Tacrine sinusoidal uptake and biliary excretion in sandwich-cultured primary rat hepatocytes

Abstract

PURPOSE. The knowledge of hepatic disposition kinetics of tacrine, a first cholinesterase inhibitor was approved by FDA for the treatment of Alzheimer's disease (AD), would help to understand its hepatotoxicity, its therapeutic effect, and improve the management of patients with AD. The current study aims to characterize tacrine hepatic transport kinetics and study the role of organic cation transporters (OCTs), P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP2) in tacrine sinusoidal uptake and biliary excretion. METHODS. Modulation of tacrine hepatic uptake and efflux, biliary excretion index (BEI%), were performed in sandwich-cultured primary rat hepatocytes (SCHs) using transporters inhibitors. Conformation of the integrity of SCHs model was established by capturing images with light-contrast and fluorescence microscopy. RESULTS. Tacrine uptake in SCHs was carrier-mediated process and saturable with apparent Km of 31.5±9.6 µM and Vmax of 908±72 pmol/min/mg protein. Tetraethyl ammonium (TEA), cimetidine and verapamil significantly reduced tacrine uptake with more pronounced effect observed with verapamil which caused 3-fold reduction in tacrine uptake, indicating role for OCTs. Tacrine has a biliary excretion in SCHs with maximum BEI% value of 22.9±1.9% at 10 min of incubation. Addition of MK571 and valspodar decreased the BEI% of tacrine by 40 and 60% suggesting roles for canalicular MRP2 and P-gp, respectively. CONCLUSIONS. Our results show that in addition to metabolism, tacrine hepatic disposition is carrier-mediated process mediated by sinusoidal OCTs, and canalicular MRP2 and P-gp.

Figure 1.

Phase-contrast and fluorescence images of SCHs cultured for 1 (a) or 4 (b & c) days. No canalicular spaces were formed on day 1 of SCHs (a), but they were formed extensively by day 4 as indicated by the yellow arrows as bright white lines (b). Canalicular spaces are shown as intense green fluorescent color (c) due to accumulation of the canalicular marker CDF. Scale bars = 10 μm.

Figure 2.

Time and concentration dependant uptake of tacrine. (a) Cellular uptake of 1 μM tacrine in SCHs at day 1. Points represent cumulative uptake for n = 4 at different treatment times, * P<0.05 compared to 2 and 5 min, based on unpaired Student’s t test. (b) Uptake rate of tacrine at different concentrations in SCHs on day 1 at 4 and 37°C showing non-linear pattern.

Figure 3.

Effect of co-incubation of different OCTs inhibitors on tacrine uptake. (a) Effect of cimetidine, verapamil, TEA, and fluovoxamine on the uptake rate of tacrine in SCHs at day 1. (b) Concentration-dependent inhibition of verapamil on the uptake rate of tacrine in SCHs at day 1. Points represent mean±SEM for triplicates from two independent experiments. * P<0.05 and *** P<0.01 compared to untreated control.

Figure 4.

Effect of fluvoxamine on tacrine uptake in SCHs at day 1. (a) Concentration-dependent inhibition of fluvoxamine on the cumulative uptake of tacrine. Hepatocytes were pre-incubated with fluvoxamine for 30 min followed by uptake of 1μM tacrine for 5 min. * P<0.05, *** P<0.01 compared to untreated control. (b) Pre-incubation of SCHs with increasing concentration of fluvoxamine for 30 min followed by uptake of 1μM tacrine for 2 h. *** P<0.01 compared to untreated control.

Figure 5.

Representative time course of tacrine biliary excretion index (BEI%) in SCHs at day 4. BEI% value of 1μM tacrine was calculated at each time point. Bars represent the mean ± SEM (n = 3), * P<0.05 compared to 5 min, based on unpaired Student’s t test.

Figure 6.

Effect of P-gp (valspodar) and MRP2 (MK571) inhibition on the cumulative uptake and BEI% of 1μM tacrine. Cumulative uptake of tacrine was determined after 10 min incubation in SCHs at day 4. Bars represent the mean ± SEM (n = 4), ns = not significant, ** P<0.01 compared to corresponding control.