A high-throughput method for predicting drug effects on gut transit time using larval zebrafish

Abstract

Introduction: Zebrafish are an attractive vertebrate model due to their small size, transparency through organogenesis, and high fecundity. The zebrafish gastrointestinal (GI) tract is similar to the mammalian GI tract in gene expression, nervous system control, and response to chemical challenges. GI intolerance is a common preclinical finding and can be a serious clinical safety concern. Mammalian GI liability tests are conducted at the expense of time, test article, and labor. We developed a high-throughput method to predict mammalian GI safety issues using larval zebrafish.

Methods: Fluorescent food is fed to larval zebrafish (7 days post fertilization). After feeding, larvae are placed individually into wells of a 96-well plate and dosed with test compounds. Fluorescence is measured from the bottom of the wells repeatedly over the course of 24h and thus fecal accumulation is tracked over time. The area under the curve is compared between treated and vehicle-treated groups.

Results: Drugs with established clinical GI effects significantly impacted zebrafish GI transit time as measured by this method; tegaserod and metoclopramide accelerated transit time, while atropine and amitriptyline slowed transit time. This method is sensitive enough to reflect dose-level associated effects as demonstrated using atropine. Using a suite of 24 compounds with known (positive or negative) mammalian GI effects, we characterized this method as having a high positive predictive value.

Discussion: Here we present an efficient assay for predicting mammalian GI transit liabilities using larval zebrafish. With this assay, an investigator can evaluate dozens of compounds in a single day using very little amount of each test article. As such, safe drug candidates can be prioritized for mammalian testing, which expedites the discovery process and provides 3 Rs impact.

Keywords: Amitriptyline (PubChem CID: 2160); Atropine (PubChem CID: 174,174); Drug; Erythromycin (PubChem CID: 12,560); Gastrointestinal; Methods; Metoclopramide (PubChem CID: 4168); Safety; Tegaserod (PubChem CID: 5,362,436); Zebrafish.