Unique Hydrolysis of an Ester-Type Prodrug of Levodopa in Human Plasma: Relay-Type Role Sharing between Alpha-1 Acid Glycoprotein and Human Serum Albumin.

ONO-2160 is a newly developed oral ester-type prodrug of levodopa for removing the problems in use of levodopa. It has a structure in which two of the same substituents are bound to levodopa. It is important to understand the pharmacokinetics and metabolic pathway for new drug candidate compounds. The aim of this study was to identify the major enzymes that contribute to the metabolism of ONO-2160 in human plasma. ONO-2160 was hydrolyzed by human serum albumin (HSA) and α1-acid glycoprotein (AGP) in human plasma, although the hydrolysis was not inhibited by various reported esterase inhibitors. The value of the intrinsic clearance per milliliter of plasma of ONO-2160 in AGP solution was greater than that in HSA solution and was comparable to that in human plasma. Therefore, AGP is responsible for the hydrolysis of ONO-2160 in human plasma. ONO-M, which is an intermediate metabolite of ONO-2160, has a structure in which one substituent is removed from ONO-2160 and was mainly generated in AGP solution, but not in human plasma or HSA solution. The hydrolysis of ONO-M by HSA was much greater than by AGP. These results indicate that ONO-M, which is mainly generated from ONO-2160 by AGP, is rapidly hydrolyzed by HSA, and that ONO-2160 generates levodopa via ONO-M in a relay-type reaction through AGP and HSA in human plasma. It has not been reported that AGP has esterase-like activity. These findings could be useful information for drug development of the ester-type prodrug.

Sandwich-Cultured Hepatocytes for Mechanistic Understanding of Hepatic Disposition of Parent Drugs and Metabolites by Transporter-Enzyme Interplay.

Functional interplay between transporters and drug-metabolizing enzymes is currently one of the hottest topics in the field of drug metabolism and pharmacokinetics. Uptake transporter-enzyme interplay is important to determine intrinsic hepatic clearance based on the extended clearance concept. Enzyme and efflux transporter interplay, which includes both sinusoidal (basolateral) and canalicular efflux transporters, determines the fate of metabolites formed in the liver. As sandwich-cultured hepatocytes (SCHs) maintain metabolic activities and form a canalicular network, the whole interplay between uptake and efflux transporters and drug-metabolizing enzymes can be investigated simultaneously. In this article, we review the utility and applicability of SCHs for mechanistic understanding of hepatic disposition of both parent drugs and metabolites. In addition, the utility of SCHs for mimicking species-specific disposition of parent drugs and metabolites in vivo is described. We also review application of SCHs for clinically relevant prediction of drug-drug interactions caused by drugs and metabolites. The usefulness of mathematical modeling of hepatic disposition of parent drugs and metabolites in SCHs is described to allow a quantitative understanding of an event in vitro and to develop a more advanced model to predict in vivo disposition.

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.

Analysis of Nonlinear Pharmacokinetics of a Highly Albumin-Bound Compound: Contribution of Albumin-Mediated Hepatic Uptake Mechanism.

The cause of nonlinear pharmacokinetics (PK) (more than dose-proportional increase in exposure) of a urea derivative under development (compound A: anionic compound [pKa: 4.4]; LogP: 6.5; and plasma protein binding: 99.95%) observed in a clinical trial was investigated. Compound A was metabolized by CYP3A4, UGT1A1, and UGT1A3 with unbound Km of 3.3-17.8 µmol/L. OATP1B3-mediated uptake of compound A determined in the presence of human serum albumin (HSA) showed that unbound Km and Vmax decreased with increased HSA concentration. A greater decrease in unbound Km than in Vmax resulted in increased uptake clearance (Vmax/unbound Km) with increased HSA concentration, the so-called albumin-mediated uptake. At 2% HSA concentration, unbound Km was 0.00657 µmol/L. A physiologically based PK model assuming saturable hepatic uptake nearly replicated clinical PK of compound A. Unbound Km for hepatic uptake estimated from the model was 0.000767 µmol/L, lower than the in vitro unbound Km at 2% HSA concentration, whereas decreased Km with increased concentration of HSA in vitro indicated lower Km at physiological HSA concentration (4%-5%). In addition, unbound Km values for metabolizing enzymes were much higher than unbound Km for OATP1B3, indicating that the nonlinear PK of compound A is primarily attributed to saturated OATP1B3-mediated hepatic uptake of compound A.

Peroxisome proliferator-activated receptor alpha (PPARalpha) agonists induce constitutive androstane receptor (CAR) and cytochrome P450 2B in rat primary hepatocytes.

The constitutive androstane receptor (CAR; NR1I3) is a key transcriptional factor that regulates genes encoding drug-metabolizing enzymes and drug transporters. However, studies on regulation of CAR target genes via up- or down-regulation of CAR are limited. In this study, we examined the effects of PPARalpha agonists (ciprofibrate, bezafibrate, fenofibrate and WY14643) on the expression of CAR and its target gene CYP2B1/2 in rat primary hepatocytes. Results from real-time PCR analysis showed that CAR and CYP2B1/2 mRNAs exhibit increases in response to all PPARalpha agonists studied (5 to 10-folds of control). Pretreatment of cells with cycloheximide, an inhibitor of protein synthesis, completely suppressed increase in CYP2B1/2 mRNA in response to ciprofibrate, suggesting that protein synthesis is required in this process. In addition, the induction of CAR by ciprofibrate on the protein level was observed with nuclear extracts as well as total cell lysates. These results indicate that CYP2B1/2 mRNAs are induced by PPARalpha agonists and that this effect is accompanied by increase in the expression of CAR gene at both mRNA and nuclear protein levels. Activated PPARalpha may increase functional CAR protein, which can induce the expression of CAR target genes such as CYP2B.