Andreas Benesic

Drug-induced liver injury: recent advances in diagnosis and risk assessment.

Idiosyncratic drug-induced liver injury (IDILI) is a rare but potentially severe adverse drug reaction that should be considered in patients who develop laboratory criteria for liver injury secondary to the administration of a potentially hepatotoxic drug. Although currently used liver parameters are sensitive in detecting DILI, they are neither specific nor able to predict the patients subsequent clinical course. Genetic risk assessment is useful mainly due to its high negative predictive value, with several human leucocyte antigen alleles being associated with DILI. New emerging biomarkers which could be useful in assessing DILI include total keratin18 (K18) and caspase-cleaved keratin18 (ccK18), macrophage colony-stimulating factor receptor 1, high mobility group box 1 and microRNA-122. From the numerous in vitro test systems that are available, monocyte-derived hepatocytes generated from patients with DILI show promise in identifying the DILI-causing agent from among a panel of coprescribed drugs. Several computer-based algorithms are available that rely on cumulative scores of known risk factors such as the administered dose or potential liabilities such as mitochondrial toxicity, inhibition of the bile salt export pump or the formation of reactive metabolites. A novel DILI cluster score is being developed which predicts DILI from multiple complimentary cluster and classification models using absorption–distribution–metabolism–elimination-related as well as physicochemical properties, diverse substructural descriptors and known structural liabilities. The provision of more advanced scientific and regulatory guidance for liver safety assessment will depend on validating the new diagnostic markers in the ongoing DILI registries, biobanks and public–private partnerships.

Intraperitoneal LPS amplifies portal hypertension in rat liver fibrosis.

Recent studies have shown that the risk of variceal bleeding in patients with liver cirrhosis increases with infections such as spontaneous bacterial peritonitis (SBP). In this study, we hypothesized that pretreatment with intraperitoneal LPS may escalate portal hypertension. In fibrotic livers (4 weeks after bile duct ligation, BDL), the activation of Kupffer cells (KCs) by zymosan (150 μg/ml) in the isolated non-recirculating liver perfusion system resulted in a transient increase in portal perfusion pressure. Pretreatment with intraperitoneal LPS (1 mg/kg body weight (b.w.) for 3 h) increased basal portal perfusion pressure, and prolonged the zymosan-induced increase from transient to a long-lasting increase that was sustained until the end of the experiments in BDL but not in sham-operated animals. Pretreatment with gadolinium chloride (10 mg/kg b.w.), MK-886 (0.6 mg/kg b.w.), Ly171883 (20 μM) or BM 13.177 (20 μM) reduced the maximal and long-lasting pressure increase in BDL animals by approximately 50–60%. The change in portal perfusion pressure was paralleled by a long-lasting production of cysteinyl leukotriene (Cys-LT) and thromboxane (TX) after LPS pretreatment. However, the response to vasoconstrictors was not altered by intraperitoneal LPS. Western blot analyses showed an increased Toll-like receptor (TLR)4 and MyD88 expression after LPS pretreatment. In vivo experiments confirmed that intraperitoneal LPS increased basal portal pressure, and extended the portal pressure increase produced by intraportal zymosan or by LPS infusion. In conclusion, upregulation of TLR4 and MyD88 expression in fibrotic livers confers hypersensitivity to LPS. This may lead to escalation of portal hypertension by production of TX and Cys-LT after endotoxin-induced KC activation. Therefore, LT inhibitors may represent a promising treatment option in addition to early administration of antibiotics in SBP.

Serum Neutrophil Gelatinase-Associated Lipocalin - A Sensitive Novel Marker of Renal Impairment in Liver Cirrhosis?

Recently, neutrophil gelatinase-associated lipocalin (NGAL) has been introduced as a sensitive and early marker of acute kidney injury [5] . NGAL in cirrhosis and chronic renal failure has not been investigated so far. We have therefore analyzed NGAL and GFR in patients with Dear Sir, Renal dysfunction is a frequent and clinically relevant problem in patients with cirrhosis of the liver [1] . While acute renal failure as in hepatorenal syndrome type 1 is characterized by a rapid increase of serum creatinine (Crea) concentration [2] , chronic renal dysfunction may be difficult to detect. For determination of the glomerular filtration rate (GFR), the use of clearance techniques is the gold standard. These techniques, however, are cumbersome and not suitable for clinical routine. Serum Crea is simple and easy to determine and therefore widely used per se or in Crea-based formulae such as MDRD (Modification of Diet in Renal Disease formula, using serum Crea, age and gender). Unfortunately, Crea is not a precise parameter of renal function in cirrhosis [3] . The overestimation of GFR by serum Crea and Crea-based equations is striking, particularly in decompensated patients with cirrhosis and normal or slightly increased Crea. Clinically it seems most important to identify renal dysfunction in these patients at an early stage before development of a hepatorenal syndrome denoting a poor prognosis. Therefore, other serum parameters such as cystatin C have been proposed to reflect renal function in cirrhosis [4] but have not been generally accepted yet. Published online: April 14, 2011

Monocyte-derived hepatocyte-like cells for causality assessment of idiosyncratic drug-induced liver injury

Background Idiosyncratic drug-induced liver injury (iDILI) is a frequent cause of acute liver injury and a serious problem in late stage drug-development. Its diagnosis is one of the most challenging in hepatology, since it is done by exclusion and relies on expert opinion. Until now no reliable in vitro test exists to support the diagnosis of iDILI. In some instances it is impossible to determine the causative drug in polymedicated patients. Aim To investigate if monocyte-derived hepatocyte-like (MH) cells might be a tool supporting clinical judgment for iDILI diagnosis and causality assessment. Methods This prospective study included 54 patients with acute liver injury and intake of at least one drug. Thirty-one patients were diagnosed with iDILI based on causality likelihood. MH cells were generated from every patient and in vitro toxicity of the respective drugs was assessed by lactate-dehydrogenase release. The results from MH cells and RUCAM, the most widely used scoring system as methods to support clinical judgement were compared. Results MH cells showed enhanced toxicity in 29 of the 31 patients with iDILI, similar to RUCAM score. MH cells exhibited negative results in the 23 non-DILI cases, whereas RUCAM indicated possible iDILI in six cases. Analysis of the comedications also showed superior specificity of MH cells. No MH cell toxicity of the drugs showing toxicity in patients with iDILI was observed in MH cells of healthy donors. Conclusions In this pilot study in vitro testing using MH cells derived from patients with acute liver injury was able to identify patients with iDILI with an excellent sensitivity and a higher specificity than RUCAM, the most widely used current causality assessment score. Therefore, MH cells could be useful to identify the causative drugs even in polymedicated patients by adding objective data to causality assessment. Trial registration number : NCT02353455

Human monocyte-derived cells with individual hepatocyte characteristics: a novel tool for personalized in vitro studies

Gender, ethnicity and individual differences in hepatic metabolism have major impact on individual drug response, adverse events and attrition rate during drug development. Therefore, there is an urgent need for reliable test systems based on human cells. Yet, the use of primary human hepatocytes (PHHs) is restricted by limited availability, invasive preparation and short-term stability in culture. All other cellular approaches proposed so far have major disadvantages. We investigated whether peripheral human monocytes after cultivation according to our novel protocol (monocyte-derived hepatocyte-like cells (MH cells)) can serve as an in vitro model for hepatocyte metabolism. Enzyme activities, synthesis parameters (coagulation factor VII and urea) and cytochrome (CY) P450 activities and induction were investigated. Furthermore, MH cells were compared with PHH from the same donor. Using our protocol, we could generate cells that exhibit hepatocyte-like properties: These cells show 71±9% of specific ALT activity, 41±3% of CYP3A4 activity and 65±13% of factor VII secretion when compared with PHHs. Consequently, CYP-mediated acetaminophen toxicity and drug interactions could be shown. Moreover, the investigated parameters were stable in culture over at least 4 weeks. Furthermore, MH cells retain gender-specific and donor-specific CYP activities and toxicity profiles, respectively. MH cells show quantitative and qualitative approximation to human hepatocytes concerning CYP-metabolism and toxicity. Our data support individual prediction of toxicity and CYP metabolism. MH cells are a novel tool to investigate long-term hepatic toxicity, metabolism and drug interactions.

Plasma Cystatin C Is a Predictor of Renal Dysfunction, Acute-on-Chronic Liver Failure, and Mortality in Patients With Acutely Decompensated Liver Cirrhosis

The development of acute‐on‐chronic liver failure (ACLF) in patients with liver cirrhosis is associated with high mortality rates. Renal failure is the most significant organ dysfunction that occurs in ACLF. So far there are no biomarkers predicting ACLF. We investigated whether cystatin C (CysC) and neutrophil gelatinase‐associated lipocalin (NGAL) can predict development of renal dysfunction (RD), hepatorenal syndrome (HRS), ACLF, and mortality. We determined the plasma levels of CysC and NGAL in 429 patients hospitalized for acute decompensation of cirrhosis in the EASL‐CLIF Acute‐on‐Chronic Liver Failure in Cirrhosis (CANONIC) study. The patients were followed for 90 days. Patients without RD or ACLF at inclusion but with development of either had significantly higher baseline concentrations of CysC and NGAL compared to patients without. CysC, but not NGAL, was found to be predictive of RD (odds ratio, 9.4; 95% confidence interval [CI], 1.8‐49.7), HRS (odds ratio, 4.2; 95% CI, 1.2‐14.8), and ACLF (odds ratio, 5.9; 95% CI, 1.3‐25.9). CysC at day 3 was not found to be a better predictor than baseline CysC. CysC and NGAL were both predictive of 90‐day mortality, with hazard ratios for CysC of 3.1 (95% CI, 2.1‐4.7) and for NGAL of 1.9 (95% CI, 1.5‐2.4). Conclusion: Baseline CysC is a biomarker of RD, HRS, and ACLF and an independent predictor of mortality in patients with acutely decompensated liver cirrhosis, though determining CysC at day 3 did not provide any benefit; while NGAL is also associated with short‐term mortality, it fails to predict development of RD, HRS, and ACLF. Baseline CysC may help to identify patients at risk earlier and improve clinical management. (Hepatology 2017;66:1232‐1241)

Plasma Cystatin C is a predictor of renal dysfunction, ACLF and mortality in patients with acutely decompensated liver cirrhosis

The development of acute‐on‐chronic liver failure (ACLF) in patients with liver cirrhosis is associated with high mortality rates. Renal failure is the most significant organ dysfunction that occurs in ACLF. So far there are no biomarkers predicting ACLF. We investigated whether cystatin C (CysC) and neutrophil gelatinase‐associated lipocalin (NGAL) can predict development of renal dysfunction (RD), hepatorenal syndrome (HRS), ACLF, and mortality. We determined the plasma levels of CysC and NGAL in 429 patients hospitalized for acute decompensation of cirrhosis in the EASL‐CLIF Acute‐on‐Chronic Liver Failure in Cirrhosis (CANONIC) study. The patients were followed for 90 days. Patients without RD or ACLF at inclusion but with development of either had significantly higher baseline concentrations of CysC and NGAL compared to patients without. CysC, but not NGAL, was found to be predictive of RD (odds ratio, 9.4; 95% confidence interval [CI], 1.8‐49.7), HRS (odds ratio, 4.2; 95% CI, 1.2‐14.8), and ACLF (odds ratio, 5.9; 95% CI, 1.3‐25.9). CysC at day 3 was not found to be a better predictor than baseline CysC. CysC and NGAL were both predictive of 90‐day mortality, with hazard ratios for CysC of 3.1 (95% CI, 2.1‐4.7) and for NGAL of 1.9 (95% CI, 1.5‐2.4). Conclusion: Baseline CysC is a biomarker of RD, HRS, and ACLF and an independent predictor of mortality in patients with acutely decompensated liver cirrhosis, though determining CysC at day 3 did not provide any benefit; while NGAL is also associated with short‐term mortality, it fails to predict development of RD, HRS, and ACLF. Baseline CysC may help to identify patients at risk earlier and improve clinical management. (Hepatology 2017;66:1232‐1241)

Development and Validation of a Test to Identify Drugs That Cause Idiosyncratic Drug-Induced Liver Injury

Background & Aims Idiosyncratic drug‐induced liver injury (iDILI) is one of the most challenging diagnoses in hepatology. It is frequently impossible to identify the agent that has caused iDILI in patients who take multiple medicines. We developed an in vitro method to identify drugs that cause liver injury in patients, based on drug toxicity to monocyte‐derived hepatocyte‐like (MH) cells from patient blood samples. We then collected data on patients who were re‐exposed to drugs found to be toxic in the MH test to validate test performance. Methods We performed a prospective study of patients referred to the University Hospital in Munich, Germany, with acute liver injury believed to be caused by medications (300 patients were enrolled in the study and we present data from 40 patients with iDILI and re‐exposure to implicated drugs). We collected data from patients on medical history, laboratory test and imaging results, findings from biopsy analyses, and medications taken. Blood samples were collected from all patients and MH cells were isolated and cultured for 10 days. MH cells were then incubated with drugs to which each patient had been exposed, and toxicity was measured based on release of lactate dehydrogenase. Agents found to be toxic to MH cells were considered as candidates for the cause of liver injury. Patients were followed up for up to 6 months after liver injury and data on drug re‐exposures and subsequent liver damage within the following 3 to 24 months were associated with findings from MH tests. Results Our test identified 10 drugs that were toxic to MH cells from 13 patients (amoxicillin/clavulanate to cells from 2 patients; diclofenac to cells from 2 patients; methylprednisolone to cells from 2 patients; and atorvastatin, metamizole, pembrolizumab, piperacillin/tazobactam, moxifloxacin, duloxetine, or sertraline each to cells from 1 patient). Thirteen patients had a recurrence of liver injury after inadvertent re‐exposure to a single drug, and the MH test correctly identified 12 of the 13 drugs that caused these liver re‐injury events. All 86 drugs that were not toxic to MH cells in our assay were safely resumed by patients and were not associated with liver re‐injury in 27 patients. Therefore, the MH test identifies drugs that cause liver injury with 92.3% sensitivity and 100% specificity (1 false‐negative and 12 true‐positive results). Conclusions We developed a test to identify drugs that cause liver injury in patients based on their toxicity to MH cells isolated from patients with DILI. We validated results from the assay and found it to identify drugs that cause DILI with 92.3% sensitivity and 100% specificity. The MH cell test could be a tool to identify causes of iDILI, even in patients taking multiple medications. ClinicalTrials.gov no: NCT 02353455.

Drug-Induced Liver Injury and Individual Cell Models

Drug-induced liver injury (DILI) is the most common cause of acute liver failure and accounts for the majority of regulatory actions on drugs. Furthermore, DILI is a relevant cause for project terminations in pharmaceutical development. The idiosyncratic form of DILI is especially a threat in late clinical development phases and postmarketing, respectively. Even the occurrence of only a few idiosyncratic DILI cases in late clinical development or postmarketing may suffice to terminate or withdraw an otherwise promising therapy. Despite advances in preclinical assessment of dose-dependent toxicity, idiosyncratic DILI is still a big challenge for in vitro research: it not only requires individualized models but also a huge number of tests. We have developed and investigated MetaHeps®, a technology involving hepatocyte-like cells generated from peripheral monocytes without genetic modifications. These cells exhibit several hepatocyte-like characteristics and show donor-specific activities of drug-metabolizing enzymes. With MetaHeps we have performed in vitro investigations in patients with DILI suspicion. By investigating MetaHeps derived from DILI patients we could show increased in vitro susceptibility to the drugs involved in the individual patients. MetaHeps testing could also rule out DILI and help to identify other causes of acute liver injury. Moreover, MetaHeps identified the causative agent in polymedicated patients. In conclusion, in vitro research of idiosyncratic DILI requires individual cell models which produce results comparable to the clinical situation. We suggest the MetaHeps technology as a novel tool to cope with these challenges of DILI.

Proteomics Analysis of Monocyte-Derived Hepatocyte-Like Cells Identifies Integrin Beta 3 as a Specific Biomarker for Drug-Induced Liver Injury by Diclofenac

Idiosyncratic drug-induced liver injury (iDILI) is a major cause of acute liver failure resulting in liver transplantation or death. Prediction and diagnosis of iDILI remain a great challenge, as current models provide unsatisfying results in terms of sensitivity, specificity, and prognostic value. The absence of appropriate tools for iDILI detection also impairs the development of reliable biomarkers. Here, we report on a new method for identification of drug-specific biomarkers. We combined the advantages of monocyte-derived hepatocyte-like (MH) cells, able to mimic individual characteristics, with those of a novel mass spectrometry-based proteomics technology to assess potential biomarkers for Diclofenac-induced DILI. We found over 2,700 proteins differentially regulated in MH cells derived from individual patients. Herefrom, we identified integrin beta 3 (ITGB3) to be specifically upregulated in Diclofenac-treated MH cells from Diclofenac-DILI patients compared to control groups. Finally, we validated ITGB3 by flow cytometry analysis of whole blood and histological staining of liver biopsies derived from patients diagnosed with Diclofenac-DILI. In summary, our results show that biomarker candidates can be identified by proteomics analysis of MH cells. Application of this method to a broader range of drugs in the future will exploit its full potential for the development of drug-specific biomarkers. Data are available via ProteomeXchange with identifier PXD008918.