAMP), through a caspase-dependent manner that is recognized by toll-like receptor (TLR) 4. This can further increase an inflammatory response.146


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Figure 3. ER Stress and UPR Activation. The endoplasmic reticulum (ER) functions to synthesize proteins and facilitate their folding into their normal conformations. Reactive


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metabolites are formed in the liver and this creates protein adducts. This can lead to an accumulation of unfolded/misfolded proteins in the ER lumen and induction of the unfolded protein response (UPR).147 The inositol-requiring enzyme 1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6) are the three major signaling proteins that are bound to immunoglobin binding protein (BiP) on the luminal domain in the inactive state. BiP dissociates from the proteins to mitigate the accumulation of unfolded/misfolded proteins in the ER lumen and assists in increased folding. In the absence of BiP, PERK and IRE1 oligodimerize and autophosphorylate to initiate a downstream signaling pathway that attempts to restore homeostasis by inhibiting protein synthesis and initiating antioxidant responses via activating transcription factor 4 (ATF4).148 However, the phosphorylation and subsequent degradation of I kappa B (IκB) by the phosphorylated alpha subunit of the eukaryotic initiation factor 2 (eIF2α) and IκB kinase (IKK) allows for the translocation of nuclear factor-kappa B (NF-κB) and expression of genes involved in the immune and inflammatory response.149 Once BiP dissociates, ATF6 enters the Golgi complex where site 1 and site 2 proteases (S1P and S2P) cleave ATF6, which releases a cytosolic component. The processed ATF6 translocates to the nucleus to upregulate the transcription of ER chaperones and foldases to aid in protein folding.147 Similarly, CREBH – a transcription factor that is highly expressed in hepatocytes - follows a similar pathway to initiate the expression of acute phase proteins.150 The double strand RNA-activated protein kinase (PKR) can be activated by DAMPs and other ER stress signals, sharing similar agonists to that of the inflammasome. Upon activation, PKR phosphorylates itself and eIF2α to contribute to an inflammatory response. There is also a role for PKR in inflammasome activation, leading to the release of the IL-1 family of cytokines and high-mobility group box 1 (HMGB1).151


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Figure 4. Mitochondrial Dysfunction. The regulation of ATP synthesis and electron flow through the electron transport chain (ETC) is under tight regulation in the mitochondria, and disruption to mitochondrial function may result in increased oxidative stress. Reactive drugs or metabolites can inhibit specific complexes of the ETC, thereby leading to increased reactive oxygen species (ROS) formation, decreased mitochondrial membrane potential (∆ψm), and lower ATP production.87 The accumulation of multiple disturbances can cause damage to vulnerable mitochondrial DNA (mtDNA), which encode for a number of complexes in the ETC. This can lead to translational mutations in the protein complex, which in turn could result in the generation of superoxide anions to inflict further damage onto mtDNA.152 The sustained opening of the mitochondrial permeability transition pore (MPTP) can lead to an unrestricted movement of small molecules and can cause necrosis/apoptosis of the cell due to the collapse of the ∆ψm. Fatty acid oxidation (FAO) can be inhibited and lead to the development of hepatosteatosis. Chronic inflammation exacerbates lipid accumulation in the liver and can also cause steatohepatitis.153, 154 However, with the exception of valproate-induced liver injury, idiosyncratic drug reactions (IDRs) do not have characteristics consistent with mitochondrial dysfunction, and drugs such as metformin that do inhibit the electron transport chain and cause lactic acidosis do not cause IDRs. Therefore, if mitochondrial injury is important to the mechanism of IDRs, it is more likely due to the production of danger signals (DAMPs) that promote an immune mediated IDR than due to direct mitochondrial injury.


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Figure 5. BSEP Inhibition. The bile salt export pump (BSEP) is an efflux transporter that exports bile salts into bile and functions to eliminate conjugated bile salts and cholate derivatives from hepatocytes. Inhibition of BSEP function by drugs can lead to the accumulation of cytotoxic bile salts that can result in cholestasis or hepatocyte injury.104, 155


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Figure 6. Immune Tolerance. Immune tolerance appears to be the major factor that prevents most patients from having an IDR. There are many cells and molecules involved in the induction of immune tolerance and only a few are illustrated in this figure. It can be inhibited through the blockade of immune checkpoints such as PD-1 and CTLA-4, as both are negative regulators of immune system activation and are both expressed on T cells. The use of a Pd-1-/- mouse along with an anti-CTLA-4 antibody to impair immune tolerance (lower part of the figure) led to an animal model of IDILI. Lymphocyte-activation protein 3 (LAG3) is expressed on regulatory T cells (Tregs) and competes for the MHC on APCs, which induces inhibitory signaling and downregulates immunostimulation. An anti-LAG3 antibody is used to also inhibit immune tolerance pathways. Myeloid-derived suppressor cells (MDSCs) inhibit APC and T cell activities, and induce a regulatory phenotype via the secretion of interleukin (IL)-10 and transforming growth factor (TGF)-β.156


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How Reactive Metabolites Induce an Immune Response That

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