RS-8359, (+/-)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]pyrimidine selectively and reversibly inhibits monoamine oxidase A (MAO-A). After oral administration of rac-RS-8359 to rats, mice, dogs, monkeys, and humans, plasma concentrations of the (R)-enantiomer were greatly higher than were those of the (S)-enantiomer in all species studied. The AUC((R)) to AUC((S)) ratios were 2.6 in rats, 3.8 in mice, 31 in dogs, and 238 in monkeys, and the (S)-enantiomer was almost negligible in human plasma. After intravenous administration of RS-8359 enantiomers to rats, the pharmacokinetic parameters showed that the (S)-enantiomer had a 2.7-fold greater total clearance (CL(t)) and a 70% shorter half-life (t(1/2)) than those for the (R)-enantiomer but had no difference in distribution volume (V(d)). No significant difference in the intestinal absorption rate was observed. The principal metabolites were the 2-keto form, possibly produced by aldehyde oxidase, the cis-diol form, and the 2-keto-cis-diol form produced by cytochrome P450 in rats, the cis-diol form in mice, RS-8359 glucuronide in dogs, and the 2-keto form in monkeys and humans. Thus, the rapid disappearance of the (S)-enantiomer from the plasma was thought to be due to the rapid metabolism of the (S)-enantiomer by different drug-metabolizing enzymes, depending on species.

The 2-oxidation activity on the pyrimidine ring of RS-8359, a MAO-A inhibitor, is the major metabolic pathway catalysed by aldehyde oxidase. This study investigated the species differences in the 2-oxidation activity by using liver cytosolic fractions from rats, mice, guinea-pigs, rabbits, dogs, monkeys and humans. The Vmax/Km value for the (S)-enantiomer of RS-8359 was extremely high in monkeys and humans, moderate in guinea-pigs, and low in rats and mice. Dogs were deficient in 2-oxidation activity. The (R)-enantiomer was only oxidized at a very low rate in guinea-pigs, monkeys and humans, and not oxidized in rats, mice and rabbits. Thus, marked species differences and enantioselectivity were obvious for the 2-oxidation of the (S)-enantiomer of RS-8359. The in vitro results were in good accordance with previously reported in vivo excretion data of the 2-keto metabolite and the non-detectable plasma concentrations of the (S)-enantiomer in monkeys and humans after administration of racemic RS-8359. Enantioselectivity was also observed for the oxidation of cinchona alkaloids catalysed by aldehyde oxidase. Among the four cinchona alkaloids studied, the oxidation activity of cinchonidine, which has no substituents at the 6-hydroxy group but bears (8S,9R)-configurations, was highest. As opposed to the (S)-enantiomer, an extremely high catalytic activity of cinchonidine was confirmed in rabbits, but not in monkeys or humans. Rabbit liver aldehyde oxidase was suggested to have characteristic properties around the active site.

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In this study, we investigated the properties of monkey liver aldehyde oxidase directed toward the clarification of species differences. The aldehyde oxidase preparation purified from male cynomolgus monkey liver cytosol showed a major 150 kDa Coomassie brilliant blue (CBB)-stained band together with a minor 130 kDa band using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both bands were identified as being aldehyde oxidase by a database search of the MS data obtained with nano-liquid chromatography, quardrupole time of flight, mass spectrometry (nano-LC Q/TOF MS). Based on the sequence coverage, the 130 kDa protein was presumed to be deficient in 20-30 kDa mass from the N-terminus. Full male cynomolgus monkey aldehyde oxidase cDNA was cloned and sequenced with the four degenerate primers designed by considering the peptide sequences containing the amino acids specific for monkey aldehyde oxidase. The deduced amino acid sequences had 96% amino acid identity with those of human enzyme. The aldehyde oxidase expressed in Escherichia coli also exhibited two immunoreactive bands on SDS-PAGE/Western blot analysis. Further, the biphasic pattern was observed for Eadie-Hofstee plots of the (S)-enantiospecific 2-oxidation activity of RS-8359 with the expressed and cytosolic monkey liver aldehyde oxidase. The results suggested that two forms of aldehyde oxidase in monkey were the expression products by a single gene. In contrast, the similarly expressed rat aldehyde oxidase showed only one immunoreactive protein and monophasic pattern. The biphasic phenomenon could be caused by the existence of two aldehyde oxidase isoforms or two active sites in a single enzyme or some other reasons. Further studies on the problems of the biphasic pattern and species differences in aldehyde oxidase are needed.

Changes in the brain lactate concentration in cerebral extracellular fluid (ECF) during intravenous infusion of glucose and local administration of glucose were investigated in adult, conscious, unrestrained rats, with a microdialysis probe in the posterior hippocampus. The rats were infused intravenously with either 25% sucrose solution or 25% glucose solution at a rate of 16.6 microliters.min-1.100 g-1 for three hours. The blood glucose concentration reached 17.0 +/- 2.6 mM at the end of the glucose infusion, and brain ECF glucose showed a parallel change with the blood glucose concentration and increased to 2.37 +/- 0.30 mM. However, blood and brain ECF glucose concentrations did not change in animals infused with the sucrose solution. On the other hand, the blood lactate concentration in the glucose-infused group also increased from 0.93 +/- 0.18 mM to 2.85 +/- 0.39 mM at the end of the glucose infusion, which was significantly higher than that measured in the sucrose-infused group. The blood lactate level in the glucose-infused group returned to the basal level by the end of the experiment. Brain ECF lactate concentrations increased from 1.21 +/- 0.06 mM to 1.69 +/- 0.11 mM in glucose-infused animals, but did not change in the sucrose-infused animals. The brain ECF lactate concentration showed a positive correlation with the brain ECF glucose concentration in glucose-infused animals. Another group of rats was administered glucose locally for 90 min after substitution of artificial cerebrospinal fluid.(ABSTRACT TRUNCATED AT 250 WORDS)