Keizo Matsuda

Structure determination of metabolites isolated from urine and bile after administration of AY4166, a novel d-phenylalanine-derivative hypoglycemic agent

Molecular structures of 10 metabolites, which were isolated from urine (M1-M8) or bile (M9 and M10) after administration of AY4166 (N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine), a novel amino acid derivative with hypoglycemic activity, have been elucidated by mass spectrometry and nuclear magnetic resonance. Four of these (M1, M2, M3 and M8) were determined to be hydroxyl derivatives of AY4166, two (M9 and M10) were carboxylate derivatives via oxidization of M2 and M3, three (M4, M5 and M6) were glucronic acid conjugates and the other (M7) was a dehydro derivative. The estimated structures for M1, M2, M3, M7, M8, M9 and M10 were confirmed by the coincidence of the retention time of HPLC, MS and 1H NMR spectra between the isolated metabolites and authentic synthesized substances. For three glucronic acid conjugates, M4, M5 and M6, structural confirmation was performed by a selective enzymatic digestion with beta-glucronidase. M1 and M2/3 were about 5-6 and 3 times less potent than AY4166, respectively, and M7 was almost as potent as AY4166.

Determination of N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine and its metabolites in human plasma and urine by column-switching high-performance liquid chromatography with ultraviolet detection

A simple, rapid and sensitive two column-switching high-performance liquid chromatographic (HPLC) method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine (AY4166, I) and its seven metabolites in human plasma and urine. Measurements of I and its metabolites were carried out by two column-switching HPLC, because metabolites were classified into two groups according to their retention times. After purification of plasma samples using solid-phase extraction and direct dilution of urinary samples, I and each metabolite were injected into HPLC. The calibration graphs for plasma and urinary samples were linear in the ranges 0.1 to 10 microg ml(-1) and 0.5 to 50 microg ml(-1), respectively. Recoveries of I and its seven metabolites were over 88% by the standard addition method and the relative standard deviations of I and its metabolites were 1-6%.

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC18, followed by HPLC. The calibration graph for I was linear in the range 0.1-20 micrograms/ml. The limit of quantitation of I, in plasma, was 0.05 microgram/ml. The recovery of spiked I (0.5 microgram/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 microgram/ml) compared to drug-free plasma was 4.3% (n = 8).