Short Communication DISPOSITION OF TACROLIMUS IN ISOLATED PERFUSED RAT LIVER: INFLUENCE OF TROLEANDOMYCIN, CYCLOSPORINE, AND GG918

The disposition of tacrolimus and the influence of cyclosporine, troleandomycin, and GF120918 (GG918, or N-[4-[2-(1,2,3,4tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl]-9,10dihydro-5-methoxy-9-oxo-4-acridine carboxamine) on its hepatic disposition were examined in the isolated perfused rat liver. Livers from groups of rats were perfused in a recirculatory manner following a bolus dose of tacrolimus (100 g), a substrate for Pglycoprotein (P-gp) and CYP3A, or with felodipine (200 g), a substrate only for CYP3A. Perfusions of each substrate were also examined in groups of rats in the presence of the inhibitors: troleandomycin (20 M, CYP3A inhibitor), GG918 (1 M, P-gp inhibitor), or cyclosporine (10 M, CYP3A and P-gp inhibitor). In all experiments, perfusate and bile were collected for 60 min. Tacrolimus, felodipine, and their primary metabolites were determined in perfusate and bile by liquid chromatography/tandem mass spectrometry. The area under the curve (AUC) from 0 to 30 min was determined. For the dual CYP3A and P-gp substrate, tacrolimus, AUC S.D. was decreased from control (2,260 430 ng min/ml) by GG918 (1,730 270 ng min/ml, P < 0.05) and was increased by troleandomycin (5,200 2,470 ng min/ml, P < 0.05) and cyclosporine (4,390 2,080 ng min/ml, P < 0.05). For the exclusive CYP3A substrate, felodipine, AUC was unchanged from control by GG918 but increased by troleandomycin and cyclosporine. It is concluded that GG918 increased the hepatic exposure of tacrolimus by inhibiting the canalicular P-gp transport, whereas GG918 has no effect on hepatic disposition of felodipine. These results support our hypothesis that the hepatic metabolic clearance of a dual substrate will be increased by inhibiting the efflux transporter. The interplay between P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) is a major factor contributing to the variable bioavailability of many therapeutically available drugs (Wacher et al., 1995; Zhang and Benet, 2001). The considerable overlap in substrate selectivity makes it difficult to differentiate the interactive roles of P-gp and CYP3A in xenobiotic disposition. In enterocytes, P-gp is located on the apical plasma membrane and CYP3A4 is on the endoplasmic reticulum inside the cells. Absorbed drug molecules may be actively extruded back into the intestinal lumen by P-gp. As we have previously reported (Benet and Cummins, 2001; Cummins et al., 2002a, 2003), through the repeated process of diffusion and active efflux, drug molecules may come in contact with CYP3A intracellularly, increasing the potential for metabolism by the enzyme. Prolonging the intracellular residence time of the parent drug decreases the rate of absorption and results in increased drug clearance by CYP3A4 relative to the parent drug traversing the intestine. We believe that P-gp, in fact, controls the access of drugs to intracellular metabolism by CYP3A4 in the intestine (Benet and Cummins, 2001), which is well supported by bidirectional transport studies using CYP3A-transfected Caco-2 cells (Cummins et al., 2002a) and mdr1 gene knockout mice (Lan et al., 2000). Our recent in vivo perfused rat intestinal studies further confirm our hypothesis (Cummins et al., 2003). In the liver, the absorbed compounds enter the hepatocytes from the sinusoidal blood, then the drugs are either biotransformed, transported back into the blood, or eliminated via biliary secretion. Since P-gp is located on the canalicular membrane of hepatocytes, drug molecules confront CYP3A before P-gp efflux. Hence, P-gp influences the access of drug molecules to CYP3A in an opposite manner to the enterocyte, where the transporter precedes the enzyme. We hypothesize that if P-gp is decreased by chemical inhibitors or via gene disruption (mdr1 knockout), the hepatic metabolic clearance of the substrates will be enhanced due to their increased availability to CYP3A. This phenomenon was implied in gene disruption studies by Lan et al. (2000) and was well illustrated for basolateral doses in our recent cellular transport studies (Cummins et al., 2002a). We further calculated that the hepatic extraction ratios of vinblastine, paclitaxel, and doxorubicin were increased in mdr1 knockout mice versus wildtype controls (Cummins et al., 2002b). The goal of the studies described here was to directly test our hypothesis of the effect of P-gp inhibition on hepatic metabolism by identifying the contribution of P-gp to CYP3A4 metabolism using an ex situ model (isolated perfused rat liver) with appropriately selected CYP3A4/P-gp substrates and inhibitors. Two compounds were tested: one a dual P-gp and CYP3A4 substrate (tacrolimus) and the other an exclusive CYP3A4 substrate (felodipine). Tacrolimus was metabolized by CYP3A4 (Lampen et al., 1995) and is a good substrate for P-gp. Felodipine is metabolized by Supported in part by National Institutes of Health Grants GM61390 and HD40543. 1 Abbreviations used are: P-gp, P-glycoprotein; GF120918 (GG918), N-[4-[2(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl]-9,10-dihydro5-methoxy-9-oxo-4-acridine carboxamine; AUC, area under the curve; MS, mass