Kazuho Okudaira

CONTRIBUTION OF OATP (ORGANIC ANION-TRANSPORTING POLYPEPTIDE) FAMILY TRANSPORTERS TO THE HEPATIC UPTAKE OF FEXOFENADINE IN HUMANS

Fexofenadine hydrochloride (FEX), a second generation H1-receptor antagonist, is mainly eliminated from the liver into bile in unchanged form. Recent studies have shown that FEX can be accepted by human MDR1 (P-glycoprotein), OATP1A2 [organic anion-transporting polypeptide (OATP)-A, and OATP2B1 (OATP-B)] expression systems. However, other transporters responsible for the hepatic uptake of FEX have not yet been identified. In the present study, we evaluated the contribution of OATP family transporters, namely OATP1B1 (OATP2/OATP-C), OATP1B3 (OATP8), and OATP2B1 (OATP-B), to FEX uptake using transporter-expressing HEK293 (human embryonic kidney) cells. The uptake of FEX in OATP1B3-expressing cells was significantly greater than that in vector-transfected cells. On the other hand, OATP1B1- or OATP2B1-mediated uptake of FEX was not statistically significant. OATP1B3-mediated transport could be explained by a one-saturable component with a Michaelis constant (Km) of 108 ± 11 μM. The inhibitory effect of FEX on the uptake of estrone-3-sulfate (E1S), cholecystokinin octapeptide (CCK-8), and 17β-estradiol-17β-d-glucuronide (E217βG) was also examined. Both OATP1B1- and OATP1B3-mediated E217βG uptake was inhibited by FEX. The Ki values were 148 ± 61 and 205 ± 72 μM for OATP1B1 and OATP1B3, respectively. FEX also inhibited OATP1B3-mediated CCK-8 uptake and OATP1B1-mediated E1S uptake with a Ki value of 83.3 ± 15.3 and 257 ± 84 μM, respectively, suggesting that FEX could not be used as a specific inhibitor for OATP1B1 and OATP1B3, although FEX was preferentially accepted by OATP1B3. In conclusion, this is, to our knowledge, the first demonstration that OATP1B3 is thought to be a major transporter involved in hepatic uptake of FEX in humans.

Involvement of reactive oxygen species in sonodynamically induced apoptosis using a novel porphyrin derivative.

In this study, we investigated the induction of apoptosis by ultrasound in the presence of the novel porphyrin derivative DCPH-P-Na(I). HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of DCPH-P-Na(I), and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Reactive oxygen species were measured by means of ESR and spin trapping technique. Cells treated with 8 μM DCPH-P-Na(I) and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound or DCPH-P-Na(I) alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and DCPH-P-Na(I) but not in cells treated with ultrasound or DCPH-P-Na(I) alone. In addition, the combination of DCPH-P-Na(I) and the same acoustical arrangement of ultrasound substantially enhanced nitroxide generation by the cells. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. These results indicate that the combination of ultrasound and DCPH-P-Na(I) induced apoptosis in HL-60 cells. The significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggests active species such as singlet oxygen are important in the sonodynamic induction of apoptosis. These experimental results support the possibility of sonodynamic treatment for cancer using the induction of apoptosis.

Species difference of metabolic clearance of bisphenol A using cryopreserved hepatocytes from rats, monkeys and humans.

In vitro metabolism of bisphenol A (BPA), an weak estrogen, was studied with cryopreserved hepatocytes from rat, monkey and human, and was compared with in vivo metabolism reported. The metabolites identified include a major metabolite, BPA glucuronide (BPAG) and BPA sulfate (BPAS). The metabolic rates of bisphenol A at 20micro the hepatocytes (BPAG plus BPAS, nmol/10(6) cells/h) followed the order of rats (48+12)>monkeys (18+4)>humans (8.6+0.8), respectively. The rate of BPAG formation was much higher than that of BPAS formation in all these species. For the BPAG formation, we have determined the apparent K(m) (microf rats (3), monkeys (7), and humans (5). V(max) (nmol/10(6) cells/h) in hepatocytes followed the order of rats (55)>monkeys (22)>humans (11). The total CL(H) for the hepatic formation of BPAG plus BPAS (L/h/kg BW) estimated by well-stirred model with low f(B) value followed the order of rats (3.0)>monkeys (0.68)>humans (0.27), correlating well with in vivo studies of BPA subcutaneously injected rats and monkeys. This study showed that the cryopreserved hepatocytes could be a useful tool for assessing BPA metabolism and predicting systemic exposure of BPA.

Correlation between the inhibitory effects of basic drugs on the uptake of cardiac glycosides and taurocholate by isolated rat hepatocytes

The role of the multispecific bile acid transporter for cardiac glycoside uptake is still controversial. This study was designed to examine the inhibitory effects of basic drugs (verapamil, dipyridamole, nifedipine, chlorpromazine, disopyramide, quinidine, propranolol, and lidocaine) on taurocholate uptake by isolated rat hepatocytes and to compare these effects with inhibition of ouabain uptake. Sodium-dependent taurocholate uptake was significantly reduced, to 50-70% of the control value, by 50 µM verapamil, dipyridamole, and nifedipine. Sodium-independent taurocholate uptake was more extensively inhibited, to 20-40%, by these basic drugs. The inhibition of ouabain uptake correlated better with sodium-independent taurocholate uptake (γ = 0.918) than with sodium-dependent taurocholate uptake (γ = 0.714). Taurocholate competitively inhibited ouabain uptake in the absence of sodium. These results indicate that the cardiac glycoside transport system is similar to the sodium-independent taurocholate transport system.

Contribution of Rat Pulmonary Metabolism to the Elimination of Lidocaine, Midazolam, and Nifedipine

The contribution of the lung to drug metabolism was investigated in rats and the possibility of prediction of in vivo metabolism from in vitro studies using rat pulmonary microsomes was assessed. Lidocaine, midazolam, or nifedipine was administered to rats at a dose of 10 mg/kg by the intra-arterial, intravenous, and intraportal routes. The pulmonary extraction ratios of lidocaine, midazolam, and nifedipine, calculated from the area under the time-plasma concentration curve (AUC) after the intra-arterial and intravenous administrations, were 39.0 ± 0.5, 18.3 ± 0.7, and 12.3 ± 0.3%, respectively. The hepatic extraction ratios of lidocaine, midazolam, and nifedipine, calculated from the AUC after the intraportal and intravenous administrations, were 68.0 ± 3.3, 52.6 ± 0.4, and 13.5 ± 0.2%, respectively. These results showed that both the liver and the lung contributed to the metabolism of these drugs. The above in vivo pulmonary extraction ratios correlated with the in vitro intrinsic clearance values, which were corrected with the protein unbound ratio in microsomes and plasma, suggesting that pulmonary extraction ratios can be predicted quantitatively from in vitro data. The pulmonary intrinsic clearance values of lidocaine, midazolam, and nifedipine in rat microsomes were lower than their hepatic intrinsic clearance, showing that there was an organ difference in metabolism between the liver and lung. Our results support the importance of the estimation of pulmonary metabolism to predict the total clearance more accurately.

ATP-Dependent Transport of a Novel Thromboxane A2 Receptor Antagonist, [2-(4-Chlorophenylsulfonylaminomethyl)-Indan-5-yl]Acetate (Z-335) and Its Xenobiotic Taurine Conjugate (Z-335-Tau) by Rat Bile Canalicular Membrane Vesicles

AbstractPurpose. The characteristics of bile canalicular transport processes for xenobiotic taurine conjugates have not yet been clarified. To elucidate the biliary excretion characteristics of xenobiotic taurine conjugates, we investigated the transport of a novel thromboxane A2 receptor antagonist, Z-335, and its taurine conjugate (Z-335-Tau) across the bile canalicular membrane. Methods. We examined the uptake of Z-335 and Z-335-Tau by isolated bile canalicular membrane vesicles (CMVs) from Sprague Dawley and Eisai-hyperbilirubinemic rats (EHBRs) which EHBRs have a hereditary defect of canalicular multidrug resistance-associated protein 2 (Mrp2) function. Also, the in vitro and in vivo kinetics of Z-335-Tau uptake and excretion were compared. Results. Z-335 uptake by CMVs from normal rats exhibited marked ATP-dependence, whereas ATP-dependent uptake of Z-335 into CMVs from EHBRs was not observed. In contrast, Z-335-Tau uptake into CMVs from both normal rats and EHBRs was ATP dependent. The initial uptake velocity was concentration-dependent, with an in vitro Michaelis constant for initial uptake of 189 μM, which was similar to the in vivo value. Conclusions. The biliary excretion of Z-335 involves Mrp2, whereas that of Z-335-Tau involves active transport systems that remain intact in EHBRs and show marked ATP dependence, which ATP-dependent transport is involved in the biliary excretion of Z-335-Tau in vivo.

Uptake of colchicine, a microtubular system disrupting agent, by isolated rat hepatocytes.

The possible mechanism of hepatic uptake of colchicine (CLC), a microtubule system disrupting agent, was examined using isolated rat hepatocytes. The existence of a carrier-mediated active transport system for CLC was demonstrated. This transport system is saturable, is affected by metabolic inhibitors (dinitrophenol, KCN) and a SH-group blocker (p-hydroxymercuribenzoic acid but not N-ethylmaleimide), and is sensitive to temperature. Ouabain, an inhibitor of Na+, K+-ATPase, does not affect the transport system of CLC.

Involvement of Reactive Oxygen Species in Sonodynamically Induced Apoptosis by 4-Formyloximeetylidene-3-Hydroxyl-2-Vinyl-euterio-Porphynyl (IX)-6-7-Diaspartic Acid (ATX-S10)

Background: In this study, we investigated the induction of apoptosis by ultrasound in the presence of the photochemically active chlorine, 4-formyloximeetylidene-3-hydroxyl-2-vinyl-deuterio-porphynyl (IX)-6-7-diaspartic acid (ATX-S10). Methods: HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of ATX-S10, and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Results: Cells treated with 80μM ATX-S10 and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound or ATX-S10 alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and ATX-S10 but not in cells treated with ultrasound or ATX-S10 alone. In addition, the combination of ATX-S10 and the same acoustical arrangement of ultrasound substantially enhanced nitroxide generation by the cells. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. Conclusions: These results indicate that the combination of ultrasound and ATX-S10 induces apoptosis in HL-60 cells. The significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggests active species such as singlet oxygen are important in the sonodynamic induction of apoptosis. General significance: The results reported in this paper are experimental, but they significantly support the possibility of sonodynamic treatment for cancer using the induction of apoptosis.