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POLYMORPHIC EXPRESSION OF CYP1A2 LEADING TO INTERINDIVIDUAL VARIABILITY IN METABOLISM OF A NOVEL BENZODIAZEPINE RECEPTOR PARTIAL INVERSE AGONIST IN DOGS

Pharmacokinetics and Physico-Chemical Property Research Laboratories (M.Mi., M.Ma., T.H., S.M., Y.T., T.F) and Safety Research Laboratories (S.Y.), Dainippon Pharmaceutical Co., Ltd., Osaka, Japan

(Received August 5, 2003; Accepted October 31, 2003)

	Abstract

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5-(3-Methoxyphenyl)-3-(5-methyl-1,2,4-oxadiazol-3-yl)-2-oxo-1,2-dihydro-1,6-naphthyridine (AC-3933) is a novel cognitive enhancer with central benzodiazepine receptor partial inverse agonistic activity. AC-3933 is predominantly metabolized to hydroxylated metabolite [SX-5745; 3-(5-hydroxymethyl-1,2,4-oxadiazol-3-yl)-5-(3-methoxyphenyl)-2-oxo-1,2-dihydro-1,6-naphthyridine] in dog. Initially, we found that there is considerable interindividual variability in AC-3933 hydroxylation in dogs and that dogs could be phenotyped as extensive metabolizer (EM) and poor metabolizer (PM). Then, to clarify the cause of AC-3933 polymorphic hydroxylation in dogs, in vitro studies were carried out using liver microsomes from EM and PM dogs. Our results show that AC-3933 hydroxylation clearance in PM dogs was much lower than that in EM dogs (0.2 versus 10.8-20.5 µl/min/mg, respectively). In addition, AC-3933 hydroxylation was significantly inhibited by -naphthoflavone, a CYP1A inhibitor, and by anti-CYP1A2 antibodies, indicating that CYP1A2 was responsible for the polymorphic hydroxylation of AC-3933 in dogs. Furthermore, immunoblotting results have shown that although CYP1A2 protein was not detected in PM dogs (<0.86 pmol/mg), CYP1A2 content in EM dogs was prominent (6.1-13.0 pmol/mg). These results indicate that AC-3933 polymorphic hydroxylation arises from the polymorphic expression of CYP1A2 in dogs, which might involve genetic polymorphism of the CYP1A2 gene.

Cytochrome P450 (P450¹) plays a decisive role in the oxidative metabolism of xenobiotics and endogenous substances (Rendic and Di Carlo, 1997). In humans, many genetic polymorphisms of P450 have been reported, and some of them are considered important factors for interindividual variability of drug metabolism and pharmacokinetics (http://www.imm.ki.se/CYPalleles/). On the other hand, in dogs several P450s have been cloned and sequenced, including CYP1A1/2 (Uchida et al., 1990), CYP2B11 (Graves et al., 1990), CYP2C21/41 (Uchida et al., 1990; Blaisdell et al., 1998), CYP2D15 (Sakamoto et al., 1995), CYP2E1 (Lankford et al., 2000), and CYP3A12/26 (Ciaccio et al., 1991; Fraser et al., 1997). However, the contribution of these P450s to the interindividual variability of pharmacokinetics in dogs is unknown.

5-(3-Methoxyphenyl)-3-(5-methyl-1,2,4-oxadiazol-3-yl)-2-oxo-1,2-dihydro-1,6-naphthyridine (AC-3933) is a novel cognitive enhancer with central benzodiazepine receptor partial inverse agonistic activity. The mechanism of AC-3933's memory-improving action is based on enhancement of the cholinergic function through the allosteric reduction of -aminobutyric acid activity. In dogs, AC-3933 is metabolized to a major hydroxylated metabolite (SX-5745) and a minor demethylated metabolite (SX-5773), and subsequently, SX-5745 is reductively metabolized to SX-6088 (Fig. 1). This report describes a polymorphism in AC-3933 pharmacokinetics in dogs, and we investigated the cause of the polymorphism using in vitro experiments.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Metabolic pathways of AC-3933.

	Materials and Methods

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Chemicals. [¹⁴C]AC-3933 (5-(3-methoxyphenyl)-3-(5-methyl-1,2,4-oxadiazol-3-yl)-2-oxo-1,2-dihydro-[7-¹⁴C]-1,6-naphthyridine) and AC-3933 were synthesized at Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan). The specific activity of [¹⁴C]AC-3933 was 3.99 MBq/mg, and radiochemical purity was >99%. Ethoxyresorfin, 7-ethoxycoumarin, and aminopyrine were purchased from Sigma-Aldrich (St. Louis, MO). Phenacetin and quinidine were purchased from Wako Pure Chemicals (Osaka, Japan). -Naphthoflavone was purchased from Nacalai Tesque (Kyoto, Japan). Sulfaphenazole and ketoconazole were purchased from Ultrafine Ltd. (Manchester, UK). Polyclonal anti-P450 antibodies were purchased from Daiichi Pure Chemicals Co., Ltd. (Tokyo, Japan). All other regents were of the highest grade commercially available.

Animals and Treatment. Beagle dogs were obtained from Covance Research Products Inc. (Kalamazoo, MI). AC-3933 at a dose of 100 mg/kg was orally administered to dogs (7-10 kg). Blood samples were collected from the cephalic vein with a heparinized syringe at 0.5, 1, 3, 6, and 24 h after AC-3933 administration, and the plasma was separated by centrifugation at 900g for 10 min. The plasma samples were stored at -20°C until use.

Quantification of AC-3933 and Its Metabolites in Dog Plasma. An aliquot of the separated plasma (0.2 ml) was diluted with 0.5 ml of 0.2 M acetic acid buffer (pH 4.0) and loaded onto a solid-phase extraction cartridge (Oasis HLB 30 mg/1 ml; Waters, Milford, MA). After washing the cartridge with 10% methanol, AC-3933 and its metabolites were eluted by 1 ml of methanol, and the eluate was evaporated to dryness. The residue was dissolved in 400 µl of the mobile phase, and an aliquot of the solution was analyzed by liquid chromatography-tandem mass spectrometry. Liquid chromatography-tandem mass spectrometry analysis was performed using a system consisting of a 2690 separation module (Waters) and a TSQ 7000 mass spectrometer (Thermo Finnigan, San Jose, CA). The separation was carried out on a reverse-phase column (Inertsil ODS-3, 2.1 x 150 mm; GL Science Inc., Tokyo, Japan) at 40°C with a flow rate of 0.15 ml/min. The mobile phase consisted of solvent A (0.05% formic acid), solvent B (acetonitrile), and solvent C (methanol), and the gradient used was as follows: 80% solvent A-10% solvent B-10% solvent C (0 min); 2%-49%-49% (0.1-9 min); 0%-0%-100% (9.01-15 min); and 80%-10%-10% (15.01-20 min). The mass spectrometer was operated in a positive electrospray ionization mode at the capillary temperature of 350°C and the spray voltage of 4.5 kV. Multiple reactions-monitoring analysis was performed with the transition of m/z 334.9 293.0 for AC-3933, m/z 350.9 293.0 for SX-5745, m/z 320.9 279.0 for SX-5773, and m/z 296.9 279.0 for SX-6088. This analytical method was validated for the specificity, linearity, and reproducibility.

Pharmacokinetic parameters in plasma for AC-3933 and its metabolites were analyzed by noncompartmental modeling using WinNonlin Professional version 4.0.1 (Pharsight, Mountain View, CA). Pharmacokinetic parameters determined for EM and PM dogs were compared by t test analysis using the SAS system version 8.02 (SAS Institute, Cary, NC).

Phenotyping. For phenotyping of dogs, we used the C_3h ratio of SX-5745 to AC-3933 after oral administration of 25 mg/kg AC-3933. After quantification of AC-3933 and SX-5745, dogs with a logarithmically transformed ratio less than zero were considered PM dogs, other dogs were considered EM dogs.

Plasma samples from 20 dogs 3 h after administration of 25 mg/kg of AC-3933 were obtained as above. An aliquot of the separated plasma (0.5 ml) was diluted with 1.5 ml of 0.1 M phosphate buffer (pH 7.4) and loaded onto the Oasis HLB 60 mg/3 ml. After washing the cartridge with 40% methanol, AC-3933 and SX-5745 were eluted by 2 ml of methanol, and the eluate was evaporated to dryness. The residue was dissolved in 50 µl of the mobile phase, and an aliquot of the solution was analyzed by HPLC (model 1090 system; Agilent Technologies, Palo Alto, CA) using a reverse-phase column (Inertsil ODS-3V, 4.6 x 250 mm) at 40°C with a flow rate of 1.0 ml/min. The mobile phase consisted of solvent A (0.05% trifluoroacetic acid) and solvent B (acetonitrile), and the gradient used was as follows: 17% solvent B (0-6 min); 17 to 40% (6-16 min); 40 to 90% (16-19 min); 90 to 17% (19-21 min); and 17% (21-25 min). The wavelength of the detector was 330 nm. With this assay method, accurate determination was possible over the concentration range of 25 to 10,000 ng/ml of AC-3933 and SX-5745.

Preparation of Dog Liver Microsomes. Livers were obtained from two EM dogs (EM1 and EM2) and two PM dogs (PM1 and PM2). Each liver was homogenized in 3 volumes of 1.15% potassium chloride/10 mM potassium phosphate buffer (pH 7.4)/0.1 mM EDTA. The homogenate was centrifuged at 9000g for 20 min, and the supernatant was further centrifuged at 105,000g for 60 min. The pellet was resuspended in 0.1 M potassium phosphate buffer (pH 7.4), and centrifuged at 105,000g for 60 min. The resulting pellet was resuspended in 50 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol and 0.1 mM EDTA and stored at -80°C until use. Microsomal protein content was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.

In Vitro Metabolism of AC-3933. [¹⁴C]AC-3933 (2.5, 5, 10, 20, 30, 40, and 50 µM) was incubated for 10 min at 37°C in 500 µl of a reaction mixture consisting of 50 mM phosphate buffer (pH 7.4), liver microsomes, and 0.8 mM NADPH. The reaction was started by adding NADPH and stopped by adding 1.5 ml of acetonitrile. After centrifugation at 900g for 10 min, the supernatant was evaporated to dryness, and the residue was dissolved in the mobile phase for HPLC analysis. HPLC was operated under the same conditions as above. AC-3933 and its metabolites (SX-5745 and SX-5773) were quantified by radioactivity using a scintillation cocktail (Ultima Flo-M, 2 ml/min; PerkinElmer Life and Analytical Sciences, Boston, MA) and a flow-scintillation detector (FLO-ONE/Beta A-515; PerkinElmer Life and Analytical Sciences). Assays were performed in triplicate.

Kinetic parameters were obtained by fitting metabolic velocity-concentration data into eq. 1 for hydroxylation (SX-5745 formation) or eq. 2 for demethylation (SX-5773 formation) using the nonlinear least-squares method and analysis software, Origin (OriginLab Corp., Northampton, MA):

(1)

(2)

where V is metabolic velocity; S, substrate concentration; V_max, maximum reaction velocity; and K_m, Michaelis-Menten constant. The intrinsic clearance (CL_int) for hydroxylation was obtained from V_max/K_m.

Identification of P450 Isozyme Responsible for AC-3933 Hydroxylation. To identify the P450 isozyme responsible for AC-3933 hydroxylation, chemical inhibition and immunoinhibition experiments were performed. The assay procedure was essentially the same as that described under In Vitro Metabolism of AC-3933, except that the AC-3933 concentration was 10 µM. For the chemical inhibition experiment, the following P450 inhibitors were used: 10 µM -naphthoflavone as a CYP1A inhibitor, 10 µM sulfaphenazole as a CYP2C inhibitor, 1 µM quinidine as a CYP2D inhibitor, and 0.5 µM ketoconazole as a CYP3A inhibitor (Roussel et al., 1998; Tasaki et al., 1998; Bogaards et al., 2000). Each inhibitor was incubated in a reaction mixture containing pooled EM dog liver microsomes. For the immunoinhibition experiment, polyclonal anti-P450 antibodies against rat CYP1A1, CYP1A2, CYP2B1, CYP2C11, and CYP3A2 were used. Before the reaction was started, antibodies (0.43-2.15 mg of IgG/mg of microsomal protein) or corresponding preimmune sera were preincubated with pooled EM dog liver microsomes for 30 min at room temperature.

Measurement of Activity of P450s. Activities of EROD and POD, both of which reflect CYP1A2 activity (Chauret et al., 1997; Graham et al., 2002), and activities of ECOD and APND, both of which reflect CYP2B and/or CYP3A activity (Nishibe and Hirata, 1995; Mae et al., 1998; Nakata et al., 2000), were determined. A reaction mixture consisting of 50 mM phosphate buffer (pH 7.4), 0.8 mM NADPH, EM or PM dog liver microsomes, and a substrate [ethoxyresorufin (0.01 mM), phenacetin (0.1 mM), ethoxycoumarin (0.1 mM), and aminopyrine (5 mM)] was incubated at 37°C for 15 min. EROD and ECOD activity was measured according to the method of Matsubara et al. (1983a,b). APND activity was measured according to the method of Nash (1953). POD activity was measured as follows: the reaction was started by adding NADPH and stopped by adding 1.5 ml of acetonitrile and 100 µl of antipyrine (10 µM) as internal standard. After centrifugation at 900g for 10 min, the supernatant was separated and evaporated to dryness, and the residue was dissolved in the mobile phase for HPLC analysis. HPLC analysis was performed under the same conditions as above. The mobile phase consisted of solvent A (0.05% trifluoroacetic acid) and solvent B (acetonitrile) and the gradient used was as follows: 10 to 50% solvent B (0-20 min); 50 to 90% (20-21 min); 90 to 10% (21-22 min); and 10% (22-25 min). The wavelength of the detector was 245 nm. For the inhibition experiment of POD, 10 µM -naphthoflavone was incubated in a reaction mixture containing PM and EM dog liver microsomes.

Immunoblotting. EM and PM dog liver microsomal proteins (5 µg) were separated by SDS-polyacrylamide gel electrophoresis and transferred electrically to a polyvinylidene difluoride membrane (Laemmli, 1970; Towbin et al., 1979). Polyclonal goat anti-CYP1A, -CYP2B, -CYP2C, and -CYP3A antibodies were used as primary antibodies. Bands that reacted with the primary antibodies were visualized using alkaline phosphatase-conjugated anti-goat IgG antibodies. CYP1A, CYP2B, CYP2C, and CYP3A content in EM and PM dog liver microsomes was measured using standard dog microsomes obtained from Daiichi Pure Chemicals Co., Ltd. Correlation coefficients of standard curves for CYP1A, CYP2B, CYP2C, and CYP3A were 0.980 (0.86-13.7 pmol/mg), 0.989 (3.28-52.5 pmol/mg), 0.942 (2.83-45.2 pmol/mg), and 0.957 (3.48-55.6 pmol/mg), respectively.

	Results

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In Vivo Pharmacokinetics. The concentrations of AC-3933, SX-5745, SX-5773, and SX-6088 in dog plasma after oral administration of 100 mg/kg AC-3933 are shown in Fig. 2, and the pharmacokinetic parameters are summarized in Table 1. There was considerable interindividual variability in AC-3933 pharmacokinetics, and the dogs could be divided into two groups. In one group designated EM, C_max and AUC_0-24 of SX-5745 were the same or higher than those of AC-3933 (Fig. 2A). In the other group designated PM, in contrast, C_max and AUC_0-24 of SX-5745 were much lower than those of AC-3933 (Fig. 2B). However, C_max ratio and AUC_0-24 ratio of SX-6088 (a subsequent metabolite from SX-5745) to SX-5745 in EM dogs (0.341 ± 0.303 and 0.336 ± 0.297, respectively) were not significantly different from those in PM dogs (0.201 ± 0.045 and 0.224 ± 0.019, respectively). On the other hand, although C_max and AUC_0-24 of SX-5773, the other primary metabolite, in PM dogs were significantly higher than those in EM dogs, there was no significant difference in the C_max ratio and AUC_0-24 ratio of SX-5773 to AC-3933 between EM and PM dogs. These results indicated that the difference in pharmacokinetics of AC-3933 and SX-5745 between EM and PM dogs is due to the difference in AC-3933 hydroxylation, but not SX-5745 metabolism and not AC-3933 demethylation. In addition, C_max ratio and AUC_0-24 ratio of SX-5745 to AC-3933 were significantly different between EM and PM dogs, indicating that those parameters reflect the phenotype. C_3h ratio of SX-5745 to AC-3933 was also significantly different between EM and PM dogs, and we decided that C_3h ratio could be used for phenotyping instead of C_max ratio and AUC_0-24 ratio.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Concentrations of AC-3933 and its metabolites in EM (A) and PM (B) dog plasma after oral administration of 100 mg/kg AC-3933. Concentrations of AC-3933 (), SX-5745 (), SX-5773 (), and SX-6088 () were measured in plasma at 0.5, 1, 3, 6, and 24 h after oral administration of 100 mg/kg of AC-3933 in dogs. Each point represents the mean ± S.E. of nine and three dogs for EM and PM, respectively.

Phenotyping. To obtain liver samples from EM and PM dogs, phenotyping test were carried out in 20 dogs using C_3h ratio of SX-5745 to AC-3933. As the histogram of logarithmically transformed C_3h ratio showed biphasic distribution (Fig. 3), the dogs could be phenotyped as EM (n = 18) and PM (n = 2).

View larger version (11K): [in this window] [in a new window]   FIG. 3. Phenotyping for AC-3933 hydroxylation in 20 dogs. AC-3933 and SX-5745 concentrations were measured in plasma 3 h after oral administration of AC-3933 (25 mg/kg) in 20 dogs. Distribution of the logarithmically transformed C3h ratios of SX-5745 to AC-3933 was plotted. Solid bars and open bars indicate EM dogs (n = 18) and PM dogs (n = 2), respectively. The values represent the average of duplicate determinations.

In Vitro Kinetics of AC-3933 Metabolism. Kinetic parameters for AC-3933 hydroxylation (SX-5745 formation) and demethylation (SX-5773 formation) in EM and PM dog liver microsomes are shown in Table 2. AC-3933 was mainly metabolized to SX-5745 in EM dog liver microsomes as well as in vivo (Fig. 4). AC-3933 hydroxylation clearance in PM dogs was 50- to 100-fold lower than that in EM dogs. On the other hand, there was no difference in AC-3933 demethylation clearance between EM and PM dogs.

View larger version (21K): [in this window] [in a new window]   FIG. 4. Typical HPLC radiochromatograms obtained from incubations of [14C]AC-3933 with EM and PM dog liver microsomes. [14C]AC-3933 (2.5 µM) was incubated with liver microsomes from EM (EM1 and EM2) and PM (PM1 and PM2) dogs.

Identification of P450 Isozyme Responsible for AC-3933 Hydroxylation. To identify the P450 isozyme responsible for AC-3933 hydroxylation in dogs, the effects of P450 inhibitors and anti-P450 antibodies on AC-3933 hydroxylation in EM dog liver microsomes were investigated (Fig. 5). In the chemical inhibition experiment, -naphthoflavone (CYP1A inhibitor) inhibited AC-3933 hydroxylation by 91%. On the other hand, sulfaphenazole (CYP2C inhibitor), quinidine (CYP2D inhibitor), and ketoconazole (CYP3A inhibitor) had no inhibitory effects on AC-3933 hydroxylation. In the immunoinhibition experiment, anti-CYP1A2 antibodies completely inhibited AC-3933 hydroxylation, whereas anti-P450 antibodies against CYP1A1, CYP2B1, CYP2C11, and CYP3A2 could not inhibit AC-3933 hydroxylation. These results strongly indicate that CYP1A2 is responsible for AC-3933 hydroxylation in dogs. Under these conditions, AC-3933 demethylation was not clearly inhibited by any of the inhibitors or antibodies.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Effects of chemical inhibitors (A) and anti-P450 antibodies (B) on AC-3933 hydroxylation in EM dog liver microsomes. A, [14C]AC-3933 (10 µM) was incubated for 15 min at 37°C with pooled EM dog liver microsomes in the presence of 10 µM -naphthoflavone (NF), 10 µM sulfaphenazole (SP), 1 µM quinidine (QD), and 0.5 µM ketoconazole (KZ). B, [14C]AC-3933 (10 µM) was incubated for 15 min at 37°C with pooled EM dog liver microsomes in the presence of polyclonal anti-P450 antibodies against rat CYP1A1 (), CYP1A2 (), CYP2B1 (), CYP2C11 (), and CYP3A2 (x). The values represent the average of duplicate determinations.

P450 Activity in EM and PM Dog Liver Microsomes. The activity of POD, EROD, ECOD, and APND in EM and PM dogs is shown in Fig. 6. POD and EROD activity (marker for CYP1A2) was 2- to 4-fold lower in PM dogs than in EM dogs, whereas ECOD and APND activity (marker for CYP2B and/or CYP3A) was the same in EM and PM dogs. To confirm the contribution of CYP1A2 to POD activity, inhibitory effects of -naphthoflavone on POD activity in EM and PM dog liver microsomes were determined (Fig. 6A). In EM dogs, POD activity was inhibited by 33 and 60%. However, in PM dogs, POD activity was activated by 50 and 55%.

View larger version (25K): [in this window] [in a new window]   FIG. 6. Activity of POD (A), EROD (B), ECOD (C), and APND (D) in EM and PM dog liver microsomes. EM (solid bars) and PM (open bars) dog liver microsomes were incubated with ethoxyresorufin (0.01 mM), phenacetin (0.1 mM), ethoxycoumarin (0.1 mM), or aminopyrine (5 mM). The lines in POD (A) represent the activity in the presence of -naphthoflavone. Each assay procedure is described under Materials and Methods. The values represent the average of duplicate determinations.

Expression Level of P450 Protein in PM and EM Dog Liver Microsomes. The expression of P450 protein in EM and PM dog liver microsomes was confirmed by immunoblotting using anti-CYP1A, -CYP2B, -CYP2C, and -CYP3A antibodies (Fig. 7). CYP1A-related protein was clearly expressed in EM dogs (6.1 and 13.0 pmol/mg). In contrast, CYP1A-related protein in PM dogs was not detected (detection limit, 0.86 pmol/mg). CYP2B-, CYP2C-, and CYP3A-related proteins, on the other hand, were equally expressed in PM and EM dogs (44.8-49.5, 34.0-46.6, and 42.2-53.5 pmol/mg, respectively).

View larger version (56K): [in this window] [in a new window]   FIG. 7. Immunoblotting for P450 expression in EM and PM dog liver microsomes. EM and PM dog liver microsomal proteins (5 µg) were separated by SDS-polyacrylamide gel electrophoresis and transferred electrically to a polyvinylidene difluoride membrane. Polyclonal goat anti-CYP1A, -CYP2B, -CYP2C, and -CYP3A antibodies were used as primary antibodies. Bands reacted with the primary antibodies were visualized using alkaline phosphatase-conjugated anti-goat IgG antibodies.

	Discussion

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In this study, we discovered that AC-3933 polymorphic hydroxylation arises from polymorphic expression of CYP1A2 in dogs. Indeed, the results of in vivo experiments indicated that the AC-3933 hydroxylation is polymorphic in dogs, and that dogs could be phenotyped as EM and PM. In vitro studies using liver microsomes from EM and PM dogs have shown a remarkable difference in the AC-3933 hydroxylation clearance between EM and PM dogs (Table 2). This difference correlated well with the difference in C_3h ratio of SX-5745 to AC-3933 in plasma. In addition, AC-3933 hydroxylation in EM dogs was strongly inhibited by -naphthoflavone, a CYP1A inhibitor, and by anti-CYP1A2 antibodies (Fig. 5). Furthermore, markers' activity for CYP1A2 in PM dogs was at least 2-fold lower than that in EM (Fig. 6). These results indicate that CYP1A2 is responsible for AC-3933 polymorphic hydroxylation in dogs and that CYP1A2 activity decreases in PM dogs. The decrease in CYP1A2 activity may be attributed to either a decrease in the expression of CYP1A2 or a decrease in its intrinsic activity. To test these assumptions, the expression levels of P450 proteins were examined using immunoblotting (Fig. 7). Remarkable differences were observed in the expression level of CYP1A-related protein between EM and PM dogs. In addition, the CYP1A-related protein detected by immunoblotting was possibly CYP1A2, because CYP1A2, but not CYP1A1, has been reported to be constitutively expressed in untreated dogs (Uchida et al., 1990; Graham et al., 2002). These results indicate that the decrease in CYP1A2 activity in PM dogs is caused by a decrease in the expression level of CYP1A2 protein and not by a decrease in its intrinsic activity.

Although CYP1A2 protein in PM dogs was undetectable in immunoblotting, CYP1A2 marker activity in PM dogs was only 2-fold lower than that in EM dogs. To resolve this discrepancy, we investigated the effects of -naphthoflavone on POD activity in EM and PM dogs (Fig. 6A). In contrast with the inhibition in EM dogs, POD activity in PM dogs was activated by -naphthoflavone, and the difference between EM and PM disappeared. It has been reported that -naphthoflavone is not only a CYP1A inhibitor but also an activator of CYP2C and CYP3A (Sai et al., 2000). Therefore, our results indicated that POD activity in PM dogs is catalyzed by other P450s except CYP1A2, and that AC-3933 is a more specific substrate for dog CYP1A2 than phenacetin.

In support of in vivo pharmacokinetics, in vitro intrinsic clearance of AC-3933 demethylation was similar in EM and PM dogs and not correlated with the expression level and marker activities of CYP1A2. These results indicated that AC-3933 demethylation is catalyzed by not CYP1A2 but other P450s, and that the P450s are not essential for polymorphic hydroxylation of AC-3933 in dogs.

Recently, Azuma et al. (2002) reported interindividual differences in CYP1A content in dogs. However, this variation in CYP1A content (about 2-fold) was much less than that found in this study. This suggests that the mechanisms of variability observed in both studies are different and that the interindividual difference in CYP1A2 content found in this study would bring about more serious variability of pharmacokinetics, which can affect toxicological and pharmacological data in dogs.

Based on our result that CYP1A2 is polymorphically expressed in dogs, it is possible that there is a genetic polymorphism in dog CYP1A2 gene or related gene(s), i.e., molecules involved in transcription, translation, and post-translational modification of CYP1A2. Polymorphism of dog CYP1A2 gene and related genes has so far not been reported. However, in humans, interindividual differences in CYP1A2 content have been reported, and it is postulated that some genetic factors along with environmental factors are associated with these interindividual differences (Pelkonen et al., 2001). Although several mutations of human CYP1A2 gene that affect the inducibility of CYP1A2 have been reported (Nakajima et al., 1999; Sachse et al., 1999), the genetic factors relevant to CYP1A2 activity are not completely understood in humans. To identify the putative genetic factors inducing polymorphic expression of dog CYP1A2 might provide clues to not only the genotyping method for selection of PM and EM dogs, but also to the mechanism of interindividual difference in human CYP1A2. Additional molecular-based studies are needed to clarify the mechanism of CYP1A2 polymorphic expression in dogs.

	Acknowledgments

 
We are very grateful to Tsutomu Nishiwaki (Dainippon Pharmaceutical Co., Ltd.) for technical assistance.

	Footnotes

 
¹ Abbreviations used are: P450, cytochrome P450; AC-3933, 5-(3-methoxyphenyl)-3-(5-methyl-1,2,4-oxadiazol-3-yl)-2-oxo-1,2-dihydro-1,6-naphthyridine; [¹⁴C]AC-3933, (5-(3-methoxyphenyl)-3-(5-methyl-1,2,4-oxadiazol-3-yl)-2-oxo-1,2-dihydro-[7-¹⁴C]-1,6-naphthyridine; SX-5745, 3-(5-hydroxymethyl-1,2,4-oxadiazol-3-yl)-5-(3-methoxyphenyl)-2-oxo-1,2-dihydro-1,6-naphthyridine; SX-5773, 5-(3-hydroxyphenyl)-3-(5-methyl-1,2,4-oxadiazol-3-yl)-2-oxo-1,2-dihydro-1,6-naphthyridine; SX-6088, 5-(3-methoxyphenyl)-2-oxo-1,2-dihydro-1,6-naphthyridine-3-carboxylic acid; EM, extensive metabolizer; PM, poor metabolizer; C_3h, concentration at 3 h after administration; HPLC, high-performance liquid chromatography; EROD, ethoxyresorufin O-deethylase; POD, phenacetin O-deethylase; ECOD, ethoxycoumarin O-deethylase; APND, aminopyrine N-demethylase; AUC_0-24, area under concentration-time curve from zero to 24 h.

Address correspondence to: Masashi Mise, Pharmacokinetics and Physico-Chemical Property Research Laboratories, Dainippon Pharmaceutical Co., Ltd., 33-94, Enoki, Suita, Osaka 564-0053, Japan. E-mail: masashi-mise{at}dainippon-pharm.co.jp

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