Dioscin protects against ANIT-induced cholestasis via regulating Oatps, Mrp2 and Bsep expression in rats.

Alpha-naphthylisothiocyanate (ANIT) is a toxicant that is widely used in rodents to model human intrahepatic cholestasis. The aim of the study is to investigate whether effects of dioscin on ANIT-induced cholestasis are related to changes in expression of hepatic transporters in rats. Effects of dioscin on cholestasis were examined by histology and biochemical marker levels. The functional changes of hepatic transporters were determined by in vitro, in situ and in vivo. qRT-PCR and western blot were used to assess the expression of hepatic transporters in cholestatic rats. Dioscin administration could ameliorate cholestasis, as evidenced by reduced biochemical markers as well as improved liver pathology. The uptakes of organic anion transporting polypeptide (Oatp) substrates were altered in liver uptake index in vivo, perfused rat liver in situ and isolated rat hepatocytes in vitro in cholestasis rats. qRT-PCR and western blot analysis indicated co-treatment of ANIT with dioscin prevented the adaptive down-regulation of Oatp1a1, 1b2, and prompted the up-regulation of Oatp1a4, multidrug resistance-associated protein (Mrp) 2 and bile salt export pump (Bsep). In addition, concerted effects on Mrp2 and Bsep occurred through up-regulation of small heterodimer partner by activating farnesoid X receptor. Dioscin might prevent impairment of hepatic function by restoring hepatic transporter expression.

Decreased liver distribution of entecavir is related to down-regulation of Oat2/Oct1 and up-regulation of Mrp1/2/3/5 in rat liver fibrosis.

AIMS: We aimed to elucidate whether entecavir was taken-up into liver by transporters and clarify the possible molecular mechanisms of changes in the distribution of entecavir in rat liver fibrosis. METHODS: Thioacetamide (TAA) was applied to induce rat liver fibrosis. Samples of liver uptake index (LUI) study and uptake of entecavir in isolated rat hepatocytes were determined by LC-MS/MS. qRT-PCR and western blotting were used to examine the expression of transporters in rat liver. RESULTS: The uptake of entecavir in hepatocytes was significantly higher at 37 °C compared to 4 °C. Furthermore, TEA and PAH could inhibit significantly the uptake of entecavir by the hepatocytes. It indicated that Oat2 and Oct1 were contributed to uptake of entecavir. Compared with control group, LUI and the uptake of entecavir, PAH and TEA in hepatocytes were significantly reduced in liver fibrosis group. Further study indicated that entecavir Vmax in liver fibrosis group was significantly decreased while the Km was not changed. These results indicated that transport capacity TAA treated isolated rat liver hepatocytes were reduced. Oat2 and Oct1 expressions were down-regulated and Mrp1/2/3/5 mRNA expressions were up-regulated in liver fibrosis group. CONCLUSIONS: The changes of these transporters were contributed to decrease liver distribution of entecavir.

The oligopeptide transporter 2-mediated reabsorption of entecavir in rat kidney.

We investigated whether entecavir is a substrate of the oligopeptide transporter 2 (PEPT2) and whether reabsorption of entecavir is mediated by PEPT2 as well as what is the contribution of PEPT2 to entecavir reabsorption during urinary excretion. Entecavir uptake in transfected cells and rat kidney slices, changes in urine entecavir concentrations following isolated kidney perfusion and in vivo entecavir plasma and urine concentrations were determined with LC-MS/MS. In hPEPT2-HELA cells, entecavir uptake was significantly higher compared to vector-HELA cells and was sharply inhibited by Gly-sar and JBP485, and there were two distinct transport systems. The Km and Vmax of entecavir were 427 (µM) and 1.60 (nmol/mg protein/30s) (low-affinity, high-velocity system) and 24.0 (µM) and 0.296 (nmol/mg protein/30s) (high-affinity, low-velocity system). In rat kidney slices, uptake of entecavir was not markedly inhibited by Gly-sar. In isolated kidney perfusion experiments, entecavir cumulative urinary excretion was statistically significant at 45 and 60 min. CLR(4 °C), CLR(37 °C Control) and CLR(37°C Experiment) were 12.6, 27.6 and 36 (ml/min/kg), respectively. CLTS and TR rate (for PEPT2) were 25.3 and 9.4 (ml/min/kg). In vivo, the cumulative urinary excretion of entecavir had statistical significance at 3 and 4h with CLR(Control) and CLR(Experiment) values of 31 and 42 (ml/min/kg), respectively. The CLTS and TR rate (for PEPT2) were 32 and 11.6 (ml/min/kg), respectively. The present study demonstrated that entecavir is a substrate of PEPT2. Moreover, reabsorption of entecavir is mediated by PEPT2, and 25% of urinary entecavir is reabsorbed by PEPT2.

OAT1 and OAT3: targets of drug-drug interaction between entecavir and JBP485.

Entecavir and JBP485 (a dipeptide) exhibit the antihepatitis activities and it is possible for the two drugs to be coadministered in the treatment of hepatitis. We aimed to elucidate whether entecavir was a substrate of OAT1, OAT3, OCT, and PEPT1 and to investigate the targets of drug-drug interactions between entecavir and JBP485. Plasma and urine concentrations of entecavir following intravenous and oral administration in vivo, uptake of entecavir in kidney slices and transfected cells in vitro, were determined by LC-MS/MS. Following intravenous co-administration of entecavir and JBP485 in rats, entecavir AUC increased 1.93-fold, t1/2ß was prolonged 2.08-fold, CLP decreased 49%, CLR decreased 73%, and accumulated urinary excretion decreased 54%. However, following oral co-administration, the entecavir Tmax and Cmax were not affected; the degree of change in other pharmacokinetic parameters (AUC, t1/2ß, CLP, and accumulated urinary excretion) was similar to that of intravenous administration. The uptake of entecavir was nearly identical in hPEPT1- as in vector-HELA cells. In rat kidney slices, uptake of entecavir was markedly inhibited by p-aminohippurate, benzylpenicillin, JBP485, and tetraethyl ammonium. In hOAT1- and hOAT3-HEK293 cells, uptake of entecavir was significantly higher compared to vector-HEK293 cells and was markedly inhibited by p-aminohippurate, benzylpenicillin, and JBP485. Km and Vmax values of entecavir were 250 µM and 0.83 nmol/mg protein/30s (OAT1) and 23 µM and 1.1 nmol/mg protein/30 s (OAT3), respectively. Entecavir is the substrate of OAT1, OAT3, and OCT. Moreover, OAT1 and OAT3 are the targets of DDI between entecavir and JBP485.