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Elucidation of the Effects of the CYP1A2 Deficiency Polymorphism in the Metabolism of 4-Cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]pyrimidine-2-(1H)-one (YM-64227), a Phosphodiesterase Type 4 Inhibitor, and Its Metabolites in Dogs

Drug Metabolism Research Laboratories, Astellas Pharma Inc., Tokyo, Japan

(Received May 27, 2006; accepted July 26, 2006)

	Abstract

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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.

In previous studies, a novel CYP1A2 1117 C>T 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 the T/T group were higher than those in the C/C and C/T groups; however, the pharmacokinetics of YM-64227 after an intravenous administration were not affected by genotype. These findings suggest that the clearance of YM-64227 in dogs is hepatic blood flow-limited, and the CYP1A2 deficiency mainly affects first pass metabolism. The metabolic profiles in the T/T group were quite distinct from the others. In the C/C and C/T groups, MM-2 had the highest AUC among the metabolites, and the AUCs of MM-1, MM-3, MM-4, and MM-5 were much lower than that of MM-2. In the T/T group, the AUCs of MM-1, MM-4, and MM-5 were significantly higher than those in the C/C group.

View larger version (13K): [in this window] [in a new window]   FIG. 1. The chemical structures of YM-64227 and its metabolites.

The purpose of this study is to determine the contribution of CYP1A2 to the metabolism of YM-64227 and its metabolites, as well as to elucidate the causes of interindividual variability in the pharmacokinetics of YM-64227 in dogs with respect to the CYP1A2 deficiency polymorphism.

	Materials and Methods

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Chemicals. YM-64227, its five metabolites (MM-1to MM-5), and 4-(3-chlorophenyl)-1-ethyl-7-(1-hydroxyethyl)pyrido[2,3-d]pyrimidin-2(1H)-one, the internal standard for HPLC analysis, were synthesized at the Chemistry Research Laboratories of Astellas Pharma Inc. (Ibaraki, Japan). -Naphthoflavone and acetaminophen were purchased from Nacalai Tesque, Inc. (Kyoto, Japan), and m-acetaminophen was purchased from Aldrich Chemical (Milwaukee, WI). Phenacetin was purchased from Wako Pure Chemical (Osaka, Japan). NADPH-generating system solutions A and B were purchased from BD (San Jose, CA), and NADPH was purchased from Roche (Mannheim, Germany). Human anti-CYP1A1/2 antibody was purchased from Nihon Nosan Kogyo Co. (Kanagawa, Japan). Purified water obtained from a Milli-Q system (Millipore Corp., Bedford, MA) was used throughout the study. All other chemicals used were commercially available and of the highest purity.

Effect of -Naphthoflavone on the Metabolism of YM-64227. The canine liver microsomes were prepared and the CYP1A2 1117 C>T SNP genotyping method was performed as described by Tenmizu et al. (2004a). YM-64227 (5 µM) was incubated in a reaction mixture (0.5 ml) consisting of 0.1 mg/ml liver microsomes, a NADPH-generating system (1.3 mM NADP, 3.3 mM glucose 6-phosphate, 0.4 U/ml glucose-6-phosphate dehydrogenase, and 3.3 mM MgCl₂), 0.1 mM EDTA, and 100 mM Na/K-phosphate buffer (pH 7.4). Pooled liver microsomes prepared from five individuals for each group genotyped as C/C, C/T, and T/T type were used. -Naphthoflavone concentrations were 1 and 10 µM. Experiments were performed in duplicate. Enzyme reactions were initiated by adding 30 µl of NADPH-generating system. The reaction was terminated after incubating at 37°C in a shaking water bath for 10 min by adding 4 ml of tert-butyl methyl ether. After stopping the metabolic reaction, the concentrations of the metabolites were quantified using HPLC (Tenmizu et al., 2004b). In brief, after the addition of the internal standards (1.5 µM), the samples were extracted with tert-butyl methyl ether for purification and enrichment. Separation was achieved on a Cosmosil-packed phenyl ethyl column (5 µm, 250 mm x 4.6 mm i.d., Nacalai Tesque, Inc.). Mobile phases A, consisting of 50 mM acetic acid and acetonitrile (80:20), and B, consisting of 50 mM acetic acid and acetonitrile (20:80), were used for gradient elution. The metabolites of YM-64227 were detected fluorimetrically at 400 nm with emission at 330 nm.

Immunoinhibition of the Metabolism of YM-64227. Immunoinhibition of the metabolism of YM-64227 was examined by preincubating 0.05 mg of pooled liver microsomes with various concentrations of human anti-CYP1A1/2 antibody at room temperature for 30 min. The other reaction conditions were the same as those described above ("Effect of -Naphthoflavone on the Metabolism of YM-64227").

Metabolism of Phenacetin. Phenacetin was incubated in a reaction mixture (0.5 ml) consisting of 0.1 mg/ml liver microsomes, 1 mM NADPH, 0.1 mM EDTA, and 100 mM Na/K-phosphate buffer (pH 7.4). Phenacetin concentrations of 1 to 500 µM were used to estimate the kinetic parameters. Individual canine liver microsomes (n = 5 per each group genotyped as C/C, C/T, and T/T types) were used. Experiments were performed in duplicate. Enzyme reactions were initiated by adding 50 µl of 10 mM NADPH. After incubating at 37°C in a shaking water bath for 10 min, the reaction was terminated by adding 5 ml of tert-butyl methyl ether. After stopping the metabolic reaction, 1 µM m-acetaminophen (internal standard) was added. After liquid extraction, the concentration of acetaminophen was quantified using an HPLC-UV method. A Chromolith SpeedROD, RP-18e (4.6 x 50 mm; Merck KGoA, Darmstadt, Germany) was used for the sample trap. Analysis was performed on a TSK gel ODS 80Ts (5 µm, 4.6 x 250 mm; Tosoh Co., Tokyo, Japan). A column switch (Campins-Falco et al., 1993; Fried and Wainer, 1997) with a minor modification was used to prevent the late-eluting substrate from interfering with the analysis. The mobile phase was a mixture of 0.5 M phosphoric acid, acetonitrile, and water (10:10:80). Acetaminophen and the internal standard were detected at 250 nm.

The kinetic data for phenacetin metabolism obtained with liver microsomes were fitted to eq. 1 using the SAS system version 8.2 (SAS Institute, Cary, NC) to estimate K_m, V_max, and CL_ns. The CL_{int, in vitro} values were calculated using eq. 2.

(1)

(2)

Metabolism of the Primary Metabolites of YM-64227. YM-64227 primary metabolites (MM-1 to MM-5, 0.7 µM) were incubated in a reaction mixture (0.5 ml) consisting of liver microsomes (2.0 mg/ml for MM-1 and 0.5 mg/ml for the other metabolites), a NADPH-generating system, 0.1 mM EDTA, and 100 mM Na/K-phosphate buffer (pH 7.4). Pooled liver microsomes for each genotype were used. Experiments were performed in duplicate. The enzyme reactions were initiated by adding 30 µl of the NADPH-generating system. The reaction was terminated after incubating at 37°C in a shaking water bath for 15, 30, 45, and 60 min by adding 4 ml of tert-butyl methyl ether. The assay method of YM-64227 metabolites was the same as described above ("Effect of -Naphthoflavone on the Metabolism of YM-64227"). The rate of metabolism for the primary YM-64227 metabolites using liver microsomes is described by eq. 3.

(3)

X(t) (ng/mg protein) is the sum of the total amounts of decreased substrate, and CL_{int, in vitro} (µl/min/mg protein) is the overall intrinsic metabolic clearance as defined in comparison with the intact substrate in the reaction mixture [C(t) (ng/ml)]. Integration of eq. 3 yields eq. 4.

(4)

AUC_0-t (ng · min/ml) represents the area under the concentration-time curve from time 0 to t for substrate in the mixture. The CL_{int, in vitro} value can be obtained from the slope of a plot of X(t) versus AUC_0-t (Iwatsubo et al., 1999).

Immunoinhibition of the Metabolism of the Primary Metabolites of YM-64227. Immunoinhibition of the primary metabolites of YM-64227 was examined by preincubating liver microsomes (1.0 mg for MM-1 and 0.25 mg for the other metabolites) with 50 µl of human anti-CYP1A1/2 antibody at room temperature for 30 min. The reaction conditions were the same as described above ("Metabolism of the Primary Metabolites of YM-64227") except that the samples were incubated for 60 min.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Effects of -naphthoflavone on the formation of YM-64227 metabolites (MM-1 to MM-5) in genotyped liver microsomes (C/C, C/T, and T/T type). YM-64227 (5.0 µM) was incubated with pooled microsomes in the presence of 1 and 10 µM -naphthoflavone. The values represent the average of duplicate determinations.

	Results

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View larger version (13K): [in this window] [in a new window]   FIG. 3. Immunoinhibition of YM-64227 by anti-CYP1A1/2 antibody in genotyped liver microsomes. YM-64227 (5.0 µM) was incubated with pooled microsomes in the presence of anti-CYP1A1/2 antibodies. The values represent the average of duplicate determinations.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Michaelis-Menten plots and Eadie-Hofstee plots for POD in genotyped liver microsomes (n = 5, mean ± S.D.). (), C/C-type microsomes; , C/T-type microsomes; , T/T-type microsomes.

Metabolism of Phenacetin. The Michaelis-Menten and Eadie-Hofstee plots of phenacetin O-deethylation (POD), a CYP1A2 activity marker substrate, are shown in Fig. 4. Since the Eadie-Hofstee plots suggested the contribution of multiple components, the following three models were compared: 1) one saturable component and one nonsaturable component, 2) two saturable components, and 3) two saturable components and one nonsaturable component. Evaluation of fit was carried out using the Akaike information criterion value (Akaike, 1969). Model 1 showed the best fit and was therefore selected for parameter calculation (Table 1). The V_max, CL_{int, s}, and CL_{int, in vitro} values for the T/T type were lower than those for the C/C type by 24, 41, and 43%, respectively. The CL_{int, ns} value did not differ significantly among the three types. The correlation between YM-64227 hydroxylation and POD are shown in Table 2 and Fig. 5. The CL_{int, in vitro} value for MM-2 formation, MM-3 formation, and the overall metabolism of YM-64227 correlated significantly with that of POD (p < 0.01).

View larger version (16K): [in this window] [in a new window]   FIG. 5. Correlation between overall YM-64227 CLint, in vitro and phenacetin CLint, in vitro. , C/C-type microsomes (n = 5); , C/T-type microsomes (n = 5); , T/T-type microsomes (n = 5).

Metabolism of the Primary Metabolites of YM-64227. The CL_{int, in vitro} values for the metabolism of MM-1, MM-2, MM-3, MM-4, and MM-5 in the T/T type were lower than that in the C/C type by 50, 73, 30, 41, and 34%, respectively (Table 3). The C/T type had CL_{int, in vitro} values similar to, to slightly higher than the C/C type for the metabolism of these metabolites.

Immunoinhibition of the Metabolism of the Primary Metabolites of YM-64227. In the C/C- and C/T-type microsomes, anti-CYP1A1/2 antibody inhibited the metabolism of MM-1, MM-3, MM-4, and MM-5 by approximately 40% of the control values (Fig. 6). The metabolic activity of MM-2 in the C/C and C/T types were not affected by anti-CYP1A1/2 antibody. In the T/T type, no inhibition of metabolism was observed at all for MM-2, MM-3, MM-4, or MM-5 in the presence of anti-CYP1A1/2 antibody. In fact, the addition of anti-CYP1A1/2 antibody increased the metabolic activity of MM-1 in the T/T group to 220% of the control.

View larger version (16K): [in this window] [in a new window]   FIG. 6. Immunoinhibition of the YM-64227 metabolites by anti-CYP1A1/2 antibody in genotyped liver microsomes. The YM-64227 metabolites (0.7 µM) were incubated with pooled microsomes in the presence of anti-CYP1A1/2 antibodies. The values represent the average of duplicate determinations.

	Discussion

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The formation rates for MM-2 and MM-3 in the T/T type were lower than those in the C/C and C/T types, whereas the formation rates of the other metabolites (MM-1, MM-4, and MM-5) were not significantly different among the three. These findings suggested that CYP1A2 plays a significant role in the formation of MM-2 and MM-3, but not in the formation of others. -Naphthoflavone, a typical CYP1A2 inhibitor (Chang et al., 1994; Mise et al., 2004b), reduced the MM-2 and MM-3 formation rates in the C/C and C/T types, but no inhibitory effects were observed in the T/T type. Thus, the metabolic activities of MM-2 and MM-3 in the presence of -naphthoflavone were comparable in the three types. The formation rates of MM-1, MM-4, and MM-5 were not inhibited by -naphthoflavone, and activation was detectable in the formation of all MM-1 types. -Naphthoflavone is a known activator of CYP3A-mediated activities (Zhao and Ishizaki, 1997; Nakajima et al., 1999), and the activation of MM-1 formation suggests the possibility that this metabolic pathway is catalyzed by homologs of CYP3A4. Although this does not fully explain the reason why MM-1 formation is activated, previous inhibition studies using -naphthoflavone suggest that CYP1A2 contributes to the formation of MM-2 and MM-3.

Results obtained from immunoinhibition studies on the C/C and C/T types support this theory. Anti-CYP1A1/2 antibody inhibited the formation of MM-2 and MM-3 without affecting the formation of other metabolites in the C/C and C/T types. No inhibitory effects caused by the anti-CYP1A1/2 antibody were detected in the T/T type. Although the direct evidence for specificity of antibodies used in this study is not confirmed, specific inhibitory effects are promising for canine CYP1A2 because of the high homology of amino acid sequence of human and canine CYP1A2. This expectation is reinforced by the similarity in inhibitory effects of anti-human CYP1A2 antibodies and -naphthoflavone, a CYP1A2 inhibitor, on the metabolism of YM-64227.

POD is known to be a human and canine CYP1A2 activity marker (Kobayashi et al., 1997; Shimada et al., 1997), and -naphthoflavone inhibits POD both in canine and human liver microsomes (Nakajima et al., 1999; Mise et al., 2004b). As is shown in Fig. 4, POD exhibited biphasic kinetics in both canine and human liver microsomes (Kobayashi et al., 1997). The T/T-type microsomes had a lower CL_{int, s} value than the C/C and C/T types, whereas the same CL_{int, ns} values were obtained for the three types. The fact that the T/T type lacks active CYP1A2 protein suggests that canine CYP1A2 is involved with the saturable component of POD, just as it is in human CYP1A2. The CL_{int, in vitro} values of POD significantly correlated with those of MM-2 and MM-3. Interestingly, a significant correlation was also observed between the POD CL_{int, in vitro} value and that of YM-64227 metabolism overall. This finding suggests that breakdown into MM-2 and MM-3 is the main way YM-642247 is eliminated. This finding is consistent with the fact that the plasma concentrations of the unchanged drug were significantly higher in the T/T type than in the C/C or C/T types.

It is known that the primary metabolites of YM-64227 (MM-1 to MM-5) undergo oxidative metabolism. The chemical structures of the further metabolites are unknown and, consequently, the CL_{int, in vitro} values for these metabolites were estimated by measuring the disappearance rate of the substrates. In a preliminary experiment, we found that the metabolic activities of these metabolites varied among individual microsomes. Some microsomes had very slow metabolic activities, and it was therefore difficult to calculate CL_{int, in vitro} accurately. Thus, pooled microsomes were used to study the metabolism of the YM-64227 metabolites. The CL_{int, in vitro} values for the metabolism of MM-1, MM-3, MM-4, and MM-5 in the T/T type were lower than those in the C/C type by less than 50%. Meanwhile, the CL_{int, in vitro} value for the metabolism of MM-2 in the T/T type was 73% of that in the C/C type. Anti-CYP1A1/2 antibody decreased the metabolic activities of MM-1, MM-3, MM-4, and MM-5 in the C/C and C/T types to less than 60% of the control values, but the metabolic activities of MM-2 were not affected by anti-CYP1A1/2 antibody. None of the metabolic activities of the primary metabolites of YM-64227 in the T/T type were inhibited by anti-CYP1A1/2 antibody. These metabolism studies suggested that CYP1A2 is involved in the metabolism of MM-1, MM-3, MM-4, and MM5, whereas MM-2 is metabolized by other P450 isozymes.

View larger version (25K): [in this window] [in a new window]   FIG. 7. Summary of the YM-64227 metabolic pathways with respect to the CYP1A2 deficiency polymorphism.

The pharmacokinetic profiles of drugs are affected by several factors, such as the induction and inhibition of metabolizing enzymes caused by drugs taken concomitantly, as well as genetic polymorphism. The effects of these factors on pharmacokinetics of metabolites need to be investigated, especially if the metabolites of the drugs are pharmacologically active or potentially toxic. In such cases, evaluation of the involvement of the P450 isozyme in both the formation and elimination of the metabolites can be very informative. Poor metabolizers of CYP2C9, 2C19, and 2D6 result in higher plasma concentrations of tolbutamide (Sullivan-Klose et al., 1996), debrisoquine (Kagimoto et al., 1990), and omeprazole (Furuta et al., 1996), respectively, compared with extensive metabolizers. Concurrent with an increased amount of the unchanged form of these drugs, the concentrations of their metabolites, hydroxy tolbutamide, 4-hydoxy debrisoquine, and 5-hydroxyomeprazole, and omeprazole sulfone, were all lower in poor metabolizers. These findings may be explained by the fact that P450 isozymes contribute to the metabolism of the parent drugs and do not eliminate their primary metabolites. In contrast, the dose-corrected steady-state plasma concentrations of mesoridazine, a primary metabolite of thioridazine, was not affected by the genetic polymorphism of CYP2D6, although the formation of mesoridazine that results from the metabolism of thioridazine is catalyzed by CYP2D6 (Berecz et al., 2003; Wojcikowski et al., 2006). CYP2D6 contributes to the metabolism of mesoridazine as well (Wojcikowski et al., 2006); therefore, both the formation and elimination of mesoridazine is regulated by CYP2D6. The decrease in the formation of mesoridazine is accompanied by a decrease in elimination; therefore, the resulting changes in the plasma concentration level are insignificant.

The contribution of CYP1A2 to the formation and elimination of the primary metabolites of YM-64227 in canine liver microsomes was investigated. The involvement of CYP1A2 in both the formation and elimination of the metabolites of YM-64227 account for the effects of the CYP1A2 deficiency polymorphism on the plasma concentration profiles in dogs. This finding is valuable as it may explain the interindividual variability seen in the metabolism of several CYP1A2 substrates in dogs.

	Acknowledgments

 
We acknowledge Shigeo Suzuki for very valuable technical assistance while conducting this study.

	Footnotes

 
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.011213.

ABBREVIATIONS: YM-64227, 4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]pyrimidine-2-(1H)-one; AUC, area under the concentration-time curve; CL_{int, in vitro}, overall intrinsic metabolic clearance estimated from the in vitro study; CL_{int, s}, intrinsic metabolic clearance of the saturable component; CL_{int, ns}, intrinsic metabolic clearance of the nonsaturable component; POD, phenacetin O-deethylation; SNP, single nucleotide polymorphism; HPLC, high-performance liquid chromatography; P450, cytochrome P450.

Address correspondence to: Daisuke Tenmizu, Drug Metabolism Research Laboratories, Astellas Pharma Inc., 1-8, Azusawa 1-Chome, Itabashi-ku, Tokyo 174-8511, Japan. E-mail: daisuke.tenmizu{at}jp.astellas.com

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