Zebrafish: A New In Vivo Model for Identifying P-Glycoprotein Efflux Modulators - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 170

Chapter 14

Zebrafish: A New In Vivo
Model for Identifying
P-Glycoprotein Efflux
Modulators

Demian Park, Maryann Haldi, and Wen Lin Seng
Phylonix, Cambridge, MA, USA

14.1 INTRODUCTION

In order to address the rapidly increasing incidence of central nervous system (CNS)
diseases, new approaches for delivering and retaining drugs in the brain are needed.
P-glycoprotein (Pgp), one of the most important members of the ABC transporter
family, has been shown to inhibit absorption and retention of more than 50% of
frequently prescribed drugs (Bauer et al., 2005). Development of Pgp inhibitors is one
strategy for increasing drug accumulation in the brain. Although drug transporters are
well described in mammals (Jeong et al., 2001), blood brain barrier (BBB) formation
and Pgp expression and function are not well characterized in zebrafish. Here we
describe development of a novel in vivo zebrafish assay to rapidly identify Pgp efflux
inhibitors. By injecting a fluorescent Pgp substrate directly into the transparent zebrafish
brain, we determined that Pgp is fully functioning by 7 days post fertilization (dpf). We
then monitored Pgp efflux kinetics by capturing images of fluorescent dye efflux from
the brain at varying time points. Using this approach, potential drug candidates can be
cotreated with Pgp substrate to visualize retention in the brain. Next, to identify
potential Pgp efflux inhibitors, we developed a quantitative functional assay that relies
on morphometric image analysis. To validate this bioassay, we confirmed that six
positive control drugs, verapamil, phenytoin, loperamide, cyclosporine, RU486, and
quinidine, inhibited Pgp efflux in zebrafish brain and one negative control drug,