Hepatic Disposition of Gemfibrozil and Its Major Metabolite Gemfibrozil 1-O-ß-Glucuronide.

Gemfibrozil (GEM), which decreases serum triglycerides and low density lipoprotein, perpetrates drug-drug interactions (DDIs) with several drugs. These DDIs are primarily attributed to the inhibition of drug transporters and metabolic enzymes, particularly cytochrome P450 (CYP) 2C8 by the major circulating metabolite gemfibrozil 1-O-ß-glucuronide (GG). Here, we characterized the transporter-mediated hepatic disposition of GEM and GG using sandwich-cultured human hepatocytes (SCHH) and transporter-transfect systems. Significant active uptake was noted in SCHH for the metabolite. GG, but not GEM, showed substrate affinity to organic anion transporting polypeptide (OATP) 1B1, 1B3, and 2B1. In SCHH, glucuronidation was characterized affinity constants (Km) of 7.9 and 61.4 µM, and biliary excretion of GG was observed. Furthermore, GG showed active basolateral efflux from preloaded SCHH and ATP-dependent uptake into membrane vesicles overexpressing multidrug resistance-associated protein (MRP) 2, MRP3, and MRP4. A mathematical model was developed to estimate hepatic uptake and efflux kinetics of GEM and GG based on SCHH studies. Collectively, the hepatic transporters play a key role in the disposition and thus determine the local concentrations of GEM and more so for GG, which is the predominant inhibitory species against CYP2C8 and OATP1B1.

Species differences in sinusoidal and canalicular efflux transport of mycophenolic acid 7-O-glucuronide in sandwich-cultured hepatocytes.

Metabolism and sinusoidal/canalicular efflux of mycophenolic acid (MPA) was investigated using sandwich-cultured hepatocytes (SCHs). After applying MPA to SCHs from humans, wild-type rats, and multidrug resistance-associated protein (Mrp) 2-deficient rats, the MPA metabolites 7-O-glucuronide (MPAG) and acyl glucuronide (AcMPAG) were detected in the intracellular compartment of the SCHs. Sinusoidal efflux of MPAG was detected in all SCH preparations including Mrp2-deficient rat SCHs, whereas canalicular efflux of MPAG was observed in wild-type rat and human SCHs but not in Mrp2-deficient rat SCHs. The ratio of canalicular efflux to net (canalicular plus sinusoidal) efflux was 37 ± 8% in wild-type rat SCHs, while the ratio in human SCHs was significantly lower (20 ± 2%, P < 0.05), indicating species differences in the direction of hepatic MPAG transport. This 20% ratio in human SCHs corresponds to a high sinusoidal MPAG efflux (80%) that can in part account for the urine-dominated recovery of MPAG in humans. Both sinusoidal and canalicular MPAG efflux in rat SCHs shows a good correspondence to urinary and biliary recovery of MPAG after MPA dosing. The sinusoidal efflux of AcMPAG in human SCHs was detected from one out of three donors, suggesting donor-to-donor variation. In conclusion, this study demonstrates the predictive value of SCHs for elucidating the interplay of metabolism and efflux transport, in addition to demonstrating a species difference between rat and human in sinusoidal and canalicular efflux of MPAG.

Quantification of biliary excretion and sinusoidal excretion of 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) in cultured hepatocytes isolated from Sprague Dawley, Wistar and Mrp2-deficient Wistar (TR(-)) rats.

Hepatic efflux of drug candidates is an important issue in pre-clinical drug development. Here we utilise a method which quantifies and distinguishes efflux of drugs at the canalicular and sinusoidal membranes in rat hepatocyte cultures. Bi-phasic kinetics of transport of 5(6)-carboxydichlorofluorescein (CDF) at the canalicular membrane was demonstrated in Sprague Dawley (SD) and Wistar (W) rat hepatocytes. The high affinity component (Km=3.2±0.8µM (SD), 9.0±3.1µM (W)) was attributed to Mrp2-mediated transport, the low affinity component (Km=192.1±291.5µM (SD), 69.2±36.2µM (W)) may be attributed to transport involving a separate Mrp2 binding site. Data from membranes (Hill coefficient (h)=2.0±0.5) and vesicles (h=1.6±0.2) expressing Mrp2 and from SD (h=1.6±0.4) and Wistar (h=4.0±0.6) hepatocytes suggests transport involves more than one binding site. In TR(-) hepatocytes, CDF efflux was predominantly over the sinusoidal membrane (Km=100.7±36.0µM), consistent with low abcc2 (Mrp2) expression and compensatory increase in abcc3 (Mrp3) expression. This report shows the potential of using this in vitro method to model changes in biliary excretion due to alterations in transporter expression.