In Vitro Hepatotoxicity of Routinely Used Opioids and Sedative Drugs.

A hepatocyte cell line was used to determine the hepatotoxicity of sedatives and opioids, as the hepatotoxicity of these drugs has not yet been well characterized. This might pose a threat, especially to critically ill patients, as they often receive high cumulative doses for daily analgosedation and often already have impaired liver function due to an underlying disease or complications during treatment. A well-established biosensor based on HepG2/C3A cells was used for the determination of the hepatotoxicity of commonly used sedatives and opioids in the intensive care setting (midazolam, propofol, s-ketamin, thiopental, fentanyl, remifentanil, and sufentanil). The incubation time was 2 × 3 days with clinically relevant (Cmax) and higher concentrations (C5× and C10×) of each drug in cell culture medium or human plasma. Afterward, we measured the cell count, vitality, lactate dehydrogenase (LDH), mitochondrial dehydrogenase activity, cytochrome P 450 1A2 (CYP1A2), and albumin synthesis. All tested substances reduced the viability of hepatocyte cells, but sufentanil and remifentanil showed more pronounced effects. The cell count was diminished by sufentanil in both the medium and plasma and by remifentanil only in plasma. Sufentanil and remifentanil also led to higher values of LDH in the cell culture supernatant. A reduction of mitochondrial dehydrogenase activity was seen with the use of midazolam and s-ketamine. Microalbumin synthesis was reduced in plasma after its incubation with higher concentrations of sufentanil and remifentanil. Remifentanil and s-ketamine reduced CYP1A2 activity, while propofol and thiopental increased it. Our findings suggest that none of the tested sedatives and opioids have pronounced hepatotoxicity. Sufentanil, remifentanil, and s-ketamine showed moderate hepatotoxic effects in vitro. These drugs should be given with caution to patients vulnerable to hepatotoxic drugs, e.g., patients with pre-existing liver disease or liver impairment as part of their underlying disease (e.g., hypoxic hepatitis or cholestatic liver dysfunction in sepsis). Further studies are indicated for this topic, which may use more complex cell culture models and global pharmacovigilance reports, addressing the limitation of the used cell model: HepG2/C3A cells have a lower metabolic capacity due to their low levels of CYP enzymes compared to primary hepatocytes. However, while the test model is suitable for parental substances, it is not for toxicity testing of metabolites.

Clinical Pattern of Tolvaptan-Associated Liver Injury in Trial Participants With Autosomal Dominant Polycystic Kidney Disease (ADPKD): An Analysis of Pivotal Clinical Trials.

RATIONALE & OBJECTIVE: Tolvaptan is associated with risk of drug-induced liver injury when used to treat autosomal dominant polycystic kidney disease (ADPKD). After this risk was described based on the clinical trials TEMPO 3:4 and TEMPO 4:4, additional data from the REPRISE trial and a long-term extension of TEMPO 4:4, REPRISE, and other tolvaptan trials in ADPKD have become available. To further characterize the hepatic safety profile of tolvaptan, an analysis of the expanded dataset was conducted. STUDY DESIGN: Analysis of safety data from prospective clinical trials of tolvaptan. SETTING & PARTICIPANTS: Multicenter clinical trials including more than 2,900 tolvaptan-treated participants, more than 2,300 with at least 18 months of drug exposure. INTERVENTION: Tolvaptan administered twice daily in split-dose regimens. OUTCOMES: Frequency of liver enzyme level increases detected by regular laboratory monitoring. RESULTS: In the placebo-controlled REPRISE trial, more tolvaptan- than placebo-treated participants (38 of 681 [5.6%] vs 8 of 685 [1.2%]) experienced alanine aminotransferase level increases to >3× the upper limit of normal (ULN), similar to TEMPO 3:4 (40 of 957 [4.4%] vs 5 of 484 [1.0%]). No participant in REPRISE or the long-term extension experienced concurrent alanine aminotransferase level increases to >3× ULN and total bilirubin increases to >2× ULN ("Hy's Law" laboratory criteria). Based on the expanded dataset, liver enzyme increases most often occurred within 18 months after tolvaptan initiation and were less frequent thereafter. Increased levels returned to normal or near normal after treatment interruption or discontinuation. Thirty-eight patients were rechallenged with tolvaptan after the initial drug-induced liver injury episode, with return of liver enzyme level increases in 30; 1 additional participant showed a clinical "adaptation" after the initial episode, with resolution of the enzyme level increases despite continuation of tolvaptan. LIMITATIONS: Retrospective analysis. CONCLUSIONS: The absence of Hy's Law cases in REPRISE and the long-term extension trial support monthly liver enzyme monitoring during the first 18 months of tolvaptan exposure and every 3 months thereafter to detect and manage enzyme level increases, as is recommended on the drug label. FUNDING: Otsuka Pharmaceutical Development & Commercialization, Inc. TRIAL REGISTRATION: Trials included in the dataset were registered at ClinicalTrials.gov with study numbers NCT00428948 (TEMPO 3:4), NCT01214421 (TEMPO 4:4), NCT02160145 (REPRISE), and NCT02251275 (long-term extension).

A computational study to identify Sesamol derivatives as NRF2 activator for protection against drug-induced liver injury (DILI).

Drug-induced liver injury can be caused by any drugs, their metabolites, or natural products due to the inefficient functioning of drug-metabolizing enzymes, resulting in reactive oxygen species generation and leading to oxidative stress-induced cell death. For protection against oxidative stress, our cell has various defense mechanisms. One of the mechanisms is NRF2 pathway, when activated, protects the cell against oxidative stress. Natural antioxidants such as Sesamol have reported pharmacological activity (hepatoprotective & cardioprotective) and signaling pathways (NRF2 & CREM) altering potential. A Computational analysis was done using molecular docking, IFD, ADMET, MM-GBSA, and Molecular dynamic simulation of the Schrödinger suite. A total of 63,345 Sesamol derivatives were downloaded for the PubChem database. The protein structure of KEAP1-NRF2 (PDB: 4L7D) was downloaded from the RCSB protein database. The molecular docking technique was used to screen compounds that can form an interaction similar to the co-crystalized ligand (1VX). Based on MM-GBSA, docking score, and interactions, ten compounds were selected for ADMET profiling and IFD. After IFD, five compounds (66867225, 46148111, 12444939, 123892179, & 94817569) were selected for molecular dynamics simulation (MDS). Protein-ligand complex stability was assessed during MDS. The selected compounds (66867225, 46148111, 12444939, 123892179, & 94817569) complex with KEAP1 protein shows good stability and bond retentions. In our study, we observed that the selected compounds show good interaction, PCA, Rg, binding free energy, and ADMET profile. We can conclude that the selected compounds can act as NRF2 activators, which should be validated using proper in-vivo/in-vitro models.

Molecular Idiosyncratic Toxicology of Drugs in the Human Liver Compared with Animals: Basic Considerations.

Drug induced liver injury (DILI) occurs in patients exposed to drugs at recommended doses that leads to idiosyncratic DILI and provides an excellent human model with well described clinical features, liver injury pattern, and diagnostic criteria, based on patients assessed for causality using RUCAM (Roussel Uclaf Causality Assessment Method) as original method of 1993 or its update of 2016. Overall, 81,856 RUCAM based DILI cases have been published until mid of 2020, allowing now for an analysis of mechanistic issues of the disease. From selected DILI cases with verified diagnosis by using RUCAM, direct evidence was provided for the involvement of the innate and adapted immune system as well as genetic HLA (Human Leucocyte Antigen) genotypes. Direct evidence for a role of hepatic immune systems was substantiated by (1) the detection of anti-CYP (Cytochrome P450) isoforms in the plasma of affected patients, in line with the observation that 65% of the drugs most implicated in DILI are metabolized by a range of CYP isoforms, (2) the DIAIH (drug induced autoimmune hepatitis), a subgroup of idiosyncratic DILI, which is characterized by high RUCAM causality gradings and the detection of plasma antibodies such as positive serum anti-nuclear antibodies (ANA) and anti-smooth muscle antibodies (ASMA), rarely also anti-mitochondrial antibodies (AMA), (3) the effective treatment with glucocorticoids in part of an unselected RUCAM based DILI group, and (4) its rare association with the immune-triggered Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) caused by a small group of drugs. Direct evidence of a genetic basis of idiosyncratic DILI was shown by the association of several HLA genotypes for DILI caused by selected drugs. Finally, animal models of idiosyncratic DILI mimicking human immune and genetic features are not available and further search likely will be unsuccessful. In essence and based on cases of DILI with verified diagnosis using RUCAM for causality evaluation, there is now substantial direct evidence that immune mechanisms and genetics can account for idiosyncratic DILI by many but not all implicated drugs, which may help understand the mechanistic background of the disease and contribute to new approaches of therapy and prevention.

Severe tyrosine-kinase inhibitor induced liver injury in metastatic renal cell carcinoma patients: two case reports assessed for causality using the updated RUCAM and review of the literature.

BACKGROUND: Sunitinib and pazopanib are both oral small molecule multityrosine kinase inhibitors (MTKI) used in the treatment of renal cell carcinoma (RCC). Hepatotoxicity or "liver injury" is the most important adverse effect of pazopanib administration, but little is known about the underlying mechanism. Liver injury may also occur in patients treated with sunitinib, but severe toxicity is extremely rare. Herein we report two new cases of severe liver injury induced by MTKI. Both cases are unique and exceptional. We assessed both cases for drug-induced liver injury (DILI) using the updated score Roussel Uclaf causality assessment method (RUCAM). The literature on potential pathogenic mechanisms and precautionary measures is reviewed. CASE PRESENTATION: A case of a metastatic RCC (mRCC) patient treated with pazopanib who had manifestation of severe liver injury is presented. These manifestations consisted of grade 4 alanine aminotransferase (ALT) increase and grade 4 hyperbilirubinemia. Alternate causes of acute or chronic liver disease were excluded. The patient gradually recovered from the liver injury and refused any further therapy for mRCC. The patient was diagnosed with acute myeloid leukemia (AML) two years later and eventually succumbed to the disease. The second case describes a mRCC patient treated with sunitinib for 3,5 years and fatal liver failure after 2 weeks of clarithromycin co-medication for acute bronchitis. CONCLUSIONS: Liver injury has been commonly observed in TKI-treated patients with unpredictable course. Management requires regular routine liver enzyme-monitoring and the collaboration of medical oncologist and hepatologist. There is an unmet medical need for a risk stratification and definition of predictive biomarkers to identify potential genetic polymorphisms or other factors associated with TKI-induced liver injury. Any potential unrecommended concomitant therapy has to be avoided.

Drug-Induced Liver Injury in Hospitalized Patients during SARS-CoV-2 Infection.

In the last few years, the world has had to face the SARS-CoV-2 infection and its multiple effects. Even though COVID-19 was first considered to be a respiratory disease, it has an extended clinical spectrum with symptoms occurring in many tissues, and it is now identified as a systematic disease. Therefore, various drugs are used during the therapy of hospitalized COVID-19 patients. Studies have shown that many of these drugs could have adverse side-effects, including drug-induced liver injury-also known as DILI-which is the focus of our review. Despite the consistent findings, the pathophysiological mechanism behind DILI in COVID-19 disease is still complex, and there are a few risk factors related to it. However, when it comes to the diagnosis, there are specific algorithms (including the RUCAM algorithm) and biomarkers that can assist in identifying DILI and which we will analyze in our review. As indicated by the title, a variety of drugs are associated with this COVID-19-related complication, including systemic corticosteroids, drugs used for the therapy of uncontrolled cytokine storm, as well as antiviral, anti-inflammatory, and anticoagulant drugs. Bearing in mind that hepatotoxicity is very likely to occur during COVID-19, especially in patients treated with multiple medications, we will also refer to the use of other drugs used for DILI therapy in an effort to control and prevent a severe and long-term outcome.

Toxicogenomics of drug induced liver injury - from mechanistic understanding to early prediction.

Despite rigorous preclinical testing, clinical attrition rates in drug development remain high with drug-induced liver injury (DILI) remaining one of the most frequent causes of project failures. To understand DILI mechanisms, major efforts are put into the development of physiologically relevant cell models and culture paradigms with the aim to enhance preclinical to clinical result translation. While the majority of toxicogenomic studies have been based on cell lines, there are emerging trends toward the predominant use of stem cell-derived organoids and primary human hepatocytes in complex 3D cell models. Such studies have been successful in disentangling diverse toxicity mechanisms, including genotoxicity, mitochondrial injury, steatogenesis and cholestasis and can aid in distinguishing hepatotoxic from nontoxic structural analogs. Furthermore, by leveraging inter-individual differences of cells from different donors, these approaches can emulate the complexity of polygenic risk scores, which facilitates personalized drug-specific DILI risk analyses. In summary, toxicogenomic studies into drug-induced hepatotoxicity have majorly contributed to our mechanistic understanding of DILI and the incorporation of organotypic human 3D liver models into the preclinical testing arsenal promises to enhance biological insights during drug discovery, increase confidence in preclinical safety and minimize the translational gap.

The application of cytokeratin-18 as a biomarker for drug-induced liver injury.

Drug-induced liver injury (DILI) is a frequent and dangerous adverse effect faced during preclinical and clinical drug therapy. DILI is a leading cause of candidate drug attrition, withdrawal and in clinic, is the primary cause of acute liver failure. Traditional diagnostic markers for DILI include alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP). Yet, these routinely used diagnostic markers have several noteworthy limitations, restricting their sensitivity, specificity and accuracy in diagnosing DILI. Consequently, new biomarkers for DILI need to be identified.A potential biomarker for DILI is cytokeratin-18 (CK18), an intermediate filament protein highly abundant in hepatocytes and cholangiocytes. Extensively researched in a variety of clinical settings, both full length and cleaved forms of CK18 can diagnose early-stage DILI and provide insight into the mechanism of hepatocellular injury compared to traditionally used diagnostic markers. However, relatively little research has been conducted on CK18 in preclinical models of DILI. In particular, CK18 and its relationship with DILI is yet to be characterised in an in vivo rat model. Such characterization of CK18 and ccCK18 responses may enable their use as translational biomarkers for hepatotoxicity and facilitate management of clinical DILI risk in drug development. The aim of this review is to discuss the application of CK18 as a biomarker for DILI. Specifically, this review will highlight the properties of CK18, summarise clinical research that utilised CK18 to diagnose DILI and examine the current challenges preventing the characterisation of CK18 in an in vivo rat model of DILI.

Protective role of c-Jun N-terminal kinase-2 (JNK2) in ibuprofen-induced acute liver injury.

Ibuprofen is a worldwide used non-steroidal anti-inflammatory drug which may cause acute liver injury (ALI) requiring liver transplantation. We aimed to unveil the molecular pathways involved in triggering ibuprofen-induced ALI, which, at present, remain elusive. First, we investigated activation of essential pathways in human liver sections of ibuprofen-induced ALI. Next, we assessed the cytotoxicity of ibuprofen in vitro and developed a novel murine model of ibuprofen intoxication. To assess the role of JNK, we used animals carrying constitutive deletion of c-Jun N-terminal kinase 1 (Jnk1-/- ) or Jnk2 (Jnk2-/- ) expression and included investigations using animals with hepatocyte-specific Jnk deletion either genetically (Jnk1Δhepa ) or by siRNA (siJnk2Δhepa ). We found in human and murine samples of ibuprofen-induced acute liver failure that JNK phosphorylation was increased in the cytoplasm of hepatocytes and other non-liver parenchymal cells (non-LPCs) compared with healthy tissue. In mice, ibuprofen intoxication resulted in a significantly stronger degree of liver injury compared with vehicle-treated controls as evidenced by serum transaminases, and hepatic histopathology. Next, we investigated molecular pathways. PKCα, AKT, JNK and RIPK1 were significantly increased 8 h after ibuprofen intoxication. Constitutive Jnk1-/- and Jnk2-/- deficient mice exhibited increased liver dysfunction compared to wild-type (WT) animals. Furthermore, siJnk2Δhepa animals showed a dramatic increase in biochemical markers of liver function, which correlated with significantly higher serum liver enzymes and worsened liver histology, and MAPK activation compared to Jnk1Δhepa or WT animals. In our study, cytoplasmic JNK activation in hepatocytes and other non-LPCs is a hallmark of human and murine ibuprofen-induced ALI. Functional in vivo analysis demonstrated a protective role of hepatocyte-specific Jnk2 during ibuprofen ALI. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Hepatotoxicty of Agents Used in the Management of Inflammatory Bowel Disease: a 2020 Update.

PURPOSE OF REVIEW: As treatment options for inflammatory bowel disease (IBD) continue to expand, the opportunity for hepatotoxicity remains a clinical concern. This review looks to update the current literature on drug-induced liver injury (DILI) and liver-related complications from current and emerging treatments for Crohn's disease (CD) and ulcerative colitis (UC). RECENT FINDINGS: An extensive literature review on currently used medications to treat IBD and their liver-related side effects that includes mesalamine, thiopurines, certain antibiotics, methotrexate, anti-TNF agents including recently introduced biosimilars, anti-integrin therapy, anti-IL 12/IL 23 therapy, and small molecule JAK inhibitors. Hepatotoxicity remains an important clinical issue when managing patients with IBD. Clinicians need to remain aware of the potential for liver-related adverse events with various medication classes and adjust their clinical monitoring as appropriate based on the agents being used.

GADD45α alleviates acetaminophen-induced hepatotoxicity by promoting AMPK activation.

As an analgesic and antipyretic drug, acetaminophen (APAP) is commonly used and known to be safe at therapeutic doses. In many countries, the overuse of APAP provokes acute liver injury and even liver failure. APAP-induced liver injury (AILI) is the most used experimental model of drug-induced liver injury (DILI). Here, we have demonstrated elevated levels of growth arrest and DNA damage-inducible 45α (GADD45α) in the livers of patients with DILI/AILI, in APAP-injured mouse livers and in APAP-treated hepatocytes. GADD45α exhibited a protective effect against APAP-induced liver injury and alleviated the accumulation of small lipid droplets in vitro and in vivo. We found that GADD45α promoted the activation of AMP-activated protein kinase α and induced fatty acid beta-oxidation, tricarboxylic acid cycle (TCA) and glycogenolysis-related gene expression after APAP exposure. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that GADD45α increased the levels of TCA cycle metabolites. Co-immunoprecipitation analysis showed that Ppp2cb, a catalytic subunit of protein phosphatase 2A, could interact directly with GADD45α. Our results indicate that hepatocyte GADD45α might represent a therapeutic target to prevent and rescue liver injury caused by APAP.

An adverse outcome pathway for immune-mediated and allergic hepatitis: a case study with the NSAID diclofenac.

Many drugs have the potential to cause drug-induced liver injury (DILI); however, underlying mechanisms are diverse. The concept of adverse outcome pathways (AOPs) has become instrumental for risk assessment of drug class effects. We report AOPs specific for immune-mediated and drug hypersensitivity/allergic hepatitis by considering genomic, histo- and clinical pathology data of mice and dogs treated with diclofenac. The findings are relevant for other NSAIDs and drugs undergoing iminoquinone and quinone reactive metabolite formation. We define reactive metabolites catalyzed by CYP monooxygenase and myeloperoxidases of neutrophils and Kupffer cells as well as acyl glucuronides produced by uridine diphosphoglucuronosyl transferase as molecular initiating events (MIE). The reactive metabolites bind to proteins and act as neo-antigen and involve antigen-presenting cells to elicit B- and T-cell responses. Given the diverse immune systems between mice and dogs, six different key events (KEs) at the cellular and up to four KEs at the organ level are defined with mechanistic plausibility for the onset and progression of liver inflammation. With mice, cellular stress response, interferon gamma-, adipocytokine- and chemokine signaling provided a rationale for the AOP of immune-mediated hepatitis. With dogs, an erroneous programming of the innate and adaptive immune response resulted in mast cell activation; their infiltration into liver parenchyma and the shift to M2-polarized Kupffer cells signify allergic hepatitis and the occurrence of granulomas of the liver. Taken together, diclofenac induces divergent immune responses among two important preclinical animal species, and the injury pattern seen among clinical cases confirms the relevance of the developed AOP for immune-mediated hepatitis.

An Evaluation of the In Vitro Roles and Mechanisms of Silibinin in Reducing Pyrazinamide- and Isoniazid-Induced Hepatocellular Damage.

Tuberculosis remains a significant infectious lung disease that affects millions of patients worldwide. Despite numerous existing drug regimens for tuberculosis, drug-induced liver injury is a major challenge that limits the effectiveness of these therapeutics. Two drugs that form the backbone of the commonly administered quadruple antitubercular regimen, that is, pyrazinamide (PZA) and isoniazid (INH), are associated with such hepatotoxicity. Yet, we lack safe and effective alternatives to the antitubercular regimen. Consequently, current research largely focuses on exploiting the hepatoprotective effect of nutraceutical compounds as complementary therapy. Silibinin, a herbal product widely believed to protect against various liver diseases, potentially provides a useful solution given its hepatoprotective mechanisms. In our study, we identified silibinin's role in mitigating PZA- and INH-induced hepatotoxicity and elucidated a deeper mechanistic understanding of silibinin's hepatoprotective ability. Silibinin preserved the viability of human foetal hepatocyte line LO2 when co-administered with 80 mM INH and decreased apoptosis induced by a combination of 40 mM INH and 10 mM PZA by reducing oxidative damage to mitochondria, proteins, and lipids. Taken together, this proof-of-concept forms the rational basis for the further investigation of silibinin's hepatoprotective effect in subsequent preclinical studies and clinical trials.

Analysis of 307 cases with drug-induced liver injury between 2010 and 2018 in Japan.

AIM: In order to know the present status of drug-induced liver injury (DILI) in Japan, we present the data of prospectively collected DILI cases between 2010 and 2018 from 27 hospitals. METHODS: Drug-induced liver injury cases diagnosed by DILI experts from 27 hospitals all over Japan have been prospectively collected since 2010. Alanine aminotransferase level ≥150 U/L and/or alkaline phosphatase ≥2× upper limit of normal were inclusion criteria. RESULTS: In total, data of 307 cases (125 male and 182 female individuals) aged between 17 and 86 years old were collected. The types of liver injury were as follows: 64% hepatocellular type, 20% mixed type, and 16% cholestatic type. A drug-induced lymphocyte stimulation test was carried out in 59% of cases, and was positive in 48% and semipositive in 3% of cases. Eosinophilia ≥6% was observed in 27% of cases. Fifty-three percent of DILI cases occurred within 30 days and 79% of DILI cases occurred within 90 days after starting drug administration. By the diagnostic scale of the Digestive Disease Week (DDW)-Japan 2004 workshop, 93.8% of cases were diagnosed as "highly probable", and 5.9% as "possible". CONCLUSIONS: Japanese DILI patients are somewhat different from those of Europe and North America. The diagnostic scale of the DDW-Japan 2004 workshop has been used in Japan. However, there are many issues to improve the causality assessment of DILI that we must investigate in the future. It is critical to elucidate the mechanisms of drug metabolism and the pathophysiology of liver injury by various drugs to prevent DILI.

Liver dysfunction induced by Levothyroxine Sodium Tablets (Euthyrox®) in a hypothyroid patient with Hashimoto's thyroiditis: case report and literature review.

A 49-year-old woman with hypothyroidism developed liver dysfunction after increasing dose of levothyroxine (L-T4) (Euthyrox®) from 25 µg to 50 µg. Viral hepatitis, autoimmune hepatitis and non-alcoholic steatohepatitis (NASH) were ruled out with examinations. She had no concurrent medication and had no history of infectious, chronic or any other autoimmune diseases. After cessation of Levothyroxine Sodium Tablets (Euthyrox®), liver enzymes gradually returned to normal. She was diagnosed levothyroxine-induced liver injury, based on criteria proposed in "Diagnosis and treatment guideline on drug-induced liver injury" issued by the Chinese Medical Association (2015). As an alternative 25 µg qod of Levothyroxine Sodium Tablets (Letrox®) was tried and increased gradually up to 75 µg daily. Since then liver enzymes have remained within normal range. The main difference of additive for both tablets is whether it contains lactose or not: Euthyrox® contains lactose which caused no liver injury, thus excluding the possibility that an additive of Euthyrox® contributed to liver injury. The relatively quicker and larger replacement with synthetic T4 for hypothyroidism inducing transient thyrotoxicosis was suspected, although thyroid function was normal. Immune-mediated drug-induced liver injury (DILI) was also not excluded. This is a rare case of drug-induced liver injury due to levothyroxine tablets. It reminded us that when replacement with synthetic T4 for hypothyroidism is done, smaller-dose initiation and slower-speed increase may be useful for treatment of cases similar to genetically susceptible individuals.

Liver safety evaluation of endothelin receptor antagonists using HepatoPac® : A single model impact assessment on hepatocellular health, function and bile acid disposition.

Marketed (bosentan, ambrisentan) and discontinued (sitaxsentan, CI-1034) endothelin receptor antagonists were examined in the human micropatterned hepatocyte co-culture (MPCC) model HepatoPac® . Differences across hepatocellular health (cellular adenosine triphosphate/glutathione content), function (urea production/albumin secretion) and taurocholic acid transport (biliary clearance/excretion index) were compared using amiodarone and ciclosporin A as positive controls. Ambrisentan had the weakest potency in all six endpoints, while sitaxsentan, bosentan and CI-1034 had more potent effects on hepatobiliary transport than health/function endpoints. Normalization to clinical Cmax gave the following relative rank order of safety based on margins for each endpoint: ambrisentan ≥ CI-1034 ~ bosentan > sitaxsentan. These data suggested impaired hepatobiliary disposition might contribute to a more prominent role in liver injury associated within sensitive human populations exposed to these compounds than direct hepatocellular toxicity. Rat, dog and monkey MPCCs also showed greater sensitivity potential to disrupted hepatobiliary disposition compared with hepatocellular health/functional endpoints. Drug metabolism competency was exhibited across all species. In vivo, rats and dogs appear more resistant to transaminase elevations and/or histological evidence of liver injury caused by these mechanisms even at exceedingly high systemic exposures relative to sensitive humans. Rats and dogs are resistant to hepatobiliary toxicants due to physiological differences in bile composition/handling. Although traditional animal testing provides adequate safety coverage for advancement of novel pharmaceuticals into clinical trials, supplemental assays employing human MPCCs may strengthen weight-of-evidence predictions for sensitive human populations. Proving the predictive value of this single impact assessment model in advance of clinical trial information for human liver injury risk is needed across more pharmaceuticals.

Hepatotoxicity induced by a second-generation combined oral contraceptive: case report and review of the literature.

Case: Second-generation combined oral contraceptives (COCs) are widely used and are believed to be safe for birth control and in the treatment of gynaecological diseases. No randomised controlled study has shown elevations in alanine transaminase (ALT) levels in relation to the use of a second-generation COC. We report a case of drug-induced liver injury (DILI) in a young, moderately obese woman, due to the use of a second-generation COC containing 30 µg ethinylestradiol and 150 µg levonorgestrel. COC use had been initiated 2 years prior to admission to our department. The diagnosis was based on elevated levels of ALT during COC use and was confirmed by re-challenge and a liver biopsy showing signs of former tissue damage after a 3 week break of COC treatment. Conclusions: To our knowledge, this is the first case of biopsy-proven DILI due to COC use in which a re-challenge was performed.

Pharmacogenomics of drug-induced liver injury (DILI): Molecular biology to clinical applications.

A 21-year old woman was admitted to hospital with a two-week history of painless jaundice, fatigue and anorexia having previously been fit and well. One month prior to presentation, the patient had taken a five-day course of amoxicillin-clavulanic acid for an infected skin cyst. Otherwise, she was only on the oral contraceptive pill and reported minimal alcohol intake. On examination, she was deeply jaundiced, but alert and oriented with no asterixis. She had no stigmata of chronic liver disease, but hepatomegaly extending 3 cm from below the right subcostal margin was evident. Investigations showed: white cell count 13.4 × 109/L (normal 3.6-9.3), haemoglobin 11.8 g/dl (normal 11-15), platelet count 356 × 109/L (normal 170-420), sodium 138 mmol/L (normal 134-144), potassium 3.5 mmol/L (normal 3.5-5.0), creatinine 32 µmol/L (normal 40-75), albumin 30 g/L (normal 35-48), alanine aminotransferase 707 IU/L (normal 15-54), alkaline phosphatase 151 IU/L (normal 30-130), bilirubin 384 µmol/L (normal 7-31) and prothrombin time 27.2 s (normal 11.7-14). Screening for hepatitis A, B, C, E, Epstein-Barr virus, cytomegalovirus and autoimmune hepatitis was negative. Tests for anti-smooth muscle, antinuclear, and anti-liver-kidney microsomal-1 antibodies were negative; immunoglobulin levels and ceruloplasmin levels were normal. Liver ultrasonography demonstrated a liver of normal contour with no biliary dilatation, a normal spleen size and patent vessels. Liver biopsy revealed severe portal interface hepatitis with lobular inflammation and scant plasma cells. Her clinical condition deteriorated in the following days with prothrombin time and bilirubin rising to 56.6 s and 470 µmol/L, respectively. At follow-up after 11 days, her alanine aminotransferase level was 1,931 IU/L. She developed grade 2 hepatic encephalopathy 14 days after presentation, and was listed for a super-urgent liver transplant. Human leucocyte antigen (HLA) typing was performed as a part of preparatory investigations and showed the patient carried the HLA haplotype HLA-DRB1∗15:02-DQB1∗06:01. Following orthotopic transplantation of a deceased donor graft her explant histology revealed severe ongoing hepatitis with multi-acinar necrosis (Fig. 1A and B). This case raised a number of important questions about the diagnosis of drug-induced liver injury and tools available for clinicians to make the best decisions for patient care: In this Grand Rounds article, we will explore these questions, describing the pathophysiology, diagnostic and prognostic biomarkers, and clinical management of drug-induced liver injury. We will also discuss ongoing areas of uncertainty.

Risk of hospital admission for liver injury in users of NSAIDs and nonoverdose paracetamol: Preliminary results from the EPIHAM study.

PURPOSE: The SALT study found similar per-user risks of acute liver failure (ALF) leading to transplantation (ALFT) between NSAIDs and a threefold higher risk in nonoverdose paracetamol (NOP) users. The objective of EPIHAM was to identify the risks of hospital admission for acute liver injury (ALI) associated with NSAIDs and NOP. METHODS: Case-population study in the 1/97 sample of the French population claims database. Acute liver injury was identified from hospital discharge summaries, from 2009 to 2013. Exposure for cases was dispensing of NSAID or NOP resulting in exposure within 30 days before admission. Population exposure was the number of patients using the drugs over the study timeframe and total number of DDD dispensed. RESULTS: Of 63 cases of ALI, 13 had been exposed to NSAIDs and 24 to NOP. Events per million DDD (95% CI) ranged from 0.46 (0.09-1.34) (ketoprofen) to 1.43 (0.04-7.97) (diclofenac combinations), 0.43 (0.23-0.73) all NSAIDs combined, 0.58 (0.37-0.86) for NOP. There was no association with average duration of treatment. Per patient risk ranged from 19.5 (5.31-49.9) (ibuprofen) per million users to 37.2 (19.8-63.6) all NSAIDs combined, 58.0 (37.2-86.3) for NOP. There was a linear relationship between average treatment duration and per-user risk (R2  = 0.51, P < .05 for NSAIDs, R2  = 0.97, P < .01 for NOP). CONCLUSIONS: Risk of hospital admission for ALI with NSAIDs and NOP was similar and indicative of a dose and duration-related effect (pharmacological) effect. Acute liver injury rates were not predictive of ALFT risk.

In vitro assessment of hepatotoxicity by metabolomics: a review.

Omics technologies, and in particular metabolomics, have received an increasing attention during the assessment of hepatotoxicity in vitro. However, at present, a consensus on good metabolomics practices has yet to be reached. Therefore, in this review, a range of experimental approaches, applied methodologies, and data processing workflows are compared and critically evaluated. Experimental designs among the studies are similar, reporting the use of primary hepatocytes or hepatic cell lines as the most frequently used cell sources. Experiments are usually conducted in short time-frames (< 48 h) at sub-toxic dosages. Applied sample preparations are protein precipitation or Bligh-and-Dyer extraction. Most analytical platforms rely on chromatographic separations with mass spectrometric detection using high-resolution instruments. Untargeted metabolomics was typically used to allow the simultaneous detection of several classes of the metabolome, including endogenous metabolites that are not initially linked to toxicity. This non-biased detection platform is a valuable tool for generating hypothesis-based mechanistic research. The most frequently reported metabolites that are altered under toxicological impulses are alanine, lactate, and proline, which are often correlated. Other unspecific biomarkers of hepatotoxicity in vitro are the down-regulation of choline, glutathione, and 3-phospho-glycerate. Disruptions on the Krebs cycle are associated with increased glutamate, tryptophan, and valine. Phospholipid alterations are described in steatosis, lipo-apoptosis, and oxidative stress. Although there is a growing trend towards quality control, data analysis procedures do often not follow good contemporary metabolomics practices, which include feature filtering, false-discovery rate correction, and reporting the confidence of metabolite annotation. The currently annotated biomarkers can be used to identify hepatotoxicity in general and provide, to a certain extent, a tool for mechanistic distinction.