Functional characterization of single nucleotide polymorphisms with amino acid substitution in CYP1A2, CYP2A6, and CYP2B6 found in the Japanese population.

As a part of the studies conducted by the Pharma SNPs Consortium (PSC), the enzyme activities of CYP1A2, CYP2A6 and CYP2B6 variants with altered amino acids as a result of single nucleotide polymorphisms (SNPs) found among the Japanese population were analyzed under a unified protocol using the same lots of reagents by the laboratories participating in the PSC. Mutations in CYP1A2, CYP2A6 and CYP2B6 were introduced by site-directed mutagenesis and the wild type and mutated CYP molecules were expressed in Escherichia coli. The expressed cytochrome P450s were purified and the enzyme activities were measured in reconstitution systems. CYP1A2 and CYP1A2Gln478His did not show any differences in 7-ethoxyresorufin O-deethylase activity. CYP2A6 and CYP2A6Glu419Asp metabolized coumarin to form 7-hydroxycoumarin in a similar manner, whereas CYP2A6Ile471Thr showed low activity compared to the wild-type CYP2A6. CYP2B6, CYP2B6Pro167Ala and CYP2B6Arg487Cys showed the same activity for 7-ethoxy-4-triflouromethyl-coumarin O-deethylation. However, CYP2B6Gln172His was roughly twice as active as CYP2B6 and the other CYP2B6 variants for 7-ethoxy-4-triflouromethylcoumarin O-deethylation activity. Although higher inter- and intra-laboratory variations were observed for the calculated Km and V(max) values because the studies were conducted in several different laboratories, the degree of variations was reduced by the increased number of analyses and the adoption of a simple analysis system.

Studies on the metabolic fate of M17055, a novel diuretic (4): species difference in metabolic pathway and identification of human CYP isoform responsible for the metabolism of M17055.

The metabolic profile of M17055, a novel diuretic, after administration to experimental animals and after incubation with human liver microsomes was investigated. 1. Extensive metabolism was observed in rats and monkeys and the structures of six metabolites (RU1, RU2, and RU3 from rat urine or liver perfusate; MU1, MU2 and MU3 from monkey urine) were assumed or identified. The clear species difference of metabolism was revealed between rats and a monkey with different structures of the isolated metabolites. 2. When these metabolites were quantified using radioactive material, RU3, RU1 and MU3 were considered to be major metabolites in rat urine, rat bile and monkey urine respectively, while in a dog, unchanged drug was observed as the major component indicating only little metabolism occurred in dog, when administered intravenously. 3. RU1 and RU2 were also generated from [(14)C]M17055 after incubation with human liver microsomes, suggesting that the metabolic pathway of M17055 in humans involves that observed in rats. 4. [(14)C]M17055 metabolism in human liver microsomes was inhibited by CYP2C8/9 and CYP3A4/5 inhibitors, and also by the antibodies that recognize CYP2C8/9/19 and CYP3A4. Significant correlations were observed between the rate of [(14)C]M17055 metabolism and the activity of testosterone 6beta-hydroxylation or tolbutamide methyl-hydroxylation. cDNA-expressed CYP3A4 and CYP2C9 could catalyze the metabolism of [(14)C]M17055. These results suggest that the metabolism of M17055 in human liver microsomes is catalyzed mainly by CYP3A4 and CYP2C9.

Studies on the metabolic fate of m17055, a novel diuretic (5): pharmacokinetics and pharmacodynamics of unchanged drug in rat and dog after intravenous administration of M17055.

The pharmacokinetics and pharamacodynamics of M17055, a novel diuretic were studied after a single intravenous administration to rats and dogs, the two species used in the pharmacological and toxicological studies. No gender dependent response to systemic exposure was observed at the high dose level in rats, in agreement with the determined LD(50). A gender difference in urinary excretion of M17055, however, was clearly observed in rats. The slower elimination and the lower total body clearance (CLtot) values of M17055 in dogs reflect the difference of the no-effect level (NOEL) between rats (0.1 mg/kg) and dogs (0.03 mg/kg) well. The diuretic response was well correlated with the urinary M17055 excretion rate by fitting to a sigmoid E(max) model in both rats and dogs. The derived ER(50) value of M17055 in dogs was approximately 10 times less than that reported for furosemide, suggesting that the intrinsic potency of M17055 is equal to or higher than those of other powerful loop diuretics. Although diuretic sensitivity was considered to be lower in dogs than in rats, the higher amount of M17055 reaching the dog kidney is likely to compensate for this. The diuretic response in female rats was predictable by using the pharmacodynamic parameters derived from male rats. These results show that the apparent high diuretic potency and the other pivotal observations for M17055 found in the pharmacological and toxicological studies can be rationalized by the pharmacokinetic and pharmacodynamic properties of the unchanged compound.