Drug-induced Liver Injury Susceptibility Research Articles

Antioxidant-Based Preventive Effect of Phytochemicals on Anticancer Drug-Induced Hepatotoxicity.

Significance: Drug-induced liver injury (DILI) or hepatotoxicity has been a hot issue to overcome on the safety and physiological function of the liver, since it is known to have biochemical, cellular, immunological, and molecular alterations in the liver mainly induced by alcohol, chemicals, drugs, heavy metals, and genetic factors. Recently efficient therapeutic and preventive strategies by some phytochemicals are of interest, targeting oxidative stress-mediated hepatotoxicity alone or in combination with anticancer drugs. Recent Advances: To assess DILI, the variety of in vitro and in vivo animal models has been developed mainly by using carbon tetrachloride, d-galactosamine, acetaminophen, and lipopolysaccharide. Also, the mechanisms on hepatotoxicity by several drugs and herbs have been explored in detail. Recent studies reveal that antioxidants including vitamins and some phytochemicals were reported to prevent against DILI. Critical Issues: Antioxidant therapy with some phytochemicals is noteworthy, since oxidative stress is critically involved in DILI via production of chemically reactive oxygen species or metabolites, impairment of mitochondrial respiratory chain, and induction of redox cycling. Future Directions: For efficient antioxidant therapy, DILI susceptibility, Human Leukocyte Antigen genetic factors, biomarkers, and pathogenesis implicated in hepatotoxicity should be further explored in association with oxidative stress-mediated signaling, while more randomized preclinical and clinical trials are required with optimal safe doses of drugs and/or phytochemicals alone or in combination for efficient clinical practice along with the development of advanced DILI diagnostic tools. Antioxid. Redox Signal. 38, 1101-1121.

Impact of SLCO1B1*5 on Flucloxacillin and Co-Amoxiclav-Related Liver Injury.

Background: Idiosyncratic drug-induced liver injury (DILI) is a serious uncommon disease that may develop as a result of the intake of certain drugs such as the antimicrobials flucloxacillin and co-amoxiclav. The reported cases showed significant associations between DILI and various human leukocyte (HLA) markers. The solute carrier organic anion transporter 1B1 (SLCO1B1), a non-HLA candidate gene, was previously reported as a risk factor for liver injury induced by rifampin and methimazole. This study presumed that SLCO1B1 may play a general role in the DILI susceptibility and therefore investigated the association of rs4149056 (SLCO1B1*5, T521C) polymorphism with flucloxacillin- and co-amoxiclav–induced liver injury. Methodology: We recruited 155 and 165 DILI cases of white ancestral origin from various European countries but mainly from the United Kingdom owing to flucloxacillin and co-amoxiclav, respectively. Only adult patients (≥18 years) who were diagnosed with liver injury and who showed i) clinical jaundice or bilirubin >2x the upper limit of normal (ULN), ii) alanine aminotransferase (ALT) >5x ULN or iii) alkaline phosphatase (ALP) >2x ULN and bilirubin > ULN were selected. The population reference sample (POPRES), a European control group (n = 282), was used in comparison with the investigated cases. TaqMan SNP genotyping custom assay designed by Applied Biosystems was used to genotype both DILI cohorts for SLCO1B1 polymorphism (rs4149056). Allelic discrimination analysis was performed using a step one real-time PCR machine. Genotype differences between cases and controls were examined using Fisher’s exact test. GraphPad Prism version 5.0 was used to determine the p-value, odds ratio, and 95% confidence interval. Compliance of the control group with Hardy–Weinberg equilibrium was proven using a web-based calculator available at https://wpcalc.com/en/equilibrium-hardy-weinberg/. Results: A small number of cases failed genotyping in each cohort. Thus, only 149 flucloxacillin and 162 co-amoxiclav DILI cases were analyzed. Genotyping of both DILI cohorts did not show evidence of association with the variant rs4149056 (T521C) (OR = 0.71, 95% CI = 0.46–1.12; p = 0.17 for flucloxacillin cases and OR = 0.87, 95% CI = 0.56–1.33; p = 0.58 for co-amoxiclav), although slightly lower frequency (22.8%) of positive flucloxacillin cases was noticed than that of POPRES controls (29.4%). Conclusion: Carriage of the examined allele SLCO1B1*5 is not considered a risk factor for flucloxacillin DILI or co-amoxiclav DILI as presumed. Testing a different allele (SLCO1B1*1B) and another family member gene (SLCO1B3) may still be needed to provide a clearer role of SLCO1B drug transporters in DILI development–related to the chosen antimicrobials.

Machine Learning to Identify Interaction of Single-Nucleotide Polymorphisms as a Risk Factor for Chronic Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) is a major cause of drug development failure and drug withdrawal from the market after approval. The identification of human risk factors associated with susceptibility to DILI is of paramount importance. Increasing evidence suggests that genetic variants may lead to inter-individual differences in drug response; however, individual single-nucleotide polymorphisms (SNPs) usually have limited power to predict human phenotypes such as DILI. In this study, we aim to identify appropriate statistical methods to investigate gene–gene and/or gene–environment interactions that impact DILI susceptibility. Three machine learning approaches, including Multivariate Adaptive Regression Splines (MARS), Multifactor Dimensionality Reduction (MDR), and logistic regression, were used. The simulation study suggested that all three methods were robust and could identify the known SNP–SNP interaction when up to 4% of genotypes were randomly permutated. When applied to a real-life DILI chronicity dataset, both MARS and MDR, but not logistic regression, identified combined genetic variants having better associations with DILI chronicity in comparison to the use of individual SNPs. Furthermore, a simple decision tree model using the SNPs identified by MARS and MDR was developed to predict DILI chronicity, with fair performance. Our study suggests that machine learning approaches may help identify gene–gene interactions as potential risk factors for better assessing complicated diseases such as DILI chronicity.

High-Fidelity Drug-Induced Liver Injury Screen Using Human Pluripotent Stem Cell–Derived Organoids

Preclinical identification of compounds at risk of causing drug induced liver injury (DILI) remains a significant challenge in drug development, highlighting a need for a predictive human system to study complicated DILI mechanism and susceptibility to individual drug. Here, we established a human liver organoid (HLO)-based screening model for analyzing DILI pathology at organoid resolution. We first developed a reproducible method to generate HLO from storable foregut progenitors from pluripotent stem cell (PSC) lines with reproducible bile transport function. The qRT-PCR and single cell RNA-seq determined hepatocyte transcriptomic state in cells of HLO relative to primary hepatocytes. Histological and ultrastructural analyses were performed to evaluate micro-anatomical architecture. HLO based drug-induced liver injury assays were transformed into a 384 well based high-speed live imaging platform. HLO, generated from 10 different pluripotent stem cell lines, contain polarized immature hepatocytes with bile canaliculi-like architecture, establishing the unidirectional bile acid transport pathway. Single cell RNA-seq profiling identified diverse and zonal hepatocytic populations that in part emulate primary adult hepatocytes. The accumulation of fluorescent bile acid into organoid was impaired by CRISPR-Cas9-based gene editing and transporter inhibitor treatment with BSEP. Furthermore, we successfully developed an organoid based assay with multiplexed readouts measuring viability, cholestatic and/or mitochondrial toxicity with high predictive values for 238 marketed drugs at 4 different concentrations (Sensitivity: 88.7%, Specificity: 88.9%). LoT positively predicts genomic predisposition (CYP2C9∗2) for Bosentan-induced cholestasis. Liver organoid-based Toxicity screen (LoT) is a potential assay system for liver toxicology studies, facilitating compound optimization, mechanistic study, and precision medicine as well as drug screening applications.

The development of a hepatic steatotic model for assessment of drug-induced liver injury susceptibility

The development of a hepatic steatotic model for assessment of drug-induced liver injury susceptibility

Candidate Risk Factors and Mechanisms for Tolvaptan-Induced Liver Injury Are Identified Using a Collaborative Cross Approach

Clinical trials of tolvaptan showed it to be a promising candidate for the treatment of Autosomal Dominant Polycystic Kidney Disease (ADPKD) but also revealed potential for idiosyncratic drug-induced liver injury (DILI) in this patient population. To identify risk factors and mechanisms underlying tolvaptan DILI, 8 mice in each of 45 strains of the genetically diverse Collaborative Cross (CC) mouse population were treated with a single oral dose of either tolvaptan or vehicle. Significant elevations in plasma alanine aminotransferase (ALT) were observed in tolvaptan-treated animals in 3 of the 45 strains. Genetic mapping coupled with transcriptomic analysis in the liver was used to identify several candidate susceptibility genes including epoxide hydrolase 2, interferon regulatory factor 3, and mitochondrial fission factor. Gene pathway analysis revealed that oxidative stress and immune response pathways were activated in response to tolvaptan treatment across all strains, but genes involved in regulation of bile acid homeostasis were most associated with tolvaptan-induced elevations in ALT. Secretory leukocyte peptidase inhibitor (Slpi) mRNA was also induced in the susceptible strains and was associated with increased plasma levels of Slpi protein, suggesting a potential serum marker for DILI susceptibility. In summary, tolvaptan induced signs of oxidative stress, mitochondrial dysfunction, and innate immune response in all strains, but variation in bile acid homeostasis was most associated with susceptibility to the liver response. This CC study has indicated potential mechanisms underlying tolvaptan DILI and biomarkers of susceptibility that may be useful in managing the risk of DILI in ADPKD patients.

Prevalence and risk factors of drug induced liver disease: a survey based study in pharmacies

The reported incidence of drug-induced liver injury (DILI) ranges from 1 in 10,000 to 1 in 100,000 patients for most drugs, but the true incidence is expected to be much higher. Several risk factors for DILI susceptibility have been suggested, however there is insufficient data to define an individual risk profile. Therefore it was our aim to study the prevalence of DILI and potential risk factors within adult pharmacy customers in Germany. We conducted two 6 week-survey studies in 30 pharmacies in 2011 and 2012, respectively, using a newly developed questionnaire comprising questions on demography, (liver) diseases, liver enzyme activities, and drug history. In each study, anonymized questionnaires were presented to non-selected adult customers taking (non-)prescription drugs. Combining the datasets from the 2011 and 2012 surveys, in total 1098 questionnaires were evaluated (mean age 57.7 ± 17.1 years; 62.6 % females, return rate 15.25 %). Overall, 141 individuals (12.8 %) reported elevated liver enzymes due to drugs, in 65 cases (5.9 %) the medication had to be stopped, and 20 customers (1.8 %) reported that they had been admitted to hospital due to DILI. Compared to individuals without adverse hepatic drug reactions (n = 957), the 141 persons with potential DILI presented more often the following risk factors in multivariate analysis: chronic liver disease (14.4 % vs. 2.4 %, odds ratio [OR] 4.2, 95 % confidence interval [CI] 2.0 - 9.0), chronic renal insufficiency (20.0 vs. 6.8 %, OR 2.2, 95 % CI 1.3 - 3.7), diabetes (34 vs. 15.3 %, OR 2.0, 95 % CI 1.3 - 3.2), family history of chronic liver disease (19.9 vs. 7.7 %, OR 2.1, 95 % CI 1.2 - 3.6), and continuous drug intake for more than 5 years (80.9 vs. 59.3 %, OR 2.1, 95 % CI 1.3 - 3.5). These studies show an unexpected high prevalence of DILI in pharmacy customers and identify multiple potential risk factors.

Circulating mitochondrial biomarkers for drug-induced liver injury.

Liver mitochondria affected by drugs can be released into circulation and serve as biomarkers for drug-induced liver injury (DILI). The tissue specificity of ALT was improved by differentiating cytosolic ALT1 and mitochondrial ALT2 isoforms released in circulation. Prior to ALT elevation, mitochondrial cytochrome c, OCT, GLDH, CPS1 and DNA were increased in circulation following DILI. The baseline expression of mt-Nd6 was predictive of individual DILI susceptibility in animals. As mitochondrial DILI biomarkers appeared to be drug or species dependent, they might have value in clinical scenarios when culprit drugs are established, but may not be ideal tools to assess DILI potentials of new drugs.

578 Interplay of Gender, Age and Drug Properties in Drug-Induced Liver Injury: Analysis of Adverse Event Reporting at Who Vigibase™

[Background] Clinical hepatotoxicity is highly associated with specific drug properties (e.g., lipophilicity, transport, and metabolism) and daily dose (>100mg). However, how these drug properties interact with age and gender in drug-induced liver injury (DILI) remains unclear. [Aim] To generate hypotheses on drug-patient interactions in DILI, we performed a data-mining analysis using the WHO VigiBaseTM to 1) identify drugs with genderand age-biased reporting frequency (RF) of liver events and 2) explore the association between the groups of identified drugs and drug properties relevant to hepatotoxicity. [Methods] Empirical Bayes Geometric Mean of relative reporting ratio of liver events (EBGM) with 90% confidence interval (CI) was calculated for 375 drugs with established clinical DILI potential (Suzuki, Drug safety 2010; Chen, Drug Discovery Today 2011), partitioning all reported cases into 4 groups by gender and age 50 yrs. Drugs with gender-biased indications were manually excluded from the analysis. Combining Medical Dictionary for Regulatory Activities terms (Hunt, Regul Tox Pharm, 2014), customized liver event terms were created for hepatocellular injury, cholestatic injury, acute liver failure, and overall liver events. Comparing EBGM and 90% CI of overall liver events among the 4 groups, drugs with significant differences in genderand age-specific RF were identified. The groups of identified drugs were then associated with distinct drug properties in the Liver Toxicity Knowledge Base (Chen, Clin Pharm & Ther, 2013). [Results] Overall liver events were 13-20% more frequently reported in women compared tomen. Compared to age-matched men, women<50 yrs exhibited a 40% increased RF of acute liver failure. Across all ages, we identified 43 drugs with an increased RF among women, which were significantly associated with transporter inhibition, mitochondrial liability, and reactive metabolite formation, compared to 186 drugs with no gender difference in RF. Fourteen drugs with an increased RF among men tended to have a higher proportion causing cholestatic injury, while 57 drugs with an increased RF among women only before age 50 yrs were associated with a higher proportion causing hepatocellular injury. Regardless of gender, seventeen drugs with an increased RF in age ≥ 50 yrs were associated with transporter inhibition, lipophilicity, high daily dose (≥100mg), and higher Cmax and plasma protein binding, yet shorter plasma elimination T1/2. [Conclusions] This data-mining analysis identified specific drug properties associated with genderand age-related RF of liver events, suggesting a potential physiologic interplay between these drug properties, gender and age. The discovered associations of gender and age-related DILI and the specific drug properties should be further evaluated for drug-patient interaction in DILI susceptibility.

Genetic basis of drug-induced liver injury: present and future.

There is considerable evidence that susceptibility to idiosyncratic drug-induced liver injury (DILI) is genetically determined. Though genetic associations with DILI have been reported since the 1980s, the development of genome-wide association studies has enabled genetic risk factors for DILI, in common with other diseases, to be detected and confirmed more confidently. Human leukocyte antigen (HLA) genotype has been demonstrated to be a strong risk factor for development of DILI with a range of drugs and the underlying mechanism, probably involving presentation of a drug-peptide complex to T cells is increasingly well understood. However, specific HLA alleles are not associated with all forms of DILI and non-HLA genetic risk factors, especially those relating to drug disposition, also appear to contribute. For some drugs, there is evidence of a dual role for HLA and drug metabolism genes. Though the associations with non-HLA genes have been less well replicated than the HLA associations, there is increasing evidence that drug metabolism genes such as NAT2 and UGT2B7 contribute to some forms of DILI. Translating current genetic findings on DILI susceptibility to the clinic has been relatively slow, but some progress is now being made. In the future, DNA sequencing may lead to the identification of rare variants that contribute to DILI. Developments in the related area of epigenomics and in the development of improved models for DILI by use of genetically defined induced pluripotent stem cells should improve understanding of the biology of DILI and inform drug development.

Selected ABCB1, ABCB4 and ABCC2 polymorphisms do not enhance the risk of drug-induced hepatotoxicity in a Spanish cohort.

Background and AimsFlawed ABC transporter functions may contribute to increased risk of drug-induced liver injury (DILI). We aimed to analyse the influence of genetic variations in ABC transporters on the risk of DILI development and clinical presentations in a large Spanish DILI cohort.MethodsA total of ten polymorphisms in ABCB1 (1236T>C, 2677G>T,A, 3435T>C), ABCB4 (1954A>G) and ABCC2 (−1774G>del, −1549A>G, −24C>T, 1249G>A, 3972C>T and 4544G>A) were genotyped using Taqman 5′ allelic discrimination assays or sequencing in 141 Spanish DILI patients and 161 controls. The influence of specific genotypes, alleles and haplotypes on the risk of DILI development and clinical presentations was analysed.ResultsNone of the individual polymorphisms or haplotypes was found to be associated with DILI development. Carriers homozygous for the ABCC2 −1774del allele were however only found in DILI patients. Hence, this genotype could potentially be associated with increased risk, though its low frequency in our Spanish cohort prevented a final conclusion. Furthermore, carriers homozygous for the ABCC2 −1774G/−1549A/−24T/1249G/3972T/4544G haplotype were found to have a higher propensity for total bilirubin elevations when developing DILI.ConclusionsOur findings do not support a role for the analysed polymorphisms in the ABCB1, ABCB4 and ABCC2 transporter genes in DILI development in Spanish patients. The ABCC2 −1774deldel genotype was however restricted to DILI cases and could potentially contribute to enhanced DILI susceptibility.

Understanding hepatotoxicity – from patients to mice to computer

Drug Induced Liver Injury (DILI) remains the major adverse drug event that leads to termination of clinical development programs and regulatory actions including failure to approve for marketing, restricted indications, and withdrawal from the marketplace. The type of DILI that is most problematic is “idiosyncratic” meaning that only a very small fraction of treated patients are susceptible to the DILI. Current preclinical models, even “humanized” ones, do not reliably identify molecules that have this liability and, conversely predict liabilities in molecule that are in fact quite safe for the liver. For example, current preclinical testing does not detect the DILI potential of xymelagatran, a drug withdrawn from worldwide markets due to idiosyncratic hepatotoxicity but would predict a high liver risk for acetaminophen (paracetamol) even when taken as directed. Reliable preclinical testing will probably not be developed until there is greater understanding of the mechanisms underlying DILI. Reasoning that the best models to study DILI are the people who have actually experienced it, we are capitalizing on the resources of the US Drug-Induced Liver Injury Network (DILIN-supported by the National Institutes of Health) that is collecting genomic DNA and other biospecimens from patients who have experienced DILI. In addition to genetic studies, we are partnering with Cellular Dynamics International to reprogram induced pleuripotent stem cells from DILIN subjects in whom we have complete exomic DNA sequence with the goal of producing patient-specific liver cultures, and ultimately, humanized mice. We are also partnering with the Shanghai Centers for Disease Control to prospectively collect a variety of biospecimens, including whole blood for transcriptome analysis, from patients treated for active tuberculosis as part of a larger biomarker discovery initiative. We are using panels of inbred mice to mimic patient population genetic diversity and to identify genes and pathways that may underlie DILI susceptibility in patients. Finally, we have established the DILIsim Initiative which is a public-private partnership that is building a computer model to synthesize the rapidly accumulating data with the goal of predicting DILI liability in drug candidates. DILIsim involves 10 pharmaceutical companies and the Food and Drug Administration.

Genetic variations in drug-induced liver injury (DILI): resolving the puzzle

Genetic variations in drug-induced liver injury (DILI): resolving the puzzle

A Genome-Wide Association Study Identifies Potential Susceptibility Loci for Hepatotoxicity Due to Various Drugs

Idiosyncratic drug-induced liver injury (DILI) is a leading cause of morbidity and mortality due to medication use yet is poorly studied, in part, due to its low incidence in the general population. Identification of genetic variants associated with these uncommon and difficult to diagnose adverse events could provide clues to underlying mechanisms. Hypothesis: Common genetic variants exist that contribute to DILI susceptibility from multiple drugs. Methods: DNA obtained from 783 individuals of European ancestry who experienced DILI attributed to >200 individual drugs were genotypedwith the Illumina 1Mor 1Mduo beadchip. Source of DNA samples included the following DILI registries: DILIN (401), DILIGEN (242), EUDRAGENE (89), and Malaga (51). Genome Wide Association (GWA) was performed using population controls (n=3001). Potential associations were tested for replication in 190 independent cases from DILIN. Results: GWA revealed a strong association within the MHC region (p 108), which did not replicate in validation cohorts composed of sufficient cases due to the same drug or class. Conclusion: Our replicated association between hepatocellular DILI and STAT4 supports the emerging role of the immune system in DILI susceptibility and suggests that common genetic variation may contribute to DILI susceptibility from multiple drugs. However, the generally negative GWA results suggest that there may be a preponderance of drug-specific genetic risk factors and/or rare genetic variation underlying DILI susceptibility. Therefore, we have begun whole exome and whole genome sequencing of DILI cases caused by the most frequent drugs in our cohorts in search of rarer, high-penetrance drug-specific risk factors.

Novel Susceptibility Loci for Primary Sclerosing Cholangitis Identified by Genome-Wide Association and Replication Analysis

Idiosyncratic drug-induced liver injury (DILI) is a leading cause of morbidity and mortality due to medication use yet is poorly studied, in part, due to its low incidence in the general population. Identification of genetic variants associated with these uncommon and difficult to diagnose adverse events could provide clues to underlying mechanisms. Hypothesis: Common genetic variants exist that contribute to DILI susceptibility from multiple drugs. Methods: DNA obtained from 783 individuals of European ancestry who experienced DILI attributed to >200 individual drugs were genotypedwith the Illumina 1Mor 1Mduo beadchip. Source of DNA samples included the following DILI registries: DILIN (401), DILIGEN (242), EUDRAGENE (89), and Malaga (51). Genome Wide Association (GWA) was performed using population controls (n=3001). Potential associations were tested for replication in 190 independent cases from DILIN. Results: GWA revealed a strong association within the MHC region (p 108), which did not replicate in validation cohorts composed of sufficient cases due to the same drug or class. Conclusion: Our replicated association between hepatocellular DILI and STAT4 supports the emerging role of the immune system in DILI susceptibility and suggests that common genetic variation may contribute to DILI susceptibility from multiple drugs. However, the generally negative GWA results suggest that there may be a preponderance of drug-specific genetic risk factors and/or rare genetic variation underlying DILI susceptibility. Therefore, we have begun whole exome and whole genome sequencing of DILI cases caused by the most frequent drugs in our cohorts in search of rarer, high-penetrance drug-specific risk factors.

Drug-Induced Liver Injury: Past, Present and Future

Drug-induced liver injury (DILI) is a rare but potentially serious idiosyncratic reaction. By using candidate gene and genome-wide association studies, replicated associations for DILI susceptibility with HLA genes and genes relevant to drug metabolism have been detected, mainly since 2000. The HLA associations include a strong association between flucloxacillin-induced injury and the class I allele B*5701 and weaker associations for co-amoxiclav and ximelagatran DILI with the class II genotype. These associations suggest an injury mechanism involving an immune response, possibly to a complex of drug or metabolite and protein. For genes relevant to drug metabolism, the best replicated association is between isoniazid DILI and NAT2 slow acetylation. Homozygosity for GSTM1 null and/or GSTT1 null alleles also seems to be a risk factor for DILI, with associations described independently for several drugs. Other not-yet-replicated associations have been described for genes relevant to drug metabolism and oxidative stress and cytokine genes.