In vitro biliary clearance of angiotensin II receptor blockers and 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors in sandwich-cultured rat hepatocytes: comparison with in vivo biliary clearance

Abstract

Previous reports have indicated that in vitro biliary clearance (Cl(biliary)) determined in sandwich-cultured hepatocytes correlates well with in vivo Cl(biliary) for limited sets of compounds. This study was designed to estimate the in vitro Cl(biliary) in sandwich-cultured rat hepatocytes (SCRHs) of angiotensin II receptor blockers and HMG-CoA reductase inhibitors that undergo limited metabolism, to compare the estimated Cl(biliary) values with published in vivo Cl(biliary) data in rats, and to characterize the mechanism(s) of basolateral uptake and canalicular excretion of these drugs in rats. The average biliary excretion index (BEI) and in vitro Cl(biliary) values of olmesartan, valsartan, pravastatin, rosuvastatin, and pitavastatin were 15, 19, 43, 45, and 20%, respectively, and 1.7, 3.2, 4.4, 46.1, and 34.6 ml/min/kg, respectively. Cl(biliary) predicted from SCRHs, accounting for plasma unbound fraction, correlated with reported in vivo Cl(biliary) for these drugs. The rank order of Cl(biliary) values predicted from SCRHs was consistent with in vivo Cl(biliary) values. Bromosulfophthalein inhibited the uptake of all drugs. BEI and Cl(biliary) values of olmesartan, valsartan, pravastatin, and rosuvastatin, known multidrug resistance-associated protein (Mrp) 2 substrates, were reduced in SCRHs from Mrp2-deficient (TR(-)) compared with wild-type (WT) rats. Although Mrp2 plays a minor role in pitavastatin biliary excretion, pitavastatin BEI and Cl(biliary) were reduced in TR(-) compared with WT SCRHs; Bcrp expression in SCRHs from TR(-) rats was decreased. In conclusion, in vitro Cl(biliary) determined in SCRHs can be used to estimate and compare in vivo Cl(biliary) of compounds in rats and to characterize transport proteins responsible for their hepatic uptake and excretion.

Figure 1

Chemical structures of ARBs (olmesartan, valsartan) and statins (pravastatin, rosuvastatin, pitavastatin), and information regarding biliary excretion in rats. ^aEisai hyperbilirubinemic rats, ^bwild-type, ^cNakagomi-Hagihara et al., 2006, ^dYamashiro et al., 2006, ^eYamazaki et al., 1997, ^fKitamura et al., 2005, ^gHirano et al., 2005.

Figure 2

Substrate concentration dependency of accumulation (cells + bile) for olmesartan (●), valsartan (■), pravastatin (▲), rosuvastatin (◆) and pitavastatin (×) in sandwich-cultured rat hepatocytes. Incubation time: 10 min. Data represent mean ± SD of three replicates from one liver. Inset: plot expanded scale of Y-axis

Figure 3

Accumulation [cells + bile (solid bars) and cells (white bars)] and biliary excretion index of olmesartan (A), valsartan (B), pravastatin (C), rosuvastatin (D) and pitavastatin (E) in sandwich-cultured rat hepatocytes. Dosing concentration: 5 μM. Data represent mean + SEM (n = 3 livers)

Figure 4

In vitro - in vivo correlation of Cl_biliary of ARBs and statins in rats. Cl_biliary predicted from in vitro data (mean ± SEM, n = 3 livers) was calculated from data generated in sandwich-cultured rat hepatocytes based on Equation (4) (A) or based on Equation (3) (B). When not visible, error bars are within the size of the symbol.

Figure 5

Effect of BSP on accumulation (cell + bile) of ARBs and statins in day 4 sandwich-cultured rat hepatocytes. Open, solid and hatched bars represent 10-min incubation with vehicle control, 30 μM BSP, or 100 μM BSP, respectively. Dosing concentration: 5 μM. Data represent mean + SEM (n = 3 livers).

Figure 6

Comparison of Mrp2 and Bcrp expression in WT and TR⁻ rat hepatocytes. Cell lysates from day 0 freshly isolated hepatocytes and day 4 sandwich-cultured rat hepatocytes were blotted for Mrp2 and Bcrp. Representative results of 2 experiments are shown (30 μg protein/well). After striping, the same blot was probed with β-actin antibody to serve as a loading control.