Eiji Takahara

Simultaneous determination of a new gastrointestinal prokinetic agent (HSR-803) and its metabolites in human serum and urine by high-performance liquid chromatography using automated column-switching

A method based on high-performance liquid chromatography using column-switching is described for the simultaneous determination of HSR-803 and its metabolites in human serum and urine. The system uses a six-port valve with a Nucleosil CN pre-column for on-line sample clean-up, and direct injection of samples. The limits of quantitation in serum and urine were 5 and 20 ng/ml for HSR-803 and 50 and 200 ng/ml for the metabolites, respectively. The coefficients of variation for the intra- and inter-day accuracies were between 0.8 and 7.1% for each compound. This method was applied to the pharmacokinetic studies in humans after oral administration of HSR-803.

Effects of oxygen concentration on the metabolism of anisole homologues by rat liver microsomes

1. The effects of oxygen concentration were studied on the metabolic pathways of anisole homologues (anisole, phenetole and isopropoxybenzene) catalysed by liver microsomes from phenobarbital-treated rats. 2. With increase of oxygen concentration, the rate of anisole o-hydroxylation reached a plateau at about 35 microM O2, while the rates of O-demethylation and aromatic p-hydroxylation were still increasing at 223 microM O2 (air). 3. The rates of all three metabolic reactions of phenetole reached plateau levels at about 80 microM O2. 4. The rates of all three metabolic reactions of iso-propoxybenzene were still increasing as 223 microM O2 (air). 5. The ratio of aromatic p-hydroxylation or O-dealkylation to aromatic o-hydroxylation decreased in anisole metabolism, and showed no uniform change in phenetole and isopropoxybenzene metabolism with decreasing oxygen concentration. 6. The ratio of aromatic p-hydroxylation to O-dealkylation was essentially constant over the range of oxygen concentration studied in anisole and phenetole metabolism, while in iso-propoxybenzene metabolism the ratio was different between higher and lower oxygen concentrations than 60 microM. 7. This series of compounds with increasing chain length did not show homologous changes in rates of product formation or O2 dependent of product formation.

Sensitive assay of trimethylamine N-oxide in liver microsomes by headspace gas chromatography with flame thermionic detection

To compare the trimethylamine N-oxygenase activity of liver microsomes from house musk shrew (Suncus murinus) and rat, a sensitive method for the quantitation of trimethylamine (TMA) N-oxide was developed using gas chromatography with flame thermionic detection. The limit of quantification was 0.5 microM and the calibration curve was linear at least up to 5 microM in incubations containing liver microsomal preparations from Suncus. The intra-day RSD values ranged from 10.4 to 12.8 at 0.5 microM and from 3.5 to 6.7 at 5 microM. The inter-day RSD values were 11.6 and 6.5 at 0.5 and 5 microM, respectively. This method provides a sensitive assay for TMA N-oxygenase activity in liver microsomes. Using this method we found that Suncus was capable of N-oxidizing trimethylamine at a very slow rate.

The house musk shrew (Suncus murinus): a unique animal with extremely low level of expression of mRNAs for CYP3A and flavin-containing monooxygenase

Expression of drug-metabolizing enzymes including cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) in various tissues of Suncus murinus (Suncus) were examined. Northern blot analysis showed that mRNAs hybridizable with cDNAs for rat CYP1A2, human CYP2A6, rat CYP2B1, human CYP2C8, human CYP2D6, rat CYP2E1, human CYP3A4 and rat CYP4A1 were expressed in various tissues from Suncus. The mRNA level of CYP2A in the Suncus lung was very high. Furthermore, it was found that the level of CYP2A mRNA in the Suncus lung was higher compared to the Suncus liver. The expression level of mRNA hybridizable with cDNA for human CYP3A4 was very low. The presence of CYP3A gene in Suncus was proven by the induction of the CYP with dexamethasone. Very low expression levels of mRNAs hybridizable with cDNAs for rat FMO1, rat FMO2, rat FMO3 and rat FMO5 were also seen in Suncus liver. No apparent hybridization band appeared when human FMO4 cDNA was used as a probe. The hepatic expression of mRNAs hybridizable with cDNAs for UDP-glucuronosyltransferase 1*6, aryl sulfotransferase, glutathione S-transferase 1, carboxyesterase and microsomal epoxide hydrolase in the Suncus were observed. These results indicate that the Suncus is a unique animal species in that mRNAs for CYP3A and FMO are expressed at very low levels.

Analysis of urinary and biliary metabolites of (+)-4-[4-(4-methylphenyl)phenylmethoxy-1-piperidinyl]butyric acid in rats by liquid chromatography—frit-fast atom bombardment mass spectrometry

The urinary and biliary metabolites of (+)-4-[4-(4-methylphenyl)phenylmethoxy-1-piperidinyl]butyric acid [(+)-MPPB] were examined in rapid-metabolizing (RM) and slow-metabolizing (SM) male Sprague-Dawley rats by means of liquid chromatography-frit-fast atom bombardment mass spectrometry. In the RM-phenotyped rats, unchanged (+)-MPPB could not be detected in urine or bile, but 4-carboxyphenyl-MPPB was detected in bile. In the SM-phenotyped rats, unchanged (+)-MPPB was detected in bile and unchanged (+)-MPPB and beta-oxidized MPPB in urine. Thus, the inter-individual difference in (+)-MPPB metabolism in rats was confirmed in vivo.

Metabolic Fate following a Dermal Application of Tulobuterol Tape (2): Absorption, Distribution and Excretion in Rats after Repeated Applications

The absorption, distribution and excretion of 14C-tulobuterol following daily dermal application at 10 mg/kg for 7 days was examined in rats. 1. Blood radioactivity reached a steady state after the 4th dose. After the final dose, blood radioactivity showed maximum values of 506.1 ng eq./ml at 5 hr and disappeared with T1/2 of 19.4 hr as also noted for T1/2 after a single application (15.7 hr). 2. After the final application, the radioactivity in most tissues was higher than after a single application. Elimination of radioactivity in tissue was basically the same as after a single application. 3. Within 168 hr after the 7th application, urinary and fecal excretion was 53.8 and 36.3% of the dose, respectively. Recovery in urine and feces and from residue in the removed tape was 98.6% of the dose.