Physiologically-Based Pharmacokinetic and Pharmacodynamic Modeling for the Inhibition of Acetylcholinesterase by Acotiamide, A Novel Gastroprokinetic Agent for the Treatment of Functional Dyspepsia, in Rat Stomach.

PURPOSE: Acotiamide, a gastroprokinetic agent used to treat functional dyspepsia, is transported to at least two compartments in rat stomach. However, the role of these stomach compartments in pharmacokinetics and pharmacodynamics of acotiamide remains unclear. Thus, the purpose of this study was to elucidate the relationship of the blood and stomach concentration of acotiamide with its inhibitory effect on acetylcholinesterase (AChE). METHODS: Concentration profiles of acotiamide and acetylcholine (ACh) were determined after intravenous administration to rats and analyzed by physiologically-based pharmacokinetic and pharmacodynamic (PBPK/PD) model containing vascular space, precursor pool and deep pool of stomach. RESULTS: Acotiamide was eliminated from the blood and stomach in a biexponential manner. Our PBPK/PD model estimated that acotiamide concentration in the precursor pool exceeded 2 µM at approximately 2 h after administration. Acotiamide inhibited AChE activity in vitro with a 50% inhibitory concentration of 1.79 µM. ACh reached the maximum concentration at 2 h after administration. CONCLUSIONS: Our PBPK model well described the profile of acotiamide and ACh concentration in the stomach in the assumption that acotiamide was distributed by carrier mediated process and inhibited AChE in the precursor pool of stomach. Thus, Acotiamide in the precursor pool plays an important role for producing the pharmacological action.

ENT1, a ribavirin transporter, plays a pivotal role in antiviral efficacy of ribavirin in a hepatitis C virus replication cell system.

We previously showed that equilibrative nucleoside transporter 1 (ENT1) is a primary ribavirin transporter in human hepatocytes. However, because the role of this transporter in the antiviral mechanism of the drug remains unclear, the present study aimed to elucidate the role of ENT1 in ribavirin antiviral action. OR6 cells, a hepatitis C virus (HCV) replication system, were used to evaluate both ribavirin uptake and efficacy. The ribavirin transporter in OR6 cells was identified by mRNA expression analyses and transport assays. Nitrobenzylmercaptopurine riboside (NBMPR) and micro-RNA targeted to ENT1 mRNA (miR-ENT1) were used to reduce the ribavirin uptake level in OR6 cells. Our results showed that ribavirin antiviral activity was associated with its accumulation in OR6 cells, which was also closely associated with the uptake of the drug. It was found that the primary ribavirin transporter in OR6 cells was ENT1 and that inhibition of ENT1-mediated ribavirin uptake by NBMPR significantly attenuated the antiviral activity of the drug as well as its accumulation in OR6 cells. The results also showed that even a small reduction in the ENT1-mediated ribavirin uptake, achieved in this case using miR-ENT1, caused a significant decrease in its antiviral activity, thus indicating that the ENT1-mediated ribavirin uptake level determined its antiviral activity level in OR6 cells. In conclusion, our results show that by facilitating its uptake and accumulation in OR6 cells, ENT1 plays a pivotal role in the antiviral effectiveness of ribavirin and therefore provides an important insight into the efficacy of the drug in anti-HCV therapy.

Characterization of ribavirin uptake systems in human hepatocytes.

BACKGROUND & AIMS: The purpose of this study was to identify the major ribavirin uptake transporter(s) in human hepatocytes and to determine if these previously unidentified transporters are involved in hepatic ribavirin uptake. Furthermore, we aimed to address what causes the difference in uptake levels among human hepatocytes. METHODS: Profiles of ribavirin uptake and nucleoside transporter mRNA expression in Caucasian hepatocytes (HH268, HH283 and HH291) were characterized by transport assay and reverse transcription-polymerase chain reaction (RT-PCR). The 5'-side of the SLC29A1 gene structure was characterized by determination of transcription start sites and by RT-PCR. RESULTS: Equilibrative nucleoside transporter 1 (ENT1)-mediated uptake was exclusively involved in ribavirin uptake in HH268 and HH283 and was responsible for the largest ribavirin uptake fraction in HH291. The level of ENT1-mediated uptake in HH291 was higher than that in HH268 and HH283. Characterization of the SLC29A1 gene structure revealed the existence of several ENT1 mRNA isoforms in the human liver, and the levels of four ENT1 mRNA isoforms in HH291 were higher than those in HH268 or HH283. No ENT2-mediated uptake was observed in any hepatocyte lines. Na(+)-dependent uptake was detected only in HH291; however, mRNA levels of concentrative nucleoside transporters (CNTs) were at trace levels in all hepatocyte lines. CONCLUSIONS: ENT1, but not ENT2 or CNTs, is a major ribavirin uptake transporter in human hepatocytes. The different ENT1-mediated ribavirin uptake levels in different hepatocyte lines are associated with different expression levels of specific isoforms of ENT1 mRNAs. Furthermore, an unidentified Na(+)-dependent ribavirin transport system might exist in human hepatocytes.

Striking species difference in the contribution of concentrative nucleoside transporter 2 to nucleoside uptake between mouse and rat hepatocytes.

Concentrative nucleoside transporter 2 (CNT2) (encoded by the SLC28A2 gene) transports various antiviral or antitumor purine nucleoside analogs to be involved in their pharmacokinetics and pharmacological actions. The results of our study showed that mouse hepatocytes hardly expressed CNT2 mRNA and no CNT2-dependent nucleoside uptake was observed, while rat hepatocytes exhibited high CNT2-dependent nucleoside uptake activity levels with abundant CNT2 mRNA expression. We concluded that CNT2 contributes considerably to nucleoside uptake in rat hepatocytes but not in mouse hepatocytes.

Functional analysis of purine nucleoside phosphorylase as a key enzyme in ribavirin metabolism.

Ribavirin is a purine nucleoside analogue that possesses potent anti-hepatitis C virus activity, and it has long been considered likely that ribavirin undergoes a first-pass metabolism at the small intestine. Although purine nucleoside phosphorylase (PNP) is assumed to be involved in this metabolism, this has not been conclusively demonstrated. Furthermore, no pharmacogenomic studies related to PNP-mediated ribavirin phosphorolysis have previously been conducted. In this study, we sought to identify the role of PNP in ribavirin phosphorolysis in the human small intestine, and to clarify the effect of the single nucleotide polymorphism (rs1049564) on PNP's ribavirin phosphorolysis activity. The results of our investigations show that PNP is abundantly expressed in the human small intestine, and that intestinal ribavirin phosphorolysis is severely inhibited by ganciclovir, a PNP-inhibitor. Therefore, PNP is likely to play a primary role in the ribavirin phosphorolysis in the human small intestine. On the other hand, the results of our attempt to clarify the function of rs1049564 show that it does not affect PNP's ribavirin phosphorolysis activity. We believe that the present study will facilitate further pharmacogenomic and biochemical characterization of PNP as a key metabolic enzyme of ribavirin.