Intravital Multiphoton Microscopy with Fluorescent Bile Salts in Rats as an In Vivo Biomarker for Hepatobiliary Transport Inhibition

Abstract

The bile salt export pump (BSEP) is expressed at the canalicular domain of hepatocytes, where it mediates the elimination of monovalent bile salts into the bile. Inhibition of BSEP is considered a susceptibility factor for drug-induced liver injury that often goes undetected during nonclinical testing. Although in vitro assays exist for screening BSEP inhibition, a reliable and specific method for confirming Bsep inhibition in vivo would be a valuable follow up to a BSEP screening strategy, helping to put a translatable context around in vitro inhibition data, incorporating processes such as metabolism, protein binding, and other exposure properties that are lacking in most in vitro BSEP models. Here, we describe studies in which methods of quantitative intravital microscopy were used to identify dose-dependent effects of two known BSEP/Bsep inhibitors, 2-[4-[4-(butylcarbamoyl)-2-[(2,4-dichlorophenyl)sulfonylamino]phenoxy]-3-methoxyphenyl]acetic acid (AMG-009) and bosentan, on hepatocellular transport of the fluorescent bile salts cholylglycyl amidofluorescein and cholyl-lysyl-fluorescein in rats. Results of these studies demonstrate that the intravital microscopy approach is capable of detecting Bsep inhibition at drug doses well below those found to increase serum bile acid levels, and also indicate that basolateral efflux transporters play a significant role in preventing cytosolic accumulation of bile acids under conditions of Bsep inhibition in rats. Studies of this kind can both improve our understanding of exposures needed to inhibit Bsep in vivo and provide unique insights into drug effects in ways that can improve our ability interpret animal studies for the prediction of human drug hepatotoxicity.

Tanshinone IIA alleviate rifampicin-induced cholestasis by regulating the expression and function of bile salt export pump

“…24 Besides, BSEP is the vital transporter to mediate the excretion of monovalent bile acids into the canaliculi. 25 BSEP gene mutation induces a variety of types of cholestasis, such as benign recurrent intrahepatic cholestasis type 2 (BRIC2) and progressive familial intrahepatic cholestasis type 2 (PFIC2). 26,27 Therefore, to further clarify the mechanism of RFP induced cholestatic DILI, we would comprehensively explore the effects of RFP on both BSEP expression and its transport activity in vitro and in vivo .…”

Spatial Temporal Analysis of Fieldwise Flow in Microvasculature

“…Due to the proximity of the diaphragm, short periods of respiration-induced motion occur, with the secured liver returning to its initial position after each breath. Securing the surgically exposed liver against the coverslip-bottomed dish using gauze, then imaging from below using an inverted microscope serves to immobilize the organ between respirations 16,17,18,19 . Second, an image acquisition speed of 100 fps is strongly recommended because the speed of flow that can be measured is a function of the speed of image acquisition and the minimum length of the segments in which flow is measured 11 .…”

Reducing target binding affinity improves the therapeutic index of anti-MET antibody-drug conjugate in tumor bearing animals

“…IVM studies were conducted either 24 hours after intravenous (IV) injection of DL488 labeled HAV or LAV (6 mg/kg), respectively, or during and for the first two hours following IV injection. Studies were conducted generally as described previously [ 28 , 29 ]. Surgical preparation was started approximately one hour prior to imaging.…”