A novel noninvasive test based on near-infrared fluorescent cholephilic probes for hepatobiliary secretory function assessment

Abstract

Routine serum biomarkers do not always accurately reflect impaired liver function. To overcome this limitation, we synthesized novel bile acid (BA) derivatives (NIRBADs) with near-infrared (NIR) fluorescence that can penetrate the abdominal wall and be detected extracorporeally. NIRBAD dynamics in the liver parenchyma were recorded through intravital imaging in mice and extracorporeally in both rats and mice. NIRBAD metabolism was analyzed using HPLC-MS/MS and fluorimetry. Transport was investigated in cells expressing BA transporters, whose interactions with NIRBADs were assessed through molecular docking and dynamics simulations. The hepatic NIRBAD clearance time (NCT) was evaluated in animal models with impaired secretory function: rats with hepatocellular cholestatic damage induced by phalloidin and mice with obstructive cholestasis caused by bile duct ligation (BDL), as well as with spontaneous development of sclerosing cholangitis (Mdr2^-/-). NIRBADs were taken up by cells expressing NTCP or OATP1B3, but minimally by OATP1B1. These findings were consistent with the NIRBAD dynamics in the liver parenchyma and in silico studies. Following intravenous administration of a non-toxic dose, the time course of NIR fluorescence in the rat liver aligned with biliary output. In mice with BDL, hepatic NIR fluorescence remained stable throughout the experimental period. Phalloidin administration impaired rat bile flow, induced a decrease in biliary NIRBAD-1 output, and caused an increase in NCT. Furthermore, the NCT was significantly longer in Mdr2^-/- than in wild-type mice. In conclusion, a novel, noninvasive, real-time test based on cholephilic probes with NIR fluorescence detectable extracorporeally serves as a valuable tool for assessing hepatobiliary secretory function.