Systems analysis of miRNA biomarkers to inform drug safety.

microRNAs (miRNAs or miRs) are short non-coding RNA molecules which have been shown to be dysregulated and released into the extracellular milieu as a result of many drug and non-drug-induced pathologies in different organ systems. Consequently, circulating miRs have been proposed as useful biomarkers of many disease states, including drug-induced tissue injury. miRs have shown potential to support or even replace the existing traditional biomarkers of drug-induced toxicity in terms of sensitivity and specificity, and there is some evidence for their improved diagnostic and prognostic value. However, several pre-analytical and analytical challenges, mainly associated with assay standardization, require solutions before circulating miRs can be successfully translated into the clinic. This review will consider the value and potential for the use of circulating miRs in drug-safety assessment and describe a systems approach to the analysis of the miRNAome in the discovery setting, as well as highlighting standardization issues that at this stage prevent their clinical use as biomarkers. Highlighting these challenges will hopefully drive future research into finding appropriate solutions, and eventually circulating miRs may be translated to the clinic where their undoubted biomarker potential can be used to benefit patients in rapid, easy to use, point-of-care test systems.

Proteomic profiling of murine biliary-derived hepatic organoids and their capacity for drug disposition, bioactivation and detoxification.

Hepatic organoids are a recent innovation in in vitro modeling. Initial studies suggest that organoids better recapitulate the liver phenotype in vitro compared to pre-existing proliferative cell models. However, their potential for drug metabolism and detoxification remains poorly characterized, and their global proteome has yet to be compared to their tissue of origin. This analysis is urgently needed to determine what gain-of-function this new model may represent for modeling the physiological and toxicological response of the liver to xenobiotics. Global proteomic profiling of undifferentiated and differentiated hepatic murine organoids and donor-matched livers was, therefore, performed to assess both their similarity to liver tissue, and the expression of drug-metabolizing enzymes and transporters. This analysis quantified 4405 proteins across all sample types. Data are available via ProteomeXchange (PXD017986). Differentiation of organoids significantly increased the expression of multiple cytochrome P450, phase II enzymes, liver biomarkers and hepatic transporters. While the final phenotype of differentiated organoids is distinct from liver tissue, the organoids contain multiple drug metabolizing and transporter proteins necessary for liver function and drug metabolism, such as cytochrome P450 3A, glutathione-S-transferase alpha and multidrug resistance protein 1A. Indeed, the differentiated organoids were shown to exhibit increased sensitivity to midazolam (10-1000 µM) and irinotecan (1-100 µM), when compared to the undifferentiated organoids. The predicted reduced activity of HNF4A and a resulting dysregulation of RNA polymerase II may explain the partial differentiation of the organoids. Although further experimentation, optimization and characterization is needed relative to pre-existing models to fully contextualize their use as an in vitro model of drug-induced liver injury, hepatic organoids represent an attractive novel model of the response of the liver to xenobiotics. The current study also highlights the utility of global proteomic analyses for rapid and accurate evaluation of organoid-based test systems.

MiR-122 and other microRNAs as potential circulating biomarkers of drug-induced liver injury.

INTRODUCTION: Drug-induced liver injury (DILI) is a severe adverse drug reaction which is of major concern to patients, clinicians and the pharmaceutical industry. Accurate and rapid detection of DILI is important for patient stratification and treatment in the clinic and benefits preclinical drug design and risk assessment. MicroRNAs (miRNAs) offer a potential new and improved class of circulating biomarkers of DILI over the current gold standard biomarkers. Areas covered: This review highlights the shortcomings of the currently used panel of biomarkers and how miRNAs, primarily miR-122, show an improved level of specificity and sensitivity in the prediction of DILI. Furthermore, the use of miRNAs as potential markers of progression of DILI and specific zonated damage within the liver is discussed. Expert commentary: MiRNAs offer more sensitive and specific markers over the current biomarkers for DILI. Combinations of different miRNAs may be able to relay the location of DILI and the progression of disease. More studies using different hepatotoxins apart from acetaminophen will ultimately strengthen the case for the clinical introduction of miRNAs as biomarkers of DILI.

Circulating levels of miR-122 increase post-mortem, particularly following lethal dosing with pentobarbital sodium: implications for pre-clinical liver injury studies.

microRNA-122 (miR-122) is increasingly being measured in pre-clinical and clinical settings due to greater sensitivity and hepatic specificity compared to the gold standard liver injury biomarker alanine aminotransferase (ALT). In pre-clinical studies, various culling methods can be employed prior to collection of blood samples, including lethal injection with pentobarbital sodium (Pentoject). However, little is known about whether such an approach could alter the circulating levels of miR-122 and compromise the interpretation of data. We therefore exposed C57BL/6J mice to saline or the model hepatotoxin paracetamol and collected blood samples pre-cull (via tail bleed) and post-cull (via cardiac puncture following exposure to a rising concentration of CO2 or intraperitoneal injection of Pentoject). Compared to pre-cull levels there was a significant increase in serum miR-122 level in mice culled with CO2 and, to a much greater extent, in mice culled with Pentoject. As a result, whilst the serum level of miR-122 increased in Pentoject-culled animals exposed to paracetamol, the higher level in saline-treated mice rendered this difference statistically non-significant, in contrast to findings in animals culled with CO2. ALT levels were unaffected by sacrifice method. Consistent with the in vivo findings, exposure of primary mouse hepatocytes to Pentoject provoked a rapid and concentration-dependent release of miR-122 into the culture media. Thus, for optimal design and interpretation of data from pre-clinical liver injury studies in which miR-122 is to be used as a biomarker, we recommend that blood samples are collected pre-cull whenever possible, and that lethal injection with Pentoject is avoided.