Publications by authors named "Jack Uetrecht"

104 Publications

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

Int J Mol Sci 2021 Mar 14;22(6). Epub 2021 Mar 14.

Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON M5S 1A8, Canada.

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.
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http://dx.doi.org/10.3390/ijms22062954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998339PMC
March 2021

Testing Possible Risk Factors for Idiosyncratic Drug-Induced Liver Injury Using an Amodiaquine Mouse Model and Co-treatment with 1-Methyl-d-Tryptophan or Acetaminophen.

ACS Omega 2021 Feb 7;6(7):4656-4662. Epub 2021 Feb 7.

Leslie Dan Faculty of Pharmacy, Department of Pharmaceutical Sciences, University of Toronto; 144 College Street, Toronto, Ontario M5S 3M2, Canada.

Idiosyncratic drug reactions are unpredictable adverse reactions. Although most such adverse reactions appear to be immune mediated, their exact mechanism(s) remain elusive. The idiosyncratic drug reaction most associated with serious consequences is idiosyncratic drug-induced liver injury (IDILI). We have developed a mouse model of amodiaquine (AQ)-induced liver injury that reflects the clinical characteristics of IDILI in humans. This was accomplished by impairing immune tolerance by using PD-1 mice and an antibody against CTLA-4. PD-1 and CTLA-4 are known negative regulators of lymphocyte activation, which promote immune tolerance. Immune checkpoint inhibitors have become important tools for the treatment of cancer. However, as in our model, immune checkpoint inhibitors increase the risk of IDILI with drugs that have an incidence of causing liver injury. Agents such as 1-methyl-d-tryptophan (D-1-MT), an inhibitor of the immunosuppressive indoleamine 2,3-dioxygenase (IDO) enzyme, have also been proposed as anti-cancer treatments. Another possible risk factor for the induction of an immune response is the release of danger-associated molecular patterns (DAMPs). Acetaminophen (APAP) is known to cause acute liver injury, and it is likely to cause the release of DAMPs. Therefore, either of these agents could increase the risk of IDILI, although through different mechanisms. If true, then this would have clinical implications. We found that co-treatment with D-1-MT paradoxically decreased liver injury in our model, and although APAP appeared to slightly increase AQ-induced liver injury, the difference was not significant. Such results highlight the complexity of the immune response, which makes potential interactions difficult to predict.
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http://dx.doi.org/10.1021/acsomega.0c05352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7905801PMC
February 2021

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

Epilepsy Behav 2021 Apr 25;117:107844. Epub 2021 Feb 25.

Department of Pharmacology & Toxicology, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Canada.

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.
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http://dx.doi.org/10.1016/j.yebeh.2021.107844DOI Listing
April 2021

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

Chem Res Toxicol 2021 Mar 22;34(3):849-856. Epub 2021 Feb 22.

Leslie Dan Faculty of Pharmacy, Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario M5S 3M2, Canada.

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.
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http://dx.doi.org/10.1021/acs.chemrestox.0c00485DOI Listing
March 2021

Investigating the Mechanism of Trimethoprim-Induced Skin Rash and Liver Injury.

Toxicol Sci 2021 Feb;180(1):17-25

Leslie Dan Faculty of Pharmacy, Faculty of Medicine, University of Toronto, Toronto M5S3M2, Canada.

Trimethoprim (TMP)-induced skin rash and liver injury are likely to involve the formation of reactive metabolites. Analogous to nevirapine-induced skin rash, 1 possible reactive metabolite is the sulfate conjugate of α-hydroxyTMP, a metabolite of TMP. We synthesized this sulfate and found that it reacts with proteins in vitro. We produced a TMP-antiserum and found covalent binding of TMP in the liver of TMP-treated rats. However, we found that α-hydroxyTMP is not a substrate for human sulfotransferases, and we did not detect covalent binding in the skin of TMP-treated rats. Although less reactive than the sulfate, α-hydroxyTMP was found to covalently bind to liver and skin proteins in vitro. Even though there was covalent binding to liver proteins, TMP did not cause liver injury in rats or in our impaired immune tolerance mouse model that has been able to unmask the ability of other drugs to cause immune-mediated liver injury. This is likely because there was much less covalent binding of TMP in the livers of TMP-treated mice than TMP-treated rats. It is possible that some patients have a sulfotransferase that can produce the reactive benzylic sulfate; however, α-hydroxyTMP, itself, has sufficient reactivity to covalently bind to proteins in the skin and may be responsible for TMP-induced skin rash. Interspecies and interindividual differences in TMP metabolism may be 1 factor that determines the risk of TMP-induced skin rash. This study provides important data required to understand the mechanism of TMP-induced skin rash and drug-induced skin rash in general.
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http://dx.doi.org/10.1093/toxsci/kfaa182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916736PMC
February 2021

Reactive metabolite of gefitinib activates inflammasomes: implications for gefitinib-induced idiosyncratic reaction.

J Toxicol Sci 2020 ;45(11):673-680

Department of Pharmaceutical Scinces, Faculty of Pharmacy, University of Toronto, Canada.

The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) have been approved for non-small cell lung cancer. Although EGFR TKIs are less toxic than traditional cytotoxic therapies, they cause many severe idiosyncratic drug reactions. Reactive metabolites can cause cellular damage with the release of danger-associated molecular patterns (DAMPs), which is thought to be involved in immune activation. Inflammasomes can be activated by DAMPs, and this may be a common mechanism by which DAMPs initiate an immune response. We tested the ability of afatinib, dacomitinib, erlotinib, gefitinib, and osimertinib to induce the release of DAMPs that activate inflammasomes. Human hepatocarcinoma functional liver cell-4 (FLC-4) cells were used for bioactivation of drugs, and the detection of inflammasome activation was performed with the human macrophage cell line, THP-1 cells. Gefitinib is known to be oxidized to a reactive iminoquinone metabolite. We found that the supernatant from the incubation of gefitinib with FLC-4 cells for 7 days led to increased caspase-1 activity and production of IL-1ß by THP-1 cells. In the supernatant of FLC-4 cells with gefitinib, the heat shock protein (HSP) 40, 70 and 90 were significantly increased. In addition, activated THP-1 cells secreted high mobility group box 1 (HMGB1) protein. These results support the hypothesis that the reactive iminoquinone metabolite can cause the release of DAMPs from hepatocytes, which in turn, can activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by gefitinib, which in some patients, can cause immune-related adverse events.
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http://dx.doi.org/10.2131/jts.45.673DOI Listing
November 2020

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

J Autoimmun 2020 11 5;114:102514. Epub 2020 Aug 5.

Indiana University School of Medicine, Indianapolis, IN, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jaut.2020.102514DOI Listing
November 2020

Mechanistic Studies of Idiosyncratic DILI: Clinical Implications.

Authors:
Jack Uetrecht

Front Pharmacol 2019 26;10:837. Epub 2019 Jul 26.

Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada.

The idiosyncratic nature of idiosyncratic drug-induced liver injury (IDILI) makes mechanistic studies very difficult, and little is known with certainty. However, the fact that the IDILI caused by some drugs is associated with specific HLA genotypes provides strong evidence that it is mediated by the adaptive immune system. This is also consistent with the histology and the general characteristics of IDILI. However, there are other mechanistic hypotheses. Various and systems have been used to test hypotheses. Two other hypotheses are mitochondrial injury and inhibition of the bile salt export pump. It is possible that these mechanisms are responsible for some cases of IDILI or that these mechanisms are complementary and are involved in initiating an immune response. In general, it is believed that the initiation of an immune response requires activation of antigen-presenting cells by molecules such as danger-associated molecular pattern molecules (DAMPs). An attractive hypothesis for the mechanism by which DAMPs induce an immune response is through the activation of inflammasomes. The dominant immune response in the liver is immune tolerance, and it is only when immune tolerance fails that significant liver injury occurs. Consistent with this concept, an animal model was developed in which immune checkpoint inhibition unmasked the ability of drugs to cause liver injury. Although it appears that the liver damage is mediated by the adaptive immune system, an innate immune response is required for an adaptive immune response. The innate immune response is not dependent on specific HLA genes or T cell receptors and may occur in most patients and animals treated with a drug that can cause IDILI. Studies of the subclinical innate immune response to drugs may provide important mechanistic clues and provide a method to screen drugs for their potential to cause IDILI.
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http://dx.doi.org/10.3389/fphar.2019.00837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6676790PMC
July 2019

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

Drug Metab Dispos 2019 10 19;47(10):1093-1096. Epub 2019 Jul 19.

Department of Cardiovascular Pharmacotherapy and Toxicology, Osaka University of Pharmaceutical Sciences, Osaka, Japan (R.K., Y.I., T.H.) and Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada (J.U.).

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-1 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.
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http://dx.doi.org/10.1124/dmd.119.087981DOI Listing
October 2019

Mechanisms of idiosyncratic drug-induced liver injury.

Authors:
Jack Uetrecht

Adv Pharmacol 2019 18;85:133-163. Epub 2019 Jan 18.

Faculties of Pharmacy and Medicine, University of Toronto, Toronto, ON, Canada. Electronic address:

Idiosyncratic drug-induced liver injury (IDILI) is a significant problem. Little is known with certainty about the mechanisms of IDILI. However, there is growing evidence that most IDILI is immune mediated and caused by reactive metabolites. The two major and complementary hypotheses that link reactive metabolite formation with the induction of an immune response that can lead to IDILI are the hapten and danger hypotheses. Specifically, a reactive metabolite can bind to proteins and make them foreign; however, without activation of antigen presenting cells (APCs), the immune response will be immune tolerance. Not all reactive metabolites are associated with the same risk of causing IDILI. If the reactive metabolite can also cause some cell damage, this can lead to the release of danger-associated molecular pattern molecules (DAMPs) that activate APCs. Other hypotheses for the mechanism of IDILI include mitochondrial injury, inhibition of the bile salt export pump, and endoplasmic reticulum stress. These mechanisms may be complementary to the danger hypothesis in that they may cause the release of DAMPs. None of these hypotheses has been adequately tested. Previous animal models have had characteristics very different from IDILI in humans and are unlikely to involve the same mechanism. However, impairment of immune tolerance has led to a model with characteristics very similar to IDILI in humans. This will make it possible to rigorously test hypotheses. A better mechanistic understanding of IDILI should lead to better methods to screen drug candidates for IDILI risk and treat IDILI when it occurs.
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http://dx.doi.org/10.1016/bs.apha.2018.12.001DOI Listing
February 2020

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.

Chem Res Toxicol 2019 07 28;32(7):1423-1431. Epub 2019 Jun 28.

Department of Pharmaceutical Sciences, Faculty of Pharmacy , University of Toronto , 144 College Street , Toronto , Ontario M5S 3M2 , Canada.

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.
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http://dx.doi.org/10.1021/acs.chemrestox.9b00116DOI Listing
July 2019

Involvement of CCL2/CCR2 macrophage recruitment in amodiaquine-induced liver injury.

J Immunotoxicol 2019 12 21;16(1):28-33. Epub 2019 Jan 21.

Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.

Evidence suggests that macrophages may play a role in the development of idiosyncratic drug-induced liver injury (IDILI). However, there has yet to be a clear link between macrophage activation and the inflammatory infiltrate that is characteristic of IDILI. A major chemokine involved in the recruitment of macrophages into the liver is C-C motif chemokine ligand 2 (CCL2)/monocyte chemoattractant protein 1 (MCP1). Therefore, we tested the effect of this chemokine in an animal model of IDILI. Specifically, amodiaquine (AQ), which is known to cause IDILI in humans, causes mild liver injury in wild-type C57BL/6 mice that resolves despite continued AQ treatment, but it causes more severe liver injury that does not resolve in PD-1 mice co-treated with anti-CTLA-4 to impair immune tolerance. CCR2 mice treated with AQ were not protected from the expected AQ-induced liver injury seen in wild-type C57BL/6 mice. In contrast, anti-CCL2 antibodies attenuated the liver injury caused by AQ in the impaired immune tolerance model. The difference in response of the two models is likely due to a difference in the IDILI mechanism; the mild injury in wild-type animals is mediated by NK cells, while the more serious injury in the impaired immune tolerance model requires CD8 T-cells. The results from these experiments provide evidence that macrophage infiltration into the liver may not be involved in mild IDILI mediated by the innate immune system, but it does appear necessary in more severe IDILI involving cytotoxic T-cells.
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http://dx.doi.org/10.1080/1547691X.2018.1516014DOI Listing
December 2019

The skin as a metabolic and immune-competent organ: Implications for drug-induced skin rash.

J Immunotoxicol 2019 12 14;16(1):1-12. Epub 2018 Oct 14.

Amgen Research, Thousand Oaks, CA, USA.

Current advances in the study of cutaneous adverse drug reactions can be attributed to the recent understanding that the skin is both a metabolically and immunologically competent organ. The ability of the skin to serve as a protective barrier with limited drug biotransformation ability, yet highly active immune function, has provided insights into its biological capability. While the immune response of the skin to drugs is vastly different from that of the liver due to evolutionary conditioning, it frequently occurs in response to various drug classes and manifests as a spectrum of hypersensitivity reactions. The skin is a common site of adverse and idiosyncratic drug reactions; drug-specific T-cells, as well as involvement of an innate immune response, appear to be key mechanistic drivers in such scenarios. Association of other factors such as human leukocyte antigen (HLA) polymorphisms may play a significant role for particular drugs. This review aims to integrate emerging findings into proposed mechanisms of drug metabolism and immunity in the skin that are likely responsible for rashes and other local allergic responses. These unique biological aspects of the skin, and their translation into implications for drug development and the use of animal models, will be discussed.
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http://dx.doi.org/10.1080/1547691X.2018.1514444DOI Listing
December 2019

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

Drug Metab Dispos 2018 11 18;46(11):1658-1669. Epub 2018 Jul 18.

Safer Medicines Trust, Kingsbridge, United Kingdom (J.G.K.); and Faculties of Pharmacy and Medicine, University of Toronto, Toronto, Ontario, Canada (J.U.)

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.
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http://dx.doi.org/10.1124/dmd.118.082719DOI Listing
November 2018

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

Chem Res Toxicol 2018 08 10;31(8):739-744. Epub 2018 Jul 10.

Department of Pharmaceutical Sciences, Faculty of Pharmacy , University of Toronto , Toronto , Ontario M5S 3H7 , Canada.

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-β), 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-β 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-β relative to AQ-treated wild-type mice. In the case of TGF-β, 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.
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http://dx.doi.org/10.1021/acs.chemrestox.8b00091DOI Listing
August 2018

Use of an animal model to test whether non-alcoholic fatty liver disease increases the risk of idiosyncratic drug-induced liver injury.

J Immunotoxicol 2018 12;15(1):90-95

a Department of Pharmaceutical Sciences, Faculty of Pharmacy , University of Toronto , Toronto , Ontario , Canada.

Clinical evidence suggests that most idiosyncratic drug-induced liver injury (IDILI) is immune-mediated. The danger hypothesis suggests that liver injury and inflammation would increase the risk of an immune response leading to IDILI. Therefore, a reasonable hypothesis would be that an underlying chronic liver disease such as non-alcoholic steatohepatitis (NASH) would increase the risk of developing IDILI due to inflammation and release of danger signals from damaged cells. In order to test this hypothesis, mice were fed a methionine-/choline-deficient (MCD) diet that produces a consistent NASH phenotype, along with amodiaquine (AQ) - a drug known to cause IDILI in humans. This study employed both wild-type C57BL/6 mice and PD-1 mice co-treated with anti-CTLA-4 antibodies. The PD-1 + anti-CTLA-4 model produces an immune-mediated liver injury very similar to the idiosyncratic liver injury observed in humans. The liver injury observed in the present experiment was dominated by the injury caused by the MCD diet; there was no significant difference between mice treated with the MCD diet alone and those also treated with AQ, whether in wild-type mice of the PD-1 model. Therefore, the MCD diet, which results in a state that mimics NASH, did not appear to increase the liver injury associated with AQ treatment. Ultimately, an animal model is just that - only a model, and cannot provide a definitive answer to clinical questions. However, given the difficulty of performing clinical studies with appropriate control populations, the present results provide important evidence to support a general clinical finding that underlying liver injury does not usually increase the risk of IDILI.
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http://dx.doi.org/10.1080/1547691X.2018.1467982DOI Listing
December 2018

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

Ultrastruct Pathol 2018 Mar-Apr;42(2):108-115. Epub 2018 Feb 9.

a Section of Histopathology , National Eye Institute, NIH , Bethesda , Maryland, USA.

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.
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http://dx.doi.org/10.1080/01913123.2017.1422831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055918PMC
August 2018

Editor's Highlight: An Impaired Immune Tolerance Animal Model Distinguishes the Potential of Troglitazone/Pioglitazone and Tolcapone/Entacapone to Cause IDILI.

Toxicol Sci 2018 02;161(2):412-420

Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 3M2, Canada.

We have developed an animal model of amodiaquine-induced liver injury that has characteristics very similar to idiosyncratic drug-induced liver injury (IDILI) in humans by impairing immune tolerance using a PD1-/- mouse and cotreatment with anti-CTLA-4. In order to test the usefulness of this model as a general model for human IDILI risk, pairs of drugs with similar structures were tested, one of which is associated with a relatively high risk of IDILI and the other not. One such pair is troglitazone and pioglitazone; troglitazone has caused fatal cases of IDILI while pioglitazone is quite safe. Another pair is tolcapone and entacapone; tolcapone can cause serious IDILI; in contrast, although entacapone has been reported to cause liver injury, it is relatively safe. PD1-/- mice treated with anti-CTLA-4 and troglitazone or tolcapone displayed liver injury as determined by ALT levels and histology, while pioglitazone and entacapone showed less signs of liver injury. One possible mechanism by which drugs could induce an immune response leading to IDILI is by causing the release of danger-associated molecular pattern molecules that activate inflammasomes. We found that the supernatants from incubations of troglitazone, tolcapone, or entacapone with hepatocytes were also able to activate inflammasomes in macrophages, while the supernatant from pioglitazone incubations did not. These results are consistent with an immune mechanism for troglitazone- and tolcapone-induced IDILI and add to the evidence that this may be a general model for IDILI.
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http://dx.doi.org/10.1093/toxsci/kfx219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837423PMC
February 2018

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

Br J Clin Pharmacol 2018 Jan 8;84(1):179-188. Epub 2017 Oct 8.

Eli Lilly and Company, Indianapolis, Indiana, USA.

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.
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http://dx.doi.org/10.1111/bcp.13423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736846PMC
January 2018

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

Chem Res Toxicol 2017 06 2;30(6):1327-1332. Epub 2017 Jun 2.

Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto , Toronto, Ontario M5S 3M2, Canada.

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-1β 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.
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http://dx.doi.org/10.1021/acs.chemrestox.7b00065DOI Listing
June 2017

Effects of immunization and checkpoint inhibition on amodiaquine-induced liver injury.

J Immunotoxicol 2017 12;14(1):89-94

a Department of Pharmaceutical Sciences, Faculty of Pharmacy , University of Toronto , Toronto , Ontario , Canada.

If idiosyncratic drug-induced liver injury (IDILI) is immune-mediated, it is possible that an individual's prior exposure to antigens may affect their susceptibility to IDILI. An individual's repertoire of memory immune cells is shaped by every past exposure to antigens. Subsequent drug-induced adverse drug reactions may therefore involve an immune cell's cross reactivity between a prior antigen and resulting drug-modified proteins. Therefore in this experiment, mice were immunized with amodiaquine (AQ)-modified hepatic proteins to mimic a previous exposure; treated with a RIBI adjuvant and anti-CD40 antibodies to stimulate an immune response; and, treated with anti-PD1 and anti-CTLA-4 antibodies prior to AQ treatment in order to overcome immune tolerance. This treatment led to greater liver injury than treatment with AQ alone. However, the mice did not develop serious liver injury. PD1 mice were then immunized and treated with AQ and anti-CTLA-4 antibodies so that immune tolerance would be impaired, both during immunization and also during AQ treatment. However, even this did not result in liver failure, and the liver injury was not significantly increased relative to un-immunized PD1 mice treated with anti-CTLA-4 and AQ. From these results we conclude that, although previous antigen exposure may affect the risk of IDILI, it appears that a very strong stimulus is required, and impairing immune tolerance remains the most effective method for producing an animal model of IDILI.
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http://dx.doi.org/10.1080/1547691X.2017.1290716DOI Listing
December 2017

Immune mechanisms of idiosyncratic drug-induced liver injury.

J Clin Transl Res 2017 Feb 12;3(1):145-156. Epub 2017 Feb 12.

Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.

Idiosyncratic drug reactions (IDRs) continue to be an important issue. Specifically, idiosyncratic drug-induced liver injury (IDILI) is the most likely IDR to lead to drug withdrawal, and it accounts for a significant portion of all cases of acute liver failure. In addition, IDRs are unpredictable and their mechanisms are not well understood. There is increasing clinical evidence that most IDILI is immune mediated. Several immune mediated mechanistic hypotheses exist such as the hapten and danger hypothesis; however, they do not completely explain the idiosyncratic nature of these reactions. Extensive mechanistic studies are needed to better understand these reactions; however, it is impossible to do controlled experiments in humans, and previous animal models did not properly model IDILI. If IDILI is immune mediated and the major factor preventing liver injury in patients is immune tolerance, then a plausible method to develop an animal model of IDILI would be to impair immune tolerance. This hypothesis has shown promise in developing valid animal models of IDILI as demonstrated by a halothane induced liver injury mouse model developed by depleting myeloid derived suppressor cells (MDSCs), as well as an amodiaquine-, isoniazid-and nevirapine-induced liver injury mouse model developed by impairing immune tolerance by blocking PD-1 and CTLA-4, two immune checkpoint inhibitors. Further characterization and validation of these models is required; however, it is likely that they will make it possible to perform mechanistic studies that have been impossible in the past. : Idiosyncratic drug-induced liver injury can be serious leading to liver transplantation or death. Their idiosyncratic nature makes mechanistic studies very difficult. However, with the development of the first animal model that is similar to the liver injury that occurs in humans, it will be possible to study the mechanisms involved. With a better mechanistic understanding it should be possible to test drug candidates and produce safer drugs. In addition, it should be possible to design better treatments when drug-induced liver injury does occur.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410666PMC
February 2017

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

Chem Res Toxicol 2017 01 8;30(1):295-314. Epub 2016 Nov 8.

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

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.
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http://dx.doi.org/10.1021/acs.chemrestox.6b00357DOI Listing
January 2017

Exploring an animal model of amodiaquine-induced liver injury in rats and mice.

J Immunotoxicol 2016 09 14;13(5):694-712. Epub 2016 Jul 14.

a Faculty of Pharmacy , University of Toronto , Toronto , Ontario , Canada ;

Amodiaquine (AQ) is associated with a relatively high incidence of idiosyncratic drug-induced liver injury (IDILI) and agranulocytosis. A previous study reported that a combination of high dose AQ and glutathione (GSH) depletion led to liver injury. However, the characteristics of this toxicity were very different from AQ-induced liver injury in humans. We developed a model of AQ-induced liver injury with characteristics similar to the injury in humans by treating mice with lower doses of AQ for several weeks. In this study we found that not only did GSH depletion not increase AQ covalent binding to hepatic proteins at this lower dose, but also it paradoxically prevented the liver injury. We extended the model to rats and found AQ treatment led to a mild delayed onset liver injury that resolved despite continued treatment with AQ. Immunohistochemistry indicated the presence of Kupffer cell activation, apoptosis and hepatocyte proliferation in the liver. There was also an increase in serum IL-2, IL-5, IL-9, IL-12, MCP-1 and TGFβ, but a decrease in leptin. Coincident with the elevated serum ALT, the number of liver CD4(+) T-cells, IL-17 secreting cells and TH17/Treg cells increased at Week 3 and decreased during continued treatment. Increases in NK1.1+ cells and activated M2 macrophages were also observed during liver injury. These results suggest that the outcome of the liver injury was determined by the balance between effector and regulatory cells. Co-treatment with cyclosporin prevented AQ-induced liver injury, which supports an immune mechanism. Retinoic acid (RA), which has been reported to enhance natural killer (NK) cell activity, exacerbated AQ-induced liver injury. These results suggest that AQ-induced IDILI is immune mediated and the subsequent adaptation appears to represent immune tolerance.
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http://dx.doi.org/10.3109/1547691X.2016.1166167DOI Listing
September 2016

Deferiprone-induced agranulocytosis: 20 years of clinical observations.

Am J Hematol 2016 10 4;91(10):1026-31. Epub 2016 Aug 4.

Departments of Medicine and Hematology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.

Use of the iron chelator deferiprone for treatment of iron overload in thalassemia patients is associated with concerns over agranulocytosis, which requires weekly absolute neutrophil counts (ANC). Here, we analyze all episodes of agranulocytosis (n = 161) and neutropenia (n = 250) during deferiprone use in clinical trials (CT) and postmarketing surveillance programs (PMSP). Rates of agranulocytosis and neutropenia in CT were 1.5% and 5.5%, respectively. Of the agranulocytosis cases, 61% occurred during the first 6 months of therapy and 78% during the first year. These events appeared to be independent of dose, and occurred three times more often in females than males. Their duration was not significantly shortened by use of G-CSF. No patient with baseline neutropenia (n = 12) developed agranulocytosis during treatment, which raises questions about the validity of prior neutropenia as a contraindication to use. Only 1/7 novel neutropenia cases in CT progressed to agranulocytosis with continued treatment, indicating that neutropenia does not necessarily lead to agranulocytosis. The agranulocytosis fatality rate was 0% in CT and 15/143 (11%) in PMSP. Rechallenge with deferiprone produced agranulocytosis in 75% of patients in whom the event had already occurred, and in 10% with previous neutropenia. Weekly ANC monitoring allows early detection and interruption of therapy, but does not prevent agranulocytosis from occurring. Its relevance appears to decrease after the first year of therapy, when agranulocytosis occurs less often. Based upon analysis of data collected over the past 20 years, it appears that patient education may be the key to minimizing agranulocytosis-associated risks during deferiprone therapy. Am. J. Hematol. 91:1026-1031, 2016. © 2016 The Authors. American Journal of Hematology Published by Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/ajh.24479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129477PMC
October 2016

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

Chem Res Toxicol 2016 06 5;29(6):1064-72. Epub 2016 May 5.

Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, Ontario M5S 3M2, Canada.

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.
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http://dx.doi.org/10.1021/acs.chemrestox.6b00098DOI Listing
June 2016

Mechanism of isoniazid-induced hepatotoxicity: then and now.

Br J Clin Pharmacol 2016 06 25;81(6):1030-6. Epub 2016 Feb 25.

Department of Medicine, Vanderbilt School of Medicine, Nashville, TN, 37232, USA.

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.
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http://dx.doi.org/10.1111/bcp.12885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4876174PMC
June 2016

The Combination of Anti-CTLA-4 and PD1-/- Mice Unmasks the Potential of Isoniazid and Nevirapine To Cause Liver Injury.

Chem Res Toxicol 2015 Dec 20;28(12):2287-91. Epub 2015 Nov 20.

Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto , Toronto, Ontario M5S 3M2, Canada.

Our laboratory recently reported what we believe is the first valid animal model of idiosyncratic drug-induced liver injury (IDILI) by treating PD1-/- mice with an anti-CTLA-4 antibody and amodiaquine (AQ). PD1 and CTLA-4 are important immune checkpoint receptors that are involved in inducing immune tolerance. This model was able to produce significant liver injury that looks very similar to the liver injury seen in humans. Although this model was shown to work with AQ, the question becomes whether blocking immune tolerance would unmask the potential of other drugs to cause IDILI. In this study, we tested isoniazid and nevirapine, both drugs with significant histories of causing IDILI in humans even though they do not cause significant injury in animals with doses that result in therapeutic blood levels. Both drugs in combination with these immune checkpoint inhibitors caused mild but significant delayed onset liver injury, which is similar to the mild injury that they can cause in humans. INH-induced liver injury in this model was associated with an increase in NK cells, while NVP-induced liver injury was associated with a greater increase in CD8 T cells. Although the liver injury caused by these drugs in this model was mild, these results suggest that impairing immune tolerance may be a general method for unmasking the potential of drugs to cause IDILI and therefore provide a screening tool for drug development.
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http://dx.doi.org/10.1021/acs.chemrestox.5b00305DOI Listing
December 2015

The Role of CD8 T Cells in Amodiaquine-Induced Liver Injury in PD1-/- Mice Cotreated with Anti-CTLA-4.

Chem Res Toxicol 2015 Aug 15;28(8):1567-73. Epub 2015 Jul 15.

Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario Canada.

The mechanism of idiosyncratic drug-induced liver injury (IDILI) remains poorly understood, in part due to the lack of a valid animal model. Clinical evidence suggests that most IDILI is immune mediated, and the major factor preventing liver injury in most patients is immune tolerance. Many attempts have been made in the past to develop an animal model of IDILI, but none had characteristics similar to those of IDILI in humans, and presumably they involved a different mechanism. Recently our laboratory reported a model of amodiaquine (AQ)-induced IDILI using PD1-/- mice and an anti-CTLA4 antibody. This may be the first valid animal model of IDILI because it mimics the characteristics of IDILI in humans. The current study extended the duration of AQ treatment to see if this model would lead to liver failure and to further characterize the associated immune response. Although AQ treatment was extended to 10 weeks and total bilirubin levels were significantly elevated compared to control, there was no further increase from weeks 7 to 10, and the animals did not develop overt liver failure. Mice treated with AQ and anti-CTLA4 had a significant increase in percentage of hepatic CD4, CD8, Th17, and Treg cells after 10 weeks of AQ treatment, as well as significantly decreased NK cells. CD8 T cells have been implicated in several serious idiosyncratic drug reactions, and we used an anti-CD8 antibody to deplete CD8 T cells to study their involvement in this liver injury. We found that depletion of CD8 T cells protected mice from AQ-induced liver injury in this model, which strongly suggests that they are responsible for the liver damage. This is consistent with the finding of CD8 T cells in liver biopsies of human IDILI and may lead the way to an effective treatment for serious IDILI.
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http://dx.doi.org/10.1021/acs.chemrestox.5b00137DOI Listing
August 2015