Early Detection of Acute Drug-Induced Liver Injury in Mice by Noninvasive Near-Infrared Fluorescence Imaging

Jeffrey D Peterson,Kristine O Vasquez

doi:10.1124/jpet.116.238378

Abstract

Hepatocellular and cholestatic forms of drug-induced liver injury (DILI) are major reasons for late-stage termination of small-molecule drug discovery research projects. Biochemical serum markers are limited in their ability to sensitively and specifically detect both of these common DILI forms in preclinical models, and tissue-specific approaches to assessing this are labor intensive, requiring extensive animal dosing, tissue preparation, and pathology assessment. In vivo fluorescent imaging offers noninvasive detection of biologic changes detected directly in the livers of living animals. Three different near-infrared fluorescent imaging probes, specific for cell death (Annexin-Vivo 750), matrix metalloproteases (MMPSense 750 FAST), and transferrin receptor (Transferrin-Vivo 750) were used to measure the effects of single bolus intraperitoneal doses of four different chemical agents known to induce liver injury. Hepatocellular injury–inducing agents, thioacetamide and acetaminophen, showed optimal injury detection with probe injection at 18–24 hours, the liver cholestasis-inducing drug rifampicin required early probe injection (2 hours), and chlorpromazine, which induces mixed hepatocellular/cholestatic injury, showed injury with both early and late injection. Different patterns of liver responses were seen among these different imaging probes, and no one probe detected injury by all four compounds. By using a cocktail of these three near-infrared fluorescent imaging probes, all labeled with 750-nm fluorophores, each of the four different DILI agents induced comparable tissue injury within the liver region, as assessed by epifluorescence imaging. A strategy of probe cocktail injection in separate cohorts at 2 hours and at 20–24 hours allowed the effective detection of drugs with either early- or late-onset injury.

Highlights

Drug-induced liver injury (DILI) is the leading reason for termination of drug discovery research projects, is a significant concern in attrition of new drug molecules reaching phase III clinical trials, is the major reason for US Food and Drug Administration withdrawal of drugs from the market after approval, and accounts for more than 50% of acute liver failure cases in the United States (Bissell et al, 2001)
One of the most obvious biologic responses to explore as a readout was cell death, so we used an Annexin V–based NIR fluorescent imaging probe (AV750) that has been used effectively in cancer treatment research (Zhao et al, 2015) and in preliminary liver injury studies (Peterson, 2016)
In vivo preclinical toxicology studies use living animals to assess potential drug-induced adverse effects, providing models in which potential toxicity is driven by the combined effects of drug distribution/metabolism, drug mechanistic efficacy, and the complex biology and physiology of a living animal

Summary

Introduction

Drug-induced liver injury (DILI) is the leading reason for termination of drug discovery research projects, is a significant concern in attrition of new drug molecules reaching phase III clinical trials, is the major reason for US Food and Drug Administration withdrawal of drugs from the market after approval, and accounts for more than 50% of acute liver failure cases in the United States (Bissell et al, 2001). Biologic pathway changes can occur in liver tissue, including those indicating direct hepatocyte injury (apoptosis/necrosis), oxidative stress, vascular or microvascular injury, induction of innate or cognate immunity/inflammation, and DNA damage Within each of these mechanisms are multiple signaling pathways and biomarkers that could serve as indicators of drug effects; a strategy of pathway/biomarker profiling has the potential for the sensitive detection of drug-induced changes for early-stage screening. This systems-based toxicology strategy utilizes in vitro experimental models and addresses dozens of biomarkers from interconnected or parallel networks; our hypothesis was that such an approach could be adapted for in vivo use in a scaled-down fashion by using simultaneous detection of a few key biomarker responses. This should retain the concept of screening for different types of adverse biologic responses, rather than relying on grosser tissue phenotypic changes, but opens up the possibility of using noninvasive rodent imaging strategies

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