Pharmacokinetics of 5 (and 6)-Carboxy-2′,7′-Dichlorofluorescein and Its Diacetate Promoiety in the Liver

Abstract

Hepatic disposition of 5 (and 6)-carboxy-2Ј,7Ј-dichlorofluorescein (CDF) and its diacetate promoiety (CDFDA) was studied in isolated perfused rat livers. Livers from Wistar wild-type and multidrug resistance-associated protein (Mrp)2-deficient (TR Ϫ ) rats were perfused with CDF in the presence or absence of probenecid. Probenecid decreased the recovery of CDF in bile ϳ4-fold in wild-type livers (65 Ϯ 8% versus 15 Ϯ 2% of dose over 2 h). In livers from TR Ϫ rats, CDF was not excreted into bile and probenecid decreased perfusate CDF concentrations in a concentration-dependent manner, in part due to inhibition of Mrp3. Plasma membrane vesicles from rat Mrp2-or Mrp3-transfected Sf9 cells were used to confirm that CDF is a substrate for Mrp2 and Mrp3; probenecid inhibited the transport of CDF by Mrp2 and Mrp3 in a concentration-dependent manner.CDF uptake in collagen sandwich-cultured rat hepatocytes was temperature-dependent and saturable (K m ϭ 22 Ϯ 10 M; V max ϭ 97 Ϯ 9 pmol/min/mg protein). Uptake of CDF in sandwich-cultured rat hepatocytes was impaired significantly by bromosulfophthalein, a substrate for organic anion-transporting polypeptides (Oatps), but was not modulated by specific Oatp2 or organic anion transporter (Oat) substrates. CDFDA uptake was not saturable, temperature-dependent, or impaired by inhibitors. The hydrolysis of CDFDA to CDF is mediated by basic pH and esterases in biological media. CDFDA passively diffuses into hepatocytes where it is hydrolyzed to CDF. In contrast, CDF appears to be taken up by Oatp-mediated transport into rat hepatocytes and effluxed via Mrp2 into bile and via Mrp3 into sinusoidal blood.

Quantitative intravital microscopy of hepatic transport

“…In this regard, the studies of Zamek-Gliszczynski and colleagues 57 may be informative. These studies found that 6-CFDA is rapidly hydrolyzed to 6-CF when incubated with 20% rat blood.…”

Intravital multiphoton microscopy can model uptake and excretion of fluorescein in hepatic ischemia-reperfusion injury

“…25 However, other members of the fluorescein family, such as 5(6)-carboxy-2'7'-dichlorofluorescein and 5(6)-carboxyfluorescein, are secreted into the blood by MRP3; thus, it is likely that fluorescein and fluorescein monoglucuronide also interact with this transporter. 29,30 The aim of this study was to investigate the transport of fluorescein in the liver following I/R injury by intravital multiphoton tomography, correlating these results with changes in biliary excretion, plasma concentrations, and expression of the transporters.…”

Differential regulation of drug transporter expression by all-trans retinoic acid in hepatoma HepaRG cells and human hepatocytes

“…As shown in Fig. 3B (Meier and Stieger, 2002;Zamek-Gliszczynski et al, 2003), at the canalicular pole of hepatocytes, appear therefore to be most likely functional in HepaRG cells. Interestingly, BEI value for CF in HepaRG cells (34.4 %) was nearly identical to CF BEI value reported in sandwich-cultured human hepatocytes (BEI=34%) (Hoffmaster et al, 2004), which suggests that MRP2 is similarly active in these primary human hepatocytes and in HepaRG cells.…”