1. The pharmacokinetics of YM-64227 (4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]-pyrimidine-2-(1H)-one), a novel and selective phosphodiesterase type 4 inhibitor, was characterized in beagle dogs. Based on the plasma parent drug to major hydroxylated metabolite ratio, 21 dogs were phenotyped as 16 extensive metabolizers (EM) and five poor metabolizers (PM).2. Nucleotide sequences of CYPs 1A2, 2B11, 2C21, 2D15, 2E1 and 3A12 were investigated in the EM and PM dogs. A CYP1A2 1117 C>T single nucleotide polymorphism was found, which resulted in an amino acid change from an Arg codon to a stop codon at position 373.

ABSTRACT:In a previous study, it was shown that the novel canine single nucleotide polymorphism (SNP) CYP1A2 1117C>T yields an inactive enzyme. In this study, the effect that this SNP has on the pharmacokinetics of 4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]pyrimidine-2-(1H)-one (YM-64227) was investigated. Plasma concentrations of the unchanged drug and five of its metabolites (MM-1 to MM-5) were determined after either intravenous or oral administration of YM-64227 to genotyped dogs (C/C, C/T, and T/T groups). Liver microsomes were prepared from these dogs to determine the in vitro metabolic clearance of YM-64227. After a single oral administration, the maximum plasma concentration and absolute bioavailability of YM-64227 in the T/T group were 17.1 times and 27.2 times higher than those in the C/C group, respectively, whereas the pharmacokinetics of YM-64227 after intravenous administration were not affected by genotype. The metabolic profiles in the T/T group were quite distinct from the others; i.e., the main metabolite was MM-2 in the C/C group, whereas MM-1 and MM-5 were the main metabolites in the T/T group. The formation clearances of MM-2 and MM-3 in the microsomes derived from T/T type dogs were significantly lower, whereas those of MM-1, MM-4, and MM-5 were not affected. A statistically significant correlation was observed between the in vivo and in vitro metabolic intrinsic clearances (r ‫؍‬ 0.82, p < 0.001). The canine CYP1A2 1117C>T SNP proved to be responsible for a substantial portion of the interindividual variability in the pharmacokinetics of YM-64227.Cytochrome P450 (P450) is a superfamily of enzymes that plays an important role in the oxidative metabolism of a wide variety of xenobiotics as well as endogenous compounds (Nelson et al., 1996). The metabolic activity of P450 is affected by several factors such as inhibition by concomitant drugs, induction, and genetic polymorphism (Pelkonen et al., 1998). The P450 polymorphisms have been investigated extensively in clinical settings, and it is well known that mutated alleles of CYP2C9, CYP2C19, and CYP2D6 cause altered plasma concentrations, which might in turn cause unforeseeable adverse reactions (Kagimoto et al., 1990;Sullivan-Klose et al., 1996;Furuta et al., 1999).A large number of P450 cDNA clones have been isolated, sequenced, and extensively studied in humans, mice, and rats (http:// drnelson.utmem.edu/CytochromeP450.html). On the other hand, only a few studies on canine P450 cDNAs have been conducted, although dogs are used extensively in pharmacological research and drug safety assessment studies. At present, a variety of canine P450 enzymes such et al., 1991;Fraser et al., 1997) have been cloned and sequenced. Although gene analysis has revealed that some of these P450s have variants, no obvious change in the plasma concentration of drug associated with single nucleotide polymorphisms (SNPs) has been reported for dogs. In fact, only a few studies have shown the possibility that interindividual variations in the pharmacokinetics of so...

ABSTRACT:The canine CYP1A2 1117 C>T single nucleotide polymorphism is responsible for a substantial portion of the interindividual variability seen in the pharmacokinetics of 4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]pyrimidine-2-(1H)-one (YM-64227). The purpose of this study is to investigate the contribution of CYP1A2 to the metabolism of YM-64227 and its five metabolites (MM-1 to MM-5), as well as to determine the interindividual variability between the pharmacokinetic profiles of the compounds with respect to the CYP1A2 deficiency polymorphism. ␣-Naphthoflavone and anti-CYP1A1/2 antibody inhibited the metabolic activities at which MM-2 and MM-3 were formed from YM-64227 in C/C-and C/T-type microsomes. In T/T type, the rate of MM-2 and MM-3 formation was lower, and ␣-naphthoflavone and anti-CYP1A1/2 antibody were shown to have no effect. A positive correlation between the overall metabolism of YM-64227 and phenacetin O-deethylation, a CYP1A2 activity marker, was observed in all the genotypes. The in vitro metabolic clearances in the T/T type of MM-1, MM-3, MM-4, and MM-5 were less than 50% lower than those in the C/C type. The anti-CYP1A1/2 antibody inhibited the metabolism of MM-1, MM-3, MM-4, and MM-5 in the C/C and C/T types. These results suggest that the formation of MM-2 and MM-3 from YM-64227 is catalyzed by CYP1A2, and that CYP1A2 contributes mainly to the subsequent metabolism of the primary metabolites of YM-64227, with the exception of MM-2. It is possible that the interindividual variability of YM-64227 with respect to the CYP1A2 deficiency polymorphism is caused by a decrease in the metabolic activities of both the unchanged drug and its metabolites. 4-) is a novel phosphodiesterase type 4 inhibitor. Unlike other phosphodiesterase type 4 inhibitors, YM-64227 does not induce the emetic effects that normally accompany the anti-inflammatory activity. In addition, it maintains the ability to inhibit antigen-induced airway responses. For this reason, YM-64227 promises to be a highly effective and safe drug when used as an antiasthmatic or antiinflammatory drug (Iwata et al., 1998).In previous studies, a novel CYP1A2 1117 CϾT single nucleotide polymorphism (SNP) was found in beagle dogs, which are used extensively in pharmacology and safety assessment studies (Tenmizu et al., 2004a;Mise et al., 2004a). This SNP resulted in an amino acid change from an Arg codon to a stop codon at position 373, yielding an inactive enzyme. The T-allele frequency was 0.39, which suggests that 10 to 15% of the dogs would not express the CYP1A2 protein. This CYP1A2 deficiency polymorphism causes significant changes in the pharmacokinetic profile of YM-64227 in beagle dogs (Tenmizu et al., 2006). The plasma concentrations of the unchanged drug and five of its metabolites (MM-1 to MM-5, Fig. 1) were determined after both intravenous and oral administration of YM-64227 to genotyped dogs (C/C, C/T, and T/T groups). After a single oral administration, the maximum plasma concentration and absolute bioavailability of YM-64227 in ...

We developed and validated a reversed-phase high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of YM-64227 [4-cyclohexyl-1-ethyl-7-methylpyrido(2,3-d)pyrimidin-2-(1H)-one], a novel and selective phosphodiesterase type 4 inhibitor, and its fi ve hydroxylated metabolites in dog plasma. The plasma samples were extracted with tert-butyl methyl ether under alkali conditions. The analytes were well separated on a phenyl ethyl column (5 microm, 250 x 4.6 mm i.d.), opreating at 40 degrees C and using an acetonitrile-acetic acid gradient at a fl ow rate of 1.0 mL/min. The fluorescence signal was monitored at an excitation and emission wavelength of 330 and 400 nm, respectively. No interfering peak was observed at the retention time of YM-64227, its metabolites or the internal standard. The validated quantitation range of the method was 0.4-200 ng/mL for all analytes using 0.5 mL of the plasma sample. The recovery of analytes in the extraction process was more than 65.5%. The intra- and inter-assay precision was less than 5.1 and 12.6%, respectively, and the intra- and inter-assay accuracy ranged from -8.1 to 11.8% and -8.0 to 9.9%, respectively. Using this assay, the plasma concentration of YM-64227 and metabolites can be determined after the oral administration of YM-64227 to beagle dogs.

UGT2B7 is the main catalyst of M11 formation in HLMs. Regarding M13 and M14 formation, UGT1A3 and UGT1A8 are strong candidates for glucuronidation, respectively.