Katherine D. Lynch

The recent ICH M12 guidance on Drug Interaction Studies encourages the use of alternate approaches for predicting drug–drug interaction (DDI) potential of new chemical entities. One approach involves the use of endogenous substrates of drug metabolizing enzymes and transporters (DMET) as biomarkers, which can assess the inhibitory potential of new chemical entities towards DMET during Phase 1 clinical studies. Thus, biomarkers could potentially eliminate the need for dedicated DDI studies with exogenous probe substrates. Metabolomics, in conjunction with in vitro and/or in vivo preclinical models or clinical studies, can be used for biomarker discovery. We developed and applied a novel metabolomics‐based DMET biomarker discovery (MDBD) approach to identify and qualify biomarkers of renal organic anion transporter 1 (OAT1) and OAT3. Untargeted metabolomics of pooled plasma and urine samples (n = 16) from a pharmacokinetic DDI study using the OAT1/3 inhibitor, probenecid, yielded 153 features identified as putative OAT1/3 biomarkers. Subsequently, in vitro transporter uptake assays using processed urine samples confirmed 57 of these features as OAT1 and/or OAT3 substrates. Finally, 23 features were clinically validated as OAT1/3 biomarkers through a detailed pharmacokinetic analysis (0–24 h) of plasma and urine samples (n = 4). These biomarkers, either alone or as part of a panel, can predict OAT1/3‐mediated DDIs and interindividual variability in the renal secretory clearance of organic anions across different populations, thereby enabling translational utility in clinical settings. The novel MDBD approach can be extended to discover biomarkers of enzymes and other transporters.

Microcystin-leucine arginine (MCLR) is one of the most common and toxic microcystin variants, a class of cyanotoxins produced by cyanobacteria. A major molecular mechanism for MCLR-elicited liver toxicity involves the dysregulation of protein phosphorylation through protein phosphatase (PP) inhibition and mitogen-activated protein kinase (MAPK) modulation. In this study, specific pharmacological MAPK inhibitors were used in HepaRG cells to examine the pathways associated with MCLR cytotoxicity. SB203580 (SB), a p38 inhibitor, rescued HepaRG cell viability, whereas treatment with SP600125 (JNK inhibitor), MK2206 (AKT inhibitor), or N-acetylcysteine (reactive oxygen species scavenger) did not. Phosphoproteomic analysis revealed that phosphosites—which were altered by the addition of SB compared to MCLR treatment alone—included proteins involved in RNA processing, cytoskeletal stability, DNA damage response, protein degradation, and cell death. A closer analysis of specific proteins in some of these pathways indicated that SB reversed the MCLR-mediated phosphorylation of the necroptosis-associated proteins, the mixed lineage kinase domain-like protein (MLKL), receptor-interacting serine/threonine kinase 1 (RIP1), DNA damage response proteins, ataxia telangiectasia and Rad3-related kinase (ATR), and checkpoint kinase 1 (CHK1). Overall, these data implicate p38/MK2, DNA damage, and necroptosis in MCLR-mediated hepatotoxicity, and suggest these pathways may be targets for prevention prior to, or treatment after, MCLR toxicity.

Silybum marianum (L.) Gaertn. (Asteraceae), commonly known as milk thistle, is a botanical natural product used to self‐treat multiple diseases such as Type 2 diabetes mellitus and nonalcoholic steatohepatitis (NASH). An extract from milk thistle seeds (achenes), termed silymarin, is comprised primarily of several flavonolignans. Systemic concentrations of these flavonolignans can influence the potential biologic effects of silymarin and the risk for pharmacokinetic silymarin‐drug interactions. The aims of this research were to determine the roles of organic anion transporting polypeptides (OATPs/Oatps) in silymarin flavonolignan disposition and in pharmacokinetic silymarin‐drug interactions. The seven major flavonolignans from silymarin were determined to be substrates for OATP1B1, OATP1B3, and OATP2B1. Sprague Dawley rats were fed either a control diet or a NASH‐inducing diet and administered pitavastatin (OATP/Oatp probe substrate), followed by silymarin via oral gavage. Decreased protein expression of Oatp1b2 and Oatp1a4 in NASH animals increased flavonolignan area under the plasma concentration‐time curve (AUC) and maximum plasma concentration. The combination of silymarin inhibition of Oatps and NASH‐associated decrease in Oatp expression caused an additive increase in plasma pitavastatin AUC in the animals. These data indicate that OATPs/Oatps contribute to flavonolignan cellular uptake and mediate the interaction between silymarin and NASH on pitavastatin systemic exposure.

Microcystin-LR (MCLR) is a hepatotoxic cyanotoxin reported to cause a phenotype similar to nonalcoholic steatohepatitis (NASH). NASH is a common progressive liver disease that advances in severity due to exogenous stressors such as poor diet and toxicant exposure. Our objective was to determine how sub-chronic MCLR toxicity affects preexisting diet-induced NASH. Sprague-Dawley rats were fed one of three diets for 10 weeks: control, methionine and choline deficient (MCD), or high fat/high cholesterol (HFHC). After six weeks of diet, animals received vehicle, 10 µg/kg, or 30 µg/kg MCLR via intraperitoneal injection every other day for the final 4 weeks. Incidence and severity scoring of histopathology endpoints suggested that MCLR toxicity drove NASH to a less fatty and more fibrotic state. In general, expression of genes involved in de novo lipogenesis and fatty acid esterification were altered in favor of decreased steatosis. The higher MCLR dose increased expression of genes involved in fibrosis and inflammation in the control and HFHC groups. These data suggest MCLR toxicity in the context of preexisting NASH may drive the liver to a more severe phenotype that resembles burnt-out NASH.