Koichi Yokogawa

P-Glycoprotein-Dependent Disposition Kinetics of Tacrolimus: Studies in mdr la Knockout Mice

AbstractPurpose. This study was performed to evaluate the involvement of P-glycoprotein in disposition kinetics of tacrolimus (FK506), a substrate of P-glycoprotein, in the body. Methods. The blood and tissue concentrations of FK506 after i.v. or p.o. administration (2 mg/kg) to normal andmdrla knockout mice were measured by competitive enzyme immunoassay. Results. The blood concentrations in knockout mice were significantly higher than those in normal mice. The value of the total clearance (CLtot) for knockout mice (19.3 mL/min/kg) was about 1/3 of that for normal mice (55.8 mL/min/kg)(P < 0.001), although there was no significant difference in the distribution volume at the steady-state (Vdss) (about 4.6 L/kg) between both types of mice. FK506 rapidly penetrated the blood-brain barrier and the brain concentration reached a maximum, which was about 10 times higher in knockout mice than in normal mice, 1 hr after administration. The brain concentration in normal mice thereafter decreased slowly, whereas in knockout mice, an extremely high concentration was maintained for 24 hr. Conclusions. The pharmacokinetic behavior of FK506 in the tissue distribution is related with the function of P-glycoprotein encoded by themdr la gene. The brain distribution of FK506 is dominated by the P-glycoprotein-mediated drug efflux and presumably also by the binding to FK-binding proteins (immunophilins) in the brain.

Relationships in the Structure–Tissue Distribution of Basic Drugs in the Rabbit

The relationship between the tissue-to-plasma partition coefficients (Kp) and drug lipophilicity was investigated using highly lipophilic drugs with apparent partition coefficients of 150 or above in an octanol–water system at pH 7.4. Ten clinically popular basic drugs with different dissociation coefficients (pKa) and lipophilicity were used. The Kp values were determined in nondisposing organs after the i.v. administration of individual drugs in rabbits. The free fraction in plasma and the blood-to-plasma concentration ratio were determined in vitro. Then the tissue-to-plasma ratios of nonionized and unbound drug concentrations (Kpfu) were calculated from Kpf (ratio of unbound drug). The true octanol–water partition coefficient of the nonionized drugs (P) was used to analyze the Kpf and Kpfu. In all tissues, log Kpfu was more highly correlated with log P than log Kpf.

Selective Delivery of Estradiol to Bone by Aspartic Acid Oligopeptide and Its Effects on Ovariectomized Mice

We have developed a novel osteotropic prodrug of estradiol (E2) conjugated with l-Asp-hexapeptide (E2·3D6), which has very low affinity for estrogen receptors, and in this study, we examined its pharmacokinetic behavior and pharmacological potential. After a single iv injection of E2·3D6 to mice, the half-time for elimination from plasma was about 100 min; however, E2 was selectively delivered to the bone and eliminated very slowly, declining to the endogenous level at about 7 days. After a single iv injection of E2, the half-time in plasma was about 70 min, whereas E2 was highly distributed to the uterus, and the bone concentration of E2 was only slightly increased at 6 h. When E2 (0.37 μmol/kg, sc, every third day) or E2·3D6 (0.11 to 1.1 μmol/kg, sc, every seventh day) was administered to OVX mice for 4 weeks, E2 increased the bone mineral density (BMD) together with weights of liver and uterus, whereas E2·3D6 increased only the BMD, in a dose-dependent manner. E2·3D6 enhanced the expression of messenge...

Effect of γ-butyrobetaine on fatty liver in juvenile visceral steatosis mice

We pharmacokinetically examined the effect of γ‐butyrobetaine, a precursor of l‐carnitine, on the change of fatty acid metabolism in juvenile visceral steatosis (JVS) mice, which have systemic l‐carnitine deficiency due to lack of l‐carnitine transporter activity. The concentrations of total free fatty acid (FFA), palmitic acid and stearic acid in the liver of JVS mice were significantly higher than those in wild‐type mice. After intravenous administration of γ‐butyrobetaine (50 mg kg−1), the concentration of l‐carnitine in the plasma of JVS mice reached about twice that of the control level and levels in the brain, liver and kidney were also significantly increased, whereas those in wild‐type mice hardly changed. Although the plasma concentrations of FFA in both types of mice were unchanged after administration of γ‐butyrobetaine, the concentrations of palmitic acid and stearic acid were significantly decreased. In particular, the liver concentration of FFA in JVS mice was decreased to the wild‐type control level, accompanied by significant decreases in long‐chain fatty acids, palmitic acid and stearic acid, whereas those in wild‐type mice were not changed. These results suggest that γ‐butyrobetaine can be taken up into organs, including the liver, of JVS mice, and transformed to l‐carnitine. Consequently, administration of γ‐butyrobetaine may be more useful than that of l‐carnitine itself for treatment of primary deficiency of carnitine due to a functional defect of the carnitine transporter.

In vivo dopamine-D2 and serotonin-5-HT2 receptor binding study of risperidone and haloperidol

An in vivo receptor binding technique was applied to evaluate the affinities of risperidone and haloperidol for dopamine-D2 receptors (D2) and serotonin-5-HT2 receptors (5-HT2) in rat brain with [3H]YM-09151-2 and [3H]ketanserin as selective ligands. Radioactivities were obtained in the striatum frontal cortex, and cerebellum of the rats treated with the ligands. Time course study of receptor occupancy at 25 to 250 min after single doses of the drugs (1 mg/kg, IP) showed higher 5-HT2 occupancy in the frontal cortex and lower D2 occupancy in the striatum by risperidone than by haloperidol. Dose-response analysis of receptor occupancy revealed risperidone demonstrated higher binding affinity for 5-HT2 than for D2, while the reverse was observed with haloperidol. It appeared that risperidone (1 mg/kg, IP), but not haloperidol (1 mg/kg, IP), demonstrated regional selectivity in D2 occupancy favouring frontal cortex more than the striatum. That risperidone displayed a higher ratio of 5-HT2 to D2 in occupancy than haloperidol is in agreement with the previous findings obtained in vitro.

Modulation of mdr1a and CYP3A gene expression in the intestine and liver as possible cause of changes in the cyclosporin A disposition kinetics by dexamethasone

We investigated the effect of dexamethasone (DEX) on the disposition kinetics of cyclosporin A (CyA) and the mechanism of this drug interaction. Rats were treated with DEX (1 or 75mg/kg per day, i.p.) once a day for 1-7 days, and the blood concentration of CyA was measured after an i.v. or p.o. dose of CyA (10mg/kg) at 1.5hr after the last DEX treatment. In rats treated with a low dose of DEX (1mg/kg), the blood concentration of CyA after i.v. administration was unchanged compared with that of untreated rats, whereas the blood concentration after oral administration was significantly decreased, and this decrease was dependent on the duration of DEX administration. The total clearance (CL(tot)) of CyA was unchanged, but the bioavailability was significantly decreased to about one-third of that in DEX-untreated rats after 7 days of DEX treatment. At this time, the expression of mdr1a mRNA and P-gp in the liver and intestine was increased, whereas CYP3A2 was unaffected at both the mRNA and protein levels. In rats treated with a high dose of DEX (75mg/kg), the blood concentration of CyA was significantly decreased after both i.v. and p.o. administrations compared with those of untreated rats. The bioavailability of CyA was decreased, and the CL(tot) was significantly increased. The P-gp and CYP3A2 in the liver and intestine were increased at both the mRNA and protein levels. Our results indicate that the drug interaction between CyA and DEX is a consequence of modulation of P-gp and CYP3A2 gene expression by DEX, with differential dose-dependence.

Effect of Meropenem on Disposition Kinetics of Valproate and Its Metabolites in Rabbits

AbstractPurpose. We investigated the effect of meropenem (MEPM) on the disposition kinetics of valproate (VPA) and its metabolites in rabbits. Methods. Rabbits were given 75 mg/kg VPA intravenously with or without 300 mg/kg MEPM. Results. The plamsa total clearance of VPA was significantly increased to about 1.5 times the control (6.09 mL/min/kg vs. 4.28 mL/min/kg) by MEPM (P < 0.05). The values of the area under the plasma concentration-time curve (AUC) of 2-en-VPA, a product of β-oxidation, and VPA-glucuronide (VPA-G) were significantly decreased to about 55% and 78% of the control, respectively (P < 0.05). The cumulative urinary excretions of VPA in the control and MEPM-treated groups were 0.54% and 0.62% of the dose, respectively, whereas those of VPA-G were 45.6% and 62.5%, respectively. The urinary excretion of VPA-G was significantly increased by MEPM (P < 0.05). Further, in the case of 33.8 mg/kg VPA-G administered intravenously the AUC value of VPA-G was unchanged by MEPM, whereas that of the generated VPA was significantly decreased to about half of the control. Conclusions. The increase of the total clearance of VPA caused by MEPM appears to be a consequence of increased renal clearance of VPA-G, as well as suppression of VPA-G hydrolysis in the liver.

Structure-activity relationship of flavonoids for inhibition of epidermal growth factor-induced transformation of JB6 Cl 41 cells.

We found that quercetin, myricetin, quercetagetin, fisetin, (−)‐epigallocatechin gallate (EGCG), and theaflavins, among 24 flavonoids examined, markedly inhibited epidermal growth factor (EGF)‐induced cell transformation of mouse epidermal JB6 Cl 41 cells. The six flavonoids suppressed the EGF‐induced activation of activator protein 1 (AP‐1). In addition, myricetin, quercetagetin, EGCG, and theaflavins directly inhibited EGF‐induced phosphatidylinositol 3‐kinase (PI3K) activation. The important structural features of flavonoids for cell transformation‐inhibitory activity are 3′‐ and 4′‐OH on the B‐ring, 3‐OH on the C‐ring, C2C3 double bond in the C‐ring, and the phenylchromone (C6C5C6) skeleton in the flavonols, and the galloyl group in EGCG and theaflavins. Our results provide new insight into possible mechanisms of the anti‐carcinogenic effects of flavonoids, and could help to provide a basis for the design of novel cancer chemopreventive agents.

Novel drug delivery system to bone using acidic oligopeptide: pharmacokinetic characteristics and pharmacological potential.

We synthesized fifteen oligopeptides consisting of Asp or Glu conjugated with a fluorescent probe, 9- fluorenylmethylchloroformate (Fmoc). In the in vitro binding assay to putative hydroxy apatite (HA), the affinities of these conjugates depended only on the number of amino acid residues, not on their optical characters (L or D) or their species (Asp or Glu). In an in vivo experiment involving a single i.v. injection of Fmoc-D-Asp oligopeptides into mice, peptides consisting of over six Asp residues were selectively distributed to the bone. Then, we synthesized estradiol-17β-succinate-(L-Asp)6 [E2-(L-Asp)6] and studied its pharmacokinetic characteristics and its antiosteoporotic effects on ovariectomized (OVX) mice. Although the distribution volume of E2-(L-Asp)6 was significantly smaller than that of E2, E2-(L-Asp)6 was selectively distributed in the bone after i.v. injection and gradually decreased during 7 days. E2-(L-Asp)6 effectively prevented OVX-induced bone loss, without altering the uterine weight, in the dosage range of 0.11 to 1.1 μmol/kg once a week, while E2 increased both the bone mineral density and uterine weight at 0.37 μmol/kg every third day. The results suggest that acidic oligopeptide may be useful for drug delivery to bone and E2-(L-Asp)6is a good candidate as an anti-osteoporosis drug without the adverse side effects of E2.

Contribution of lysosomes to the subcellular distribution of basic drugs in the rat liver

AbstractPurpose. We examined the subcellular distribution of the basic drugs, chlorpromazine (CPZ), imipramine (IMP) and biperiden (BP), in rat liver, and evaluated the contribution of lysosome (Lys) to their intracellular distribution in comparison with that of mitochondria (Mit). Methods. In an in vivo distribution, the concentrations of CPZ, IMP and BP in the liver subcellular fractions were determined. In an in vitro study, uptake of [3H]IMP into Lys and Mit fractions was determined in the presence or absence of several agents. Results. The distribution of these drugs 10 min after administration was quite similar. However, the relative specific contents (the drug concentration per protein of each fraction divided by that of the total homogenate) in Lys were 7.3, 9.6 and 4.2, respectively for CPZ, IMP and BP, whereas those in the other organella were only 0.4 ~ 1.7. In an in vitro uptake study, the dose response of IMP uptake into Lys was biphasic, while that into Mit fractions was monophasic. The binding of IMP to the high affinity sites of Lys was pH dependent and disappeared in 50 mM NH4C1 or 50 µM CPZ, both of which increased the intralysosomal pH. the low affinity sites were not affected by these drugs. Conclusions. The results indicated that Lys has the highest affinity for the basic drugs in the liver and that its contribution to their subcellular distribution depends on the intralysosomal pH, which is also affected by these drugs. The importance of these effects may become significant in combination therapy using various basic drugs.