Involvement of Extracellular Vesicles in the Proinflammatory Response to Clozapine: Implications for Clozapine-Induced Agranulocytosis.

Most idiosyncratic drug reactions (IDRs) appear to be immune-mediated, but mechanistic events preceding severe reaction onset remain poorly defined. Damage-associated molecular patterns (DAMPs) may contribute to both innate and adaptive immune phases of IDRs, and changes in extracellular vesicle (EV) cargo have been detected post-exposure to several IDR-associated drugs. To explore the hypothesis that EVs are also a source of DAMPs in the induction of the immune response preceding drug-induced agranulocytosis, the proteome and immunogenicity of clozapine- (agranulocytosis-associated drug) and olanzapine- (non-agranulocytosis-associated drug) exposed EVs were compared in two preclinical models: THP-1 macrophages and Sprague-Dawley rats. Compared with olanzapine, clozapine induced a greater increase in the concentration of EVs enriched from both cell culture media and rat serum. Moreover, treatment of drug-naïve THP-1 cells with clozapine-exposed EVs induced an inflammasome-dependent response, supporting a potential role for EVs in immune activation. Proteomic and bioinformatic analyses demonstrated an increased number of differentially expressed proteins with clozapine that were enriched in pathways related to inflammation, myeloid cell chemotaxis, wounding, transforming growth factor-ß signaling, and negative regulation of stimuli response. These data indicate that, although clozapine and olanzapine exposure both alter the protein cargo of EVs, clozapine-exposed EVs carry mediators that exhibit significantly greater immunogenicity. Ultimately, this supports the working hypothesis that drugs associated with a risk of IDRs induce cell stress, release of proinflammatory mediators, and early immune activation that precedes severe reaction onset. Further studies characterizing EVs may elucidate biomarkers that predict IDR risk during development of drug candidates. SIGNIFICANCE STATEMENT: This work demonstrates that clozapine, an idiosyncratic drug-induced agranulocytosis (IDIAG)-associated drug, but not olanzapine, a safer structural analogue, induces an acute proinflammatory response and increases extracellular vesicle (EV) release in two preclinical models. Moreover, clozapine-exposed EVs are more immunogenic, as measured by their ability to activate inflammasomes, and contain more differentially expressed proteins, highlighting a novel role for EVs during the early immune response to clozapine and enhancing our mechanistic understanding of IDIAG and other idiosyncratic reactions.

The Emerging Role of the Innate Immune Response in Idiosyncratic Drug Reactions.

Idiosyncratic drug reactions (IDRs) range from relatively common, mild reactions to rarer, potentially life-threatening adverse effects that pose significant risks to both human health and successful drug discovery. Most frequently, IDRs target the liver, skin, and blood or bone marrow. Clinical data indicate that most IDRs are mediated by an adaptive immune response against drug-modified proteins, formed when chemically reactive species of a drug bind to self-proteins, making them appear foreign to the immune system. Although much emphasis has been placed on characterizing the clinical presentation of IDRs and noting implicated drugs, limited research has focused on the mechanisms preceding the manifestations of these severe responses. Therefore, we propose that to address the knowledge gap between drug administration and onset of a severe IDR, more research is required to understand IDR-initiating mechanisms; namely, the role of the innate immune response. In this review, we outline the immune processes involved from neoantigen formation to the result of the formation of the immunologic synapse and suggest that this framework be applied to IDR research. Using four drugs associated with severe IDRs as examples (amoxicillin, amodiaquine, clozapine, and nevirapine), we also summarize clinical and animal model data that are supportive of an early innate immune response. Finally, we discuss how understanding the early steps in innate immune activation in the development of an adaptive IDR will be fundamental in risk assessment during drug development. SIGNIFICANCE STATEMENT: Although there is some understanding that certain adaptive immune mechanisms are involved in the development of idiosyncratic drug reactions, the early phase of these immune responses remains largely uncharacterized. The presented framework refocuses the investigation of IDR pathogenesis from severe clinical manifestations to the initiating innate immune mechanisms that, in contrast, may be quite mild or clinically silent. A comprehensive understanding of these early influences on IDR onset is crucial for accurate risk prediction, IDR prevention, and therapeutic intervention.

Potential Involvement of Sulfotransferase in the Mechanism of Lamotrigine-induced Skin Rash.

The mechanism of drug-induced skin rash is not well understood. Circumstantial evidence suggests that the covalent binding of a reactive metabolite is involved in the mechanism of most idiosyncratic drug reactions. However, there is a limited quantity of drug metabolizing enzymes in the skin, except for sulfotransferases. It is possible that some drugs are metabolized to reactive sulfate metabolites that are responsible for skin rashes. For example, nevirapine-induced skin rash involves metabolism of nevirapine to 12-hydroxy-nevirapine, which is further metabolized by sulfotransferase in the skin to a reactive benzylic sulfate that covalently binds to proteins. The working hypothesis is that lamotrigine, valdecoxib, and sertraline skin rashes involve the formation of reactive sulfate in the skin. Lamotrigine-N-oxide, hydroxy-valdecoxib, and hydroxy-sertraline were tested as substrates with known human sulfotransferases. Hydroxy-valdecoxib and the benzylic alcohol metabolite of sertraline were not substrates for human sulfotransferases. Therefore, this pathway is presumably not involved in the mechanism by which they cause skin rashes. In contrast, lamotrigine-N-oxide is a substrate for several human sulfotransferases and the sulfate is chemically reactive. Furthermore, lamotrigine-N-sulfate not only alkylates proteins as we described previously but also forms the sulfate of tyrosine, suggesting another possible mechanism for protein modification. This study has further added to the understanding of the potential of the sulfotransferase pathways and protein sulfation to play a role in drug-induced skin rash.

The Role of Myeloperoxidase in Clozapine-Induced Inflammation: A Mechanistic Update for Idiosyncratic Drug-Induced Agranulocytosis.

The risk of idiosyncratic drug-induced agranulocytosis (IDIAG) markedly constrains the use of clozapine, a neuroleptic with unparalleled efficacy. Most clozapine patients experience an early inflammatory response, likely a necessary step in IDIAG onset. However, most patients do not progress to IDIAG, presumably because of the requirement of specific human leukocyte antigen (HLA) haplotypes, T cell receptors, and other unknown factors. We established that clozapine activates inflammasomes and that myeloperoxidase bioactivation of clozapine generates neoantigens, but the connection between these early mechanistic events remained unknown and, thus, was the aim of this work. We found that the myeloperoxidase inhibitor PF-1355 attenuated myeloperoxidase activity in phorbol myristate acetate (PMA)-differentiated THP-1 macrophages, and it also attenuated clozapine-induced release of inflammatory mediators (e.g., IL-1ß, CXCL1, and C-reactive protein). In vivo, pretreatment of Sprague Dawley rats with PF-1355 significantly attenuated clozapine-induced increases in neutrophil mobilization from the bone marrow to the blood and spleen, as determined using differential blood counts and flow cytometry. Moreover, the clozapine-triggered release of inflammatory mediators (e.g., IL-1ß, calprotectin, CXCL1, and α-1-acid glycoprotein) from the liver, spleen, and bone marrow was dampened by myeloperoxidase inhibition. These data support the working hypothesis that oxidation of clozapine to a reactive metabolite by myeloperoxidase is critical for induction of the inflammatory response to clozapine. Ultimately, a better mechanistic understanding of the early events involved in the immune response to clozapine may elucidate ways to prevent IDIAG, enabling safer, more frequent therapeutic use of this and potentially other highly efficacious drugs.

Idiosyncratic Drug Reactions: A 35-Year Chemical Research in Toxicology Perspective.

When Larry Marnett founded Chemical Research in Toxicology, the study of idiosyncratic drug reactions (IDRs) was in its infancy. There was evidence that IDRs involve chemically reactive metabolites, and many of the papers in Chemical Research in Toxicology investigated the bioactivation of drugs. However, it became clear that not all drugs that form reactive metabolites are associated with a high risk of IDRs, and some drugs that do not appear to form reactive metabolites do cause IDRs. Some of the early Chemical Research in Toxicology papers investigated involvement of the adaptive immune system in the mechanism of IDRs, and HLA associations provided strong evidence for an immune mechanism of IDRs. This led to the question of how reactive metabolites might induce an immune response. The classic hapten hypothesis provided an obvious explanation, but a new hypothesis the danger hypothesis, added another dimension. Although there are common features to IDRs, it is becoming increasingly clear that there are also many differences in the mechanisms caused by different drugs. Other pharmacological effects of drugs may also play a role in the mechanism, and that is obviously true of IDRs caused by biological agents. The requirement for specific HLA and T-cell receptors is presumably the major factor that makes IDRs idiosyncratic. However, an innate immune response is required to prime the adaptive immune response. In contrast to the adaptive immune response, the innate immune response is unlikely to be idiosyncratic, and studies of the innate immune response to drugs may provide a much more accurate way to screen drugs for their potential to cause IDRs. For essential drugs that are known to cause IDRs, it may be possible to markedly decrease risk by a slow dose titration to induce immune tolerance. Significant progress has been made in the study of IDRs, but there is still a long way to go.

Liver Injury Caused by Green Tea Extract in PD-1-/- Mice: An Impaired Immune Tolerance Model for Idiosyncratic Drug-Induced Liver Injury.

Idiosyncratic drug-induced liver injury (IDILI) is an idiosyncratic drug reaction that is specific to an individual and can lead to liver failure and even death. The mechanism of IDILI remains poorly understood, but most IDILI appears to be immune-mediated. We have developed the first validated animal model by using a PD-1-/- mouse model in combination with anti-CTLA-4 to block immune checkpoints and impair immune tolerance. Treatment of these mice with drugs that cause IDILI in humans led to delayed-onset liver injury with characteristics similar to IDILI in humans. The current study investigates the effects of green tea extract, a weight-loss dietary supplement that has been reported to cause IDILI in humans. Green tea extracts contain a highly variable content of catechins including (-)-epigallocatechin gallate, the major catechin in green tea formulations. If the liver injury caused by green tea extract in humans is immune-mediated, it may occur in our impaired immune tolerance model. Female PD-1-/- mice treated with anti-CTLA-4 antibody and green tea extract (500 mg/kg), a dose that is considered a no-observed-adverse-effect level for liver in rodents, produced a delayed onset increase in serum alanine transaminase levels and an increase in hepatic CD8+ T cells. In contrast, the response in male PD-1-/- mice was less pronounced, and there was no evidence of liver injury in wild-type mice. These findings are consistent with the hypothesis that the IDILI caused by green tea extract is immune-mediated and is similar to IDILI caused by medications that are associated with IDILI.

Severe cutaneous adverse reaction associated with antiseizure medications: Diagnosis, management, and prevention.

Severe cutaneous adverse reactions (SCARs) are potentially life-threatening, with considerable morbidity and mortality. They are nonimmediate hypersensitivity reactions that occur in specifically predisposed patients with delayed T-cell-mediated hypersensitivity reaction. Antiseizure medications (ASMs) are among the drugs that can induce SCAR. Increased awareness of SCAR among clinicians treating patients with ASMs is critically important for early recognition of symptoms, prompt identification and removal of the causal drug, and early intervention to reduce SCAR-related acute and long-term morbidity and mortality. The diagnosis, management, and prevention of Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and drug reaction with eosinophilia and systemic symptoms (DRESS) are reviewed, along with the current understanding of the pathomechanisms and role of genetics in SCAR development. Supportive care and immunomodulating treatments for SCAR are discussed.

Idiosyncratic Drug-Induced Liver Injury: Mechanistic and Clinical Challenges.

Idiosyncratic drug-induced liver injury (IDILI) remains a significant problem for patients and drug development. The idiosyncratic nature of IDILI makes mechanistic studies difficult, and little is known of its pathogenesis for certain. Circumstantial evidence suggests that most, but not all, IDILI is caused by reactive metabolites of drugs that are bioactivated by cytochromes P450 and other enzymes in the liver. Additionally, there is overwhelming evidence that most IDILI is mediated by the adaptive immune system; one example being the association of IDILI caused by specific drugs with specific human leukocyte antigen (HLA) haplotypes, and this may in part explain the idiosyncratic nature of these reactions. The T cell receptor repertoire likely also contributes to the idiosyncratic nature. Although most of the liver injury is likely mediated by the adaptive immune system, specifically cytotoxic CD8+ T cells, adaptive immune activation first requires an innate immune response to activate antigen presenting cells and produce cytokines required for T cell proliferation. This innate response is likely caused by either a reactive metabolite or some form of cell stress that is clinically silent but not idiosyncratic. If this is true it would make it possible to study the early steps in the immune response that in some patients can lead to IDILI. Other hypotheses have been proposed, such as mitochondrial injury, inhibition of the bile salt export pump, unfolded protein response, and oxidative stress although, in most cases, it is likely that they are also involved in the initiation of an immune response rather than representing a completely separate mechanism. Using the clinical manifestations of liver injury from a number of examples of IDILI-associated drugs, this review aims to summarize and illustrate these mechanistic hypotheses.

Best practices for detection, assessment and management of suspected immune-mediated liver injury caused by immune checkpoint inhibitors during drug development.

Immune checkpoint inhibitors (ICIs) have shown significant efficacy in patients with various malignancies, however, they are associated with a wide range of immune-related toxicities affecting many organs, including the liver. Immune-mediated liver injury caused by checkpoint inhibitors (ILICI) is a distinctive form of drug induced liver injury (DILI), that differs from most DILI types in presumed underlying mechanism, incidence, and response to therapeutic interventions. Despite increased awareness of ILICI and other immune-related adverse effects of ICIs reflected by recent guidelines for their management in post marketing clinical practice, there is lack of uniform best practices to address ILICI risk during drug development. As efforts to develop safer and more effective ICIs for additional indications grow, and as combination therapies including ICIs are increasingly investigated, there is a growing need for consistent practices for ILICI in drug development. This publication summarizes current best practices to optimize the monitoring, diagnosis, assessment, and management of suspected ILICI in clinical trials using ICI as a single agent and in combination with other ICIs or other oncological agents. It is one of several publications developed by the IQ DILI Initiative in collaboration with DILI experts from academia and regulatory agencies. Recommended best practices are outlined pertaining to hepatic inclusion and exclusion criteria, monitoring of liver tests, ILICI detection, approach to a suspected ILICI signal, causality assessment, hepatic discontinuation rules and additional medical treatment.

The 2-Hydroxyiminostilbene Metabolite of Carbamazepine or the Supernatant from Incubation of Hepatocytes with Carbamazepine Activates Inflammasomes: Implications for Carbamazepine-Induced Hypersensitivity Reactions.

Although the pathophysiology of carbamazepine-induced idiosyncratic or hypersensitivity reactions is unclear, they are presumed to be immune mediated, involving a complex interaction between drug metabolism and activation of the immune system. Cell stress can be caused by reactive metabolites, and this has the potential to release damage-associated molecular patterns (DAMPs), which are responsible for activation of the immune system. Idiosyncratic drug reactions occur mainly in the liver because of its role in drug metabolism and reactive metabolite formation. DAMPs can activate inflammasomes, which may be a common mechanism by which DAMPs lead to an immune response. In the present study, we investigated whether carbamazepine induces the release of DAMPs by using human hepatocarcinoma functional liver cell-4 (FLC-4) cells for bioactivation of carbamazepine. THP-1 cells, a human macrophage cell line, were used for detecting inflammasome activation. We found that increased caspase-1 activity and production of interleukin-1ß by THP-1 cells were caused by the supernatant from the incubation of carbamazepine with FLC-4 cells. In the supernatant, heat shock protein 60 was significantly increased. In addition, 2-hydroxyiminostilbene, which is a metabolite of carbamazepine, activated inflammasomes. These results suggest that the reactive iminoquinone metabolite can directly activate inflammasomes or that stressed hepatocytes cause the release of DAMPs, which are responsible for inflammasome activation. The activation of inflammasomes may be an important step in the immune system activation by carbamazepine, which can lead to hypersensitivity reactions in some patients. SIGNIFICANCE STATEMENT: A metabolite of carbamazepine, 2-hydroxyiminostilbene itself, and the damage-associated molecular patterns released from hepatocytes incubated with carbamazepine activated inflammasomes. The activation of inflammasomes may be an important step in the immune system activation by carbamazepine, which can lead to hypersensitivity reactions in some patients.

Rotenone Increases Isoniazid Toxicity but Does Not Cause Significant Liver Injury: Implications for the Hypothesis that Inhibition of the Mitochondrial Electron Transport Chain Is a Common Mechanism of Idiosyncratic Drug-Induced Liver Injury.

Idiosyncratic drug reactions (IDRs) significantly increase the risk of failure in drug development. The major IDR leading to drug candidate failure is idiosyncratic drug-induced liver injury (IDILI). Although most evidence suggests that IDRs are mediated by the immune system, there are other hypotheses, such as mitochondrial dysfunction. Many pharmaceutical companies routinely screen for mitochondrial toxicity in an attempt to "derisk" drug candidates. However, the basic hypothesis has never been rigorously tested. A major assay used for this screening involves measurement of inhibition of the mitochondrial electron transport chain. One study found that the combination of rotenone and isoniazid, which inhibit mitochondrial complex I and II, respectively, were synergistic in causing hepatocyte toxicity in vitro and suggested the combination of another drug that inhibited complex I would increase the risk of isoniazid-induced liver injury in patients. We tested this hypothesis in vivo where wild-type and PD-1-/- mice administered anti-CTLA-4, our impaired immune tolerance mouse model, were given 0.02% (w/v) rotenone in water or 0.1%, 0.05%, and 0.01% (w/w) rotenone alone or in combination with isoniazid in food. The cotreatment led to lethality in 100% of the animals receiving 0.1% rotenone and 0.2% isoniazid and 83% of the animals cotreated with 0.05% rotenone and 0.2% isoniazid in food. Nevertheless, there was no significant increase in GLDH or histological evidence of liver injury. No signs of toxicity were observed in any of the mice given rotenone or isoniazid alone. Even though inhibition of the mitochondrial electron transport chain did not lead to significant liver toxicity, it could provide danger signals that promote immune-mediated liver injury. However, rotenone did not significantly increase the liver injury induced by isoniazid in our impaired immune tolerance model. Overall, we conclude that inhibition of the mitochondrial electron transport chain is not a significant mechanism of IDILI.

Do In Vitro Assays Predict Drug Candidate Idiosyncratic Drug-Induced Liver Injury Risk?

In vitro assays are commonly used during drug discovery to try to decrease the risk of idiosyncratic drug-induced liver injury (iDILI). But how effective are they at predicting risk? One of the most widely used methods evaluates cell cytotoxicity. Cytotoxicity assays that used cell lines that are very different from normal hepatocytes, and high concentrations of drug, were not very accurate at predicting idiosyncratic drug reaction risk. Even cytotoxicity assays that use more biologically normal cells resulted in many false-positive and false-negative results. Assays that quantify reactive metabolite formation, mitochondrial injury, and bile salt export pump (BSEP) inhibition have also been described. Although evidence suggests that reactive metabolite formation and BSEP inhibition can play a role in the mechanism of iDILI, these assays are not very accurate at predicting risk. In contrast, inhibition of the mitochondrial electron transport chain appears not to play an important role in the mechanism of iDILI, although other types of mitochondrial injury may do so. It is likely that there are many additional mechanisms by which drugs can cause iDILI. However, simply measuring more parameters is unlikely to provide better predictive assays unless those parameters are actually involved in the mechanism of iDILI. Hence, a better mechanistic understanding of iDILI is required; however, mechanistic studies of iDILI are very difficult. There is substantive evidence that most iDILI is immune mediated; therefore, the most accurate assays may involve those that determine immune responses to drugs. New methods to manipulate immune tolerance may greatly facilitate development of more suitable methods.

The Effects of Immune Modulators on Amodiaquine-Induced Liver Injury.

If idiosyncratic drug-induced liver injury (IDILI) is immune mediated, then it is logical that immune modulators may be able to affect liver injury caused by a drug. We have previously shown that modulating the immune system by impairing programmed cell death protein (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) signaling, both receptors involved in immune tolerance, was capable of producing an animal model of amodiaquine (AQ) IDILI with characteristics very similar to IDILI in humans. Other immune modulators may also increase liver injury caused by drugs that cause IDILI in humans. In this study, myeloid derived suppressor cells (MDSCs), transforming growth factor beta (TGF-ß), and lymphocyte-activation gene 3 (LAG3) were targeted with antibodies, with and without PD-1 and CTLA-4 impairment. We found that anti-Gr1 antibodies used to deplete MDSCs led to a significant increase in AQ-induced liver injury in wild-type mice; however, the injury was actually less in PD-1-/- mice, with or without anti-CTLA-4, and it was less than we have previously observed in PD-1-/- mice combined with anti-CTLA-4 without anti-Gr1. Addition of anti-LAG3 or anti-TGF-ß antibodies produced a small increase ALT in AQ-treated wild-type mice. There was a significant increase in ALT in PD-1-/- mice co-treated with anti-LAG3 or anti-TGF-ß relative to AQ-treated wild-type mice. In the case of TGF-ß, this was further increased by the addition of anti-CTLA-4, but if anything, there appeared to be a paradoxical decrease when anti-CTLA-4 was combined with anti-LAG3. Overall, the results from this study were not always as expected, and they highlight the complexity of the immune response, in particular immune tolerance, which appears to be the dominant immune response to drugs that cause IDILI.

Dose-dependent acute liver injury with hypersensitivity features in humans due to a novel microsomal prostaglandin E synthase 1 inhibitor.

AIMS: LY3031207, a novel microsomal prostaglandin E synthase 1 inhibitor, was evaluated in a multiple ascending dose study after nonclinical toxicology studies and a single ascending dose study demonstrated an acceptable toxicity, safety and tolerability profile. METHODS: Healthy subjects were randomized to receive LY3031207 (25, 75 and 275 mg), placebo or celecoxib (400 mg) once daily for 28 days. The safety, tolerability and pharmacokinetic and pharmacodynamic profiles of LY3031207 were evaluated. RESULTS: The study was terminated when two subjects experienced drug-induced liver injury (DILI) after they had received 225 mg LY3031207 for 19 days. Liver biopsy from these subjects revealed acute liver injury with eosinophilic infiltration. Four additional DILI cases were identified after LY3031207 dosing had been stopped. All six DILI cases shared unique presentations of hepatocellular injury with hypersensitivity features and demonstrated a steep dose-dependent trend. Prompt discontinuation of the study drug and supportive medical care resulted in full recovery. Metabolites from metabolic activation of the imidazole ring were observed in plasma and urine samples from all subjects randomized to LY3031207 dosing. CONCLUSIONS: This study emphasized the importance of careful safety monitoring and serious adverse events management in phase I trials. Metabolic activation of the imidazole ring may be involved in the development of hepatotoxicity of LY3031207.

Nevirapine-induced liver lipid-SER inclusions and other ultrastructural aberrations.

Nevirapine (NVP) therapy is associated with a high risk of serious liver injury and skin rash. Treatment of Brown Norway rats with NVP causes an immune-mediated skin rash. Even though NVP does not cause serious liver injury in wildtype animals, incubation of hepatocytes with NVP leads to the release of presumably danger-associated molecular pattern molecules (DAMPs), which activate macrophages. In this study, we examined the liver biopsies of Brown Norway rats treated with NVP to determine the histologic correlate to the release of DAMPs by hepatocytes. In vivo, debris from necrotic hepatocytes and endothelial cells were present in the liver sinusoids, a condition that can trigger an immune response. In addition to mitochondrial, hepatocytic, and endothelial damage, the drug induced large hepatocytic inclusions composed of lipid droplets surrounded by concentric whorls of smooth endoplasmic reticulum (SER) cisternae-lipid-SER (LSER) inclusions, which were deposited in the sinusoids. NVP is lipid soluble, and these LSER inclusions may be sinks of NVP or its metabolites. LSERs are deposited in the blood stream where they may be picked up by lymph nodes and contribute to initiation of an immune response leading to serious liver injury or skin rash. LSERs migration from liver to the blood stream may signify a novel mechanism of drug exocytosis.

Supernatant from Hepatocyte Cultures with Drugs That Cause Idiosyncratic Liver Injury Activates Macrophage Inflammasomes.

There is increasing evidence that most idiosyncratic drug-induced liver injury (IDILI) is immune mediated, and in most cases, reactive metabolites appear to be responsible for the induction of this immune response. Reactive metabolites can cause cell damage with the release of damage-associated molecular patterns (DAMPs), which is thought to be involved in immune activation. Presumably, the reason that the liver is a common target of idiosyncratic drug reactions is because it is the major site of drug metabolism and reactive metabolite formation. Inflammasomes can be activated by DAMPs, and this may be a common mechanism by which DAMPs initiate an immune response. In this study, we tested the ability of drugs to induce the release of DAMPs that activate inflammasomes. The drugs tested were amodiaquine and nevirapine; both are associated with significant incidences of severe IDILI. The hepatocytes were a human hepatocarcinoma functional liver cell-4 (FLC-4) cell line. For the detection of inflammasome activation, we used the human macrophage cell line, THP-1 cells. We found that the supernatant from the incubation of both drugs with FLC-4 cells for 7 days led to increased caspase-1 activity and production of IL-1ß by THP-1 cells. However, amodiaquine alone also directly activated THP-1 cells. This is presumably because the myeloperoxidase in THP-1 cells can bioactivate amodiaquine to a reactive metabolite. In contrast, nevirapine requires cytochromes P450 for reactive metabolite formation and therefore required incubation with hepatocytes. These results support the hypothesis that reactive metabolites of drugs can cause the release of DAMPs, which in turn can activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by drugs, which in some patients can lead to IDILI. Our in vitro model is simple and convenient for evaluating inflammasome activation, and this may be a method to screen drugs for IDILI risk.

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

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 exceptions, 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.

Treatment of PD-1(-/-) mice with amodiaquine and anti-CTLA4 leads to liver injury similar to idiosyncratic liver injury in patients.

UNLABELLED: The mechanism of idiosyncratic drug-induced liver injury (IDILI) remains poorly understood, to a large degree because of the lack of a valid animal model. Recently, we reported an animal model in which treatment of female C57BL/6 mice with amodiaquine (AQ) resulted in mild liver injury with a delayed onset and resolution despite continued treatment. Such adaptation is a common outcome in the IDILI caused by drugs that can cause liver failure. We had hypothesized that most IDILI is immune-mediated and adaptation represents immune tolerance. In this study we found that AQ treatment of Cbl-b(-/-) and PD-1(-/-) mice, which have impaired immune tolerance, resulted in a slightly greater injury. Cotreatment of C57BL/6 with AQ and anti-CTLA4 also resulted in a greater increase in ALT than treatment with AQ alone; however, these mice also had an increase in T regulatory (Treg) cells and T helper cells expressing PD-1 and CTLA4. The increase in these cells implies the induction of immune tolerance, and the alanine aminotransferase (ALT) activity in these mice returned to normal despite continued treatment. Cotreatment of PD-1(-/-) mice with anti-CTLA4 antibody and AQ resulted in the greatest increase in ALT (200-300 U/L), and necroinflammatory responses characterized by portal infiltration of lymphocytes with interface hepatitis. The lymphocyte infiltration included T and B cells, and the CD8(+) T cells produced perforin and granzyme. In addition, the ALT activity in PD-1(-/-) mice cotreated with anti-CTLA4 antibody and AQ did not return to normal, as it had in other mice. CONCLUSION: We report here the first animal model of IDILI that is similar to the IDILI that occurs in humans, and it was accomplished by inhibiting immune tolerance.

Protein Targets of Isoniazid-Reactive Metabolites in Mouse Liver in Vivo.

Isoniazid (INH) has been a first-line drug for the treatment of tuberculosis for more than 40 years. INH is well-tolerated by most patients, but some patients develop hepatitis that can be severe in rare cases or after overdose. The mechanisms underlying the hepatotoxicity of INH are not known, but covalent binding of reactive metabolites is known to occur in animals and is suspected in human cases. A major unresolved question is the identity of the liver proteins that are modified by INH metabolites. Treating mice with INH leads to accumulation of isonicotinoyl-lysine residues on numerous proteins in the hepatic S9 fraction. Analysis of this fraction by SDS-PAGE followed by tryptic digestion of bands and LC-MS/MS revealed a single adducted peptide derived from d-dopachrome decarboxylase. When a tryptic digest of whole S9 was applied to anti-INH antibody immobilized on beads, only 12 peptides were retained, 5 of which clearly contained isonicotinoyl-lysine adducts and could be confidently assigned to 5 liver proteins. In another experiment, undigested S9 fractions from INA-treated and untreated (UT) mice were adsorbed in parallel on anti-INA beads and the retained proteins were digested and analyzed by LC-MS/MS. The INA-S9 digest showed 1 adducted peptide that was associated with a unique protein whose identity was corroborated by numerous nonadducted peptides in the digest and 13 other proteins identified only by multiple nonadducted peptides. None of these 14 proteins was associated with any peptides present in the UT-S9 fraction. Overall, we identified 7 mouse liver proteins that became adducted by INH metabolites in vivo. Of these 7 INH target proteins, only 2 have been previously reported as targets of any reactive metabolite in vivo.

Mechanism of isoniazid-induced hepatotoxicity: then and now.

Isoniazid (INH) remains a mainstay for the treatment of tuberculosis despite the fact that it can cause liver failure. Previous mechanistic hypotheses have classified this type of drug-induced liver injury (DILI) as 'metabolic idiosyncrasy' which was thought not to involve an immune response and was mainly due to the bioactivation of the acetylhydrazine metabolite. However, more recent studies support an alternative hypothesis, specifically, that INH itself is directly bioactivated to a reactive metabolite, which in some patients leads to an immune response and liver injury. Furthermore, there appear to be two phenotypes of INH-induced liver injury. Most cases involve mild liver injury, which resolves with immune tolerance, while other cases appear to have a more severe phenotype that is associated with the production of anti-drug/anti-CYP P450 antibodies and can progress to liver failure.