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Pharmacokinetics Research Laboratories, Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan
(Received May 15, 2008; Accepted June 20, 2008)
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Abstract |
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). For example, CYP3A4 is the major isoform expressed in human liver, whereas CYP2C is the most abundant isoform in rat liver (Cheng and Schenkman, 1982). Moreover, it has been reported that S-mephenytoin 4'-hydroxylation is catalyzed by human CYP2C19 but not rat orthologous isoforms (CYP2C6/11/12/13) (Kobayashi et al., 2002). Therefore, for prediction of human metabolism from animal data, it is important to determine the species differences in the P450 functions.
In the pharmaceutical industry, beagle dogs are commonly used as nonrodent animals for toxicological and pharmacological studies of drug candidates. Dog pharmacokinetic data along with in vitro metabolic data can be very useful for the prediction of human in vivo pharmacokinetics and interpretation of toxicity and efficacy results in both species. In dogs, several P450s have been cloned and sequenced, including CYP1A1/2 (Uchida et al., 1990; Fukuta et al., 1992), 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). For almost all these P450s, heterologous expression, functional characterization, and comparison of substrate specificity with corresponding human P450s have been investigated. However, functional characterization of dog CYP1A2 has not been reported. CYP1A2 is constitutively expressed in human and animal liver and is responsible for the metabolism of many drugs, including caffeine, phenacetin, and theophylline in humans (Rendic and Di Carlo, 1997). In addition, it plays a major role in the metabolic activation of carcinogens, such as polycyclic aromatic hydrocarbons and aromatic amines (Guengerich, 1997; Rendic and Di Carlo, 1997). For reliable and effective preclinical studies using dogs, it is necessary to characterize the species differences in CYP1A2 function between dogs and humans.
Recently, we reported that beagle dogs have a nonsense mutation (C1117T) in CYP1A2 with an allele frequency of 37% (Mise et al., 2004a). The deduced amino acid sequence of the mutant is 140 amino acids shorter than that of the wild-type and lacks the heme-binding region. Therefore, homozygotes for the T allele are CYP1A2-deficient dogs (genotype frequency of 11%). Generally, a knockout animal for a specific gene is a useful model for the characterization of the corresponding protein. To study for the function of CYP1A2, CYP1A2 knockout mice have been produced and analyzed (Liang et al., 1996). Similarly, the CYP1A2-deficient dog is likely to be very useful for the study of CYP1A2 function. By comparing metabolic activity between deficient and wild-type dog liver microsomes, the contribution of CYP1A2 to the metabolism can be determined.
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; Yamazaki et al., 1998; Zhang et al., 2000; Facciolá et al., 2001). |
Materials and Methods |
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-Naphthoflavone (ANF) was purchased from Nacalai Tesque (Kyoto, Japan). Bufuralol hydrochloride, 1'-hydroxybufuralol, and 1'-hydroxymidazolam were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). All the other reagents were of the highest grade commercially available.
Genotyping. Beagle dogs were obtained from Covance Research Products Inc. (Kalamazoo, MI). A polymerase chain reaction (PCR)-restriction fragment length polymorphism was used to detect the C1117T mutation (Mise et al., 2004a). Genomic DNA was extracted from dog whole blood using a DNAeasy tissue kit (Qiagen, Hilden, Germany). Forward and reverse primers were 5'-GACACGGTGATTGGCAGGGC-3' and 5'-CTGTGGGGGATGGTGAAGGGGATAAAGGAGGTGTCT-3', respectively. Genomic DNA was added to a PCR mixture containing PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 µM of each primer, and 1 U TaKaRa Taq (Takara Shuzo, Shiga, Japan) in a final volume of 50 µl. PCR conditions were 94°C for 2 min; 35 cycles with 94°C for 20 s, 52°C for 10 s, 72°C for 10 s; and 72°C for 1 min. PCR products (5 µl) were incubated with 5 U of DdeI (New England Biolabs, Inc., Beverly, MA) and NEBuffer 3 for 2 h at 37°C in 20 µl of a total reaction volume. Digested products were analyzed by 3% agarose gel electrophoresis. For the mutant allele (T allele), the PCR product of 124 base pair (bp) was digested to fragments of 86 and 38 bp.
Liver Microsomes. Dog livers were obtained from C/C (numbers 1–5), C/T (numbers 6–10), and T/T (numbers 11–15) genotyped dogs (Fig. 1). 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 then 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 then 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. Total P450 content was determined by the method of Omura and Sato (1964). Human liver microsomes (mixed gender, pool of 50 individuals) were purchased from XenoTech LLC (Lenexa, KS).
Aniline Hydroxylase Assay. Aniline (5 mM) was incubated for 30 min at 37°C in 1 ml of a reaction mixture consisting of 50 mM phosphate buffer, pH 7.4, 1 mg/ml dog or human liver microsomes, and 0.8 mM NADPH. p-Aminophenol, hydroxylated metabolite, was measured according to the method of Imai et al. (1966).
Bufuralol 1'-Hydroxylase and Midazolam 1'-Hydroxylase Assay. Bufuralol (20 µM) or midazolam (4 µM) was incubated for 10 or 5 min at 37°C in 150 µl of reaction mixture consisting of 50 mM phosphate buffer, pH 7.4, 0.25 or 0.1 mg/ml liver microsomes, and 0.8 mM NADPH, respectively. The reaction was started by adding NADPH and stopped by adding 150 µl of acetonitrile, with 100 µl of 0.5 µM dextrorphan as an internal standard. After centrifugation at 900g for 10 min, 1'-hydroxybufuralol and 1'-hydroxymidazolam were quantified by liquid chromatography/tandem mass spectrometry (LC/MS/MS) consisting of a 1100 liquid chromatography (Agilent Technologies, Palo Alto, CA) and an API2000 mass spectrometer (MDS Sciex, Concord, ON, Canada). Separation was performed on a reverse-phase column (Capcell Pak C18 AQ 2.0 x 35 mm; Shiseido, Tokyo, Japan) at 40°C with a flow rate of 0.3 ml/min. Mobile phase consisted of solvent A (0.1% formic acid) and solvent B (methanol). The gradient used was as follows: 0 to 50% solvent B (0–0.5 min), 50% (0.5–2.5 min), and 0% (2.51–7 min). The mass spectrometer was operated in positive electrospray ionization mode at a capillary temperature of 550°C and spray voltage of 5 kV. Multiple reaction monitoring analysis was performed with a transition of m/z 278.2
186.2 for 1'-hydroxybufuralol, m/z 342.1168.2 for 1'-hydroxymidazolam, and m/z 258.0157.1 for dextrorphan. Intra-assay coefficients of variation (CVs) for 0.3 µM 1'-hydroxybufuralol and 0.3 µM 1'-hydroxymidazolam were 5.0 and 14.3%, respectively.
Tacrine 1-Hydroxylase Assay. Tacrine (25 µM) was incubated for 15 min at 37°C in 500 µl of reaction mixture consisting of 50 mM phosphate buffer, pH 7.4, 0.8 mg/ml dog or human liver microsomes, and 0.8 mM NADPH. The reaction was started by adding NADPH and stopped by adding 1.5 ml of acetonitrile, with 25 µl of 50 µM antipyrine as an internal standard. After centrifugation at 900g for 10 min, the supernatant was evaporated to dryness, and then the residue was dissolved in the mobile phase for high-performance liquid chromatography (HPLC) analysis. 1-Hydroxytacrine was quantified by HPLC (1100 liquid chromatography) using a reverse-phase column (Inertsil ODS-3, 3.0 x 100 mm) at 40°C with a flow rate of 0.4 ml/min. Mobile phase consisted of solvent A (0.05% trifluoroacetic acid) and solvent B (acetonitrile). The gradient used was as follows: 13% solvent B (0–5 min), 13 to 25% (5–10 min), 25 to 80% (10–10.5 min), and 13% (11–15 min). Wavelength of the detector was 250 nm. Intra-assay and interassay CVs for 0.5 µM 1-hydroxytacrine were 0.9 and 3.1%, respectively.
Caffeine 7-Demethylase, 3-Demethylase, 1-Demethylase, and 8-Hydroxylase Assay. Caffeine (1 mM) was incubated for 30 min at 37°C in 500 µl of a reaction mixture consisting of 50 mM phosphate buffer, pH 7.4, 1 mg/ml dog or human liver microsomes, and 0.8 mM NADPH. The reaction was started by adding NADPH and stopped by adding 1.5 ml of acetonitrile, with 50 µl of 20 µM enprofylline as an internal standard. After centrifugation at 900g for 10 min, the supernatant was evaporated to dryness, and then the residue was dissolved in the mobile phase for HPLC analysis. Theophylline (7-demethylated metabolite), paraxanthine (3-demethylated metabolite), theobromine (1-demethylated metabolite), and trimethyluric acid (8-hydroxylated metabolite) were quantified using HPLC under the same conditions as the tacrine 1-hydroxylase (TC) assay. Mobile phase consisted of solvent A (0.05% trifluoroacetic acid) and solvent B (acetonitrile). The gradient used was as follows: 7% solvent B (0–6 min), 7 to 20% (6–10 min), 20 to 80% (10–10.5 min), 80 to 7% (10.5–11 min), and 7% (11–15 min). Wavelength of the detector was 280 nm. Intra-assay and interassay CVs were 1.7 and 3.8% for 0.5 µM theophylline, 1.9 and 4.1% for 0.5 µM paraxanthine, 1.7 and 6.4% for 0.5 µM theobromine, and 1.7 and 1.4% for 0.5 µM trimethyluric acid, respectively.
Melatonin 6-Hydroxylase Assay. Melatonin 6-hydroxylase assay was performed as described previously with several modifications (Ma et al., 2005). Melatonin (25 µM) was incubated for 15 min at 37°C in 100 µl of reaction mixture consisting of 50 mM phosphate buffer, pH 7.4, 0.5 mg/ml dog or human liver microsomes, and 0.8 mM NADPH. For human liver microsomes, inhibitory effects of 1 µM ANF, human CYP1A2 inhibitor, were also evaluated. The reaction was started by adding NADPH and stopped by adding 100 µl of acetonitrile, with 50 µl of 2 µM 6-chloromelatonin as an internal standard. After centrifugation at 900g for 10 min, 6-hydroxymelatonin was quantified by LC/MS/MS. LC/MS/MS analysis was performed under the same conditions as above. Mobile phase consisted of solvent A (0.1% formic acid) and solvent B (acetonitrile). The gradient used was as follows: 5 to 50% solvent B (0–4 min), 100% (4.1 min), and 5% (4.2–8 min). The mass spectrometer was operated in positive electrospray ionization mode at a capillary temperature of 550°C and spray voltage of 5 kV. Multiple reaction monitoring analysis was performed with a transition of m/z 249.0190.2 for 6-hydroxymelatonin and m/z 267.0208.1 for 6-chloromelatonin. Intra-assay and interassay CVs for 0.5 µM 6-hydroxymelatonin were 3.2 and 3.8%, respectively.
9-cis-Retinal Oxidase Assay. 9-cis-Retinal oxidase assay was performed as described previously with several modifications (Zhang et al., 1998). 9-cis-Retinal (100 µM) was incubated for 20 min at 37°C in 500 µl of reaction mixture consisting of 50 mM phosphate buffer, pH 7.4, 1 mg/ml dog or human liver microsomes, and 0.8 mM NADPH. For human liver microsomes, inhibitory effects of 1 µM ANF were also evaluated. The reaction was stopped by adding 2 ml of ethyl acetate containing 0.005% butylated hydroxytoluene. Metabolites were extracted three times with ethyl acetate. Extracts were pooled, dried under a nitrogen stream, and then dissolved in methanol. 9-cis-Retinoic acid was quantified using HPLC under the same conditions as the TC assay. Mobile phase consisted of solvent A (0.05% trifluoroacetic acid) and solvent B (acetonitrile). The gradient used was as follows: 50 to 85% solvent B (0–5 min), 85% (5–15 min), and 50% (15.5–20 min). Wavelength of the detector was 350 nm. All the procedures were carried out in the absence of an overhead light. Intra-assay and interassay CVs for 0.5 µM 9-cis-retinoic acid were 2.6 and 8.5%, respectively.
Estradiol 2-Hydroxylase Assay. Estradiol (20 µM) was incubated for 20 min at 37°C in 500 µl of reaction mixture consisting of 50 mM phosphate buffer, pH 7.4, 0.8 mg/ml dog or human liver microsomes, and 0.8 mM NADPH. For human liver microsomes, inhibitory effects of 1 µM ANF were also evaluated. The reaction was stopped by adding 2 ml of ethyl acetate, with 50 µl of 20 µM chlorzoxazone as an internal standard. After centrifugation at 900g for 10 min, the extract was evaporated to dryness, and then the residue was dissolved in methanol. 2-Hydroxyestradiol was quantified using HPLC under the same conditions as the TC assay. Mobile phase consisted of solvent A (0.05% trifluoroacetic acid) and solvent B (acetonitrile). The gradient used was as follows: 30% solvent B (0–5 min), 30 to 90% (5–15 min), and 30% (15.5–20 min). Wavelength of the detector was 280 nm. Intra-assay and interassay CVs for 0.5 µM 2-hydroxyestradiol were 2.4 and 4.7%, respectively.
Other Assays. Ethoxyresorufin O-deethylase (EROD), phenacetin O-deethylase (POD), and aminopyrine N-demethylase (AP) assays were performed as described previously (Mise et al., 2004b). Substrate concentrations used were 10, 100, and 5 mM, respectively.
Statistical Analysis. Statistical significance was tested using Dunnett's multiple comparison procedure (significance levels: 5%, two-tailed test) between C/C, C/T, and T/T genotyped dogs.
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Results |
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Metabolism of Exogenous Substrates for Human CYP1A2. Activities of POD, EROD, and TC in genotyped dog and human liver microsomes are shown in Fig. 2. These activities were significantly lower in T/T dogs than in C/C dogs. In addition, EROD activity was significantly lower in C/T dogs than in C/C dogs. These significant differences between genotypes were also observed in activities per nanomole of P450, indicating that the difference is not caused by difference in total P450 content. POD and TC activities in C/T dogs tended to be lower than those in C/C dogs, but this finding is not statistically significant. These results indicate that dog CYP1A2 is responsible for these activities.
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Activities of caffeine 7-demethylase (13X), caffeine 3-demethylase (17X), caffeine 1-demethylase (37X), and caffeine 8-hydroxylase (137U) in genotyped dog and human liver microsomes are shown in Table 2. There was no difference in caffeine metabolism between C/C, C/T, and T/T dogs, indicating that, unlike human, dog CYP1A2 is not involved in these reactions. In comparisons with humans, although 17X pathway was the main metabolic pathway both in humans and dogs, the contribution of 13X and 137U to caffeine metabolism in dogs was 15.4 and 18.4%, respectively, higher than those in humans (4.7 and 3.9%, respectively). These results suggest that the metabolic profile of caffeine in dogs is different from that in humans.
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Metabolism of Endogenous Substrates for Human CYP1A2. Activities of melatonin 6-hydroxylase (MLT), 9-cis-retinal oxidase (RAL), and estradiol 2-hydroxylase (E2) in genotyped dog and human liver microsomes are shown in Fig. 3. MLT activity in T/T dogs was 30% lower than that in C/C dogs (P < 0.05). These results indicate that dog CYP1A2 is only partially involved in MLT activity. In human liver microsomes, MLT activity was strongly inhibited by CYP1A2 inhibitor, ANF (94% inhibition), indicating that CYP1A2 is responsible for MLT activity.
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Discussion |
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), direct evidence, such as immunoinhibition studies, has not been reported. The current study produced direct evidence that dog CYP1A2 is largely responsible for POD activity in dog liver microsomes. Moreover, as the major metabolite of dog urine after administration of phenacetin is acetaminophen, an O-deethylated metabolite (Podder et al., 1988), dog CYP1A2 is the major P450 isoform involved in phenacetin metabolism in dogs. EROD activity is used as an in vitro CYP1A1/2 marker activity not only in humans but also in dogs (Graham et al., 2002). Although heterologously expressed dog CYP1A1 and CYP1A2 catalyzed EROD activity, the contribution of CYP1A2 in EROD activity in dog liver microsomes has been unknown (Gibson et al., 2005). Our results suggested that CYP1A2 is largely responsible for EROD activity in dog liver microsomes. TC in humans is CYP1A2 (Madden et al., 1993); however, the P450 isoform responsible for TC activity in dogs has been unknown. In this study, CYP1A2 was shown to be responsible for TC activity in dog liver microsomes. Because the major metabolite of dog urine after administration of [14C]tacrine is 1-hydroxyl tacrine (Pool et al., 1997), dog CYP1A2 is the major P450 isoform involved in tacrine metabolism in dogs. Based on results from the current study and previous reports, POD and TC are useful marker activities for CYP1A2 in dogs, both in vitro and in vivo. Therefore, for example, phenacetin and tacrine are available for in vivo drug-drug interaction study in dogs as a CYP1A2 probe substrate. If in vivo drug-drug interaction data along with in vitro data are able to be obtained in dogs, it is very useful to predict drug-drug interaction based on CYP1A2 in humans by in vitro/in vivo extrapolation methods.
In contrast, dog CYP1A2 was not involved in AP, AN, BF, and MDZ activities (Table 1). AP and AN are used as marker activities for CYP2B/3A and CYP2E1, respectively, in dogs (Tanaka et al., 1998; Nakata et al., 2000). In addition, it has been reported that dog CYP2D15 and CYP3A12 have BF and MDZ activity, respectively (Roussel et al., 1998; Carr et al., 2006). Our results are consistent with these previous findings.
17X activity, the major metabolic pathway of caffeine, is commonly used as a CYP1A2 marker activity in both humans and dogs (Tanaka et al., 1998). Moreover, 17X activity is catalyzed by isoforms induced by β-naphthoflavone in dogs (Aldridge and Neims, 1979); however, it has not been determined whether CYP1A2 is involved in 17X activity in the uninduced state. In the current study, there was no difference in caffeine metabolism between C/C, C/T, and T/T dogs, indicating that dog CYP1A2 is not involved in 17X activity at a substrate concentration of 1 mM (Table 2). Generally, in the in vitro caffeine metabolism study, the substrate concentration is at least 1 mM (Tanaka et al., 1998). Therefore, it should be recognized that 17X activity is not a marker activity for CYP1A2 in dog liver microsomes at a substrate concentration of 1 mM. The metabolic profile in dog liver microsomes was also different from human liver microsomes, with the contribution of 13X to caffeine metabolism higher in dogs than humans (Table 2). These results are supported by previous reports describing 3- and/or 7-demethylation as the primary routes of caffeine metabolism in dogs (Aldridge and Neims, 1979).
Some endogenous substances are substrates for human CYP1A2 (e.g., melatonin, retinoids, estrogens, and uroporphyrinogen). The pineal hormone melatonin is metabolized to 6-hydroxymelatonin mainly by human CYP1A2, and clearance of p.o. administered melatonin is significantly correlated with the caffeine clearance, a marker activity for human CYP1A2 (Facciolá et al., 2001; Härtter et al., 2001). 9-cis-Retinal is oxidized by human CYP1A2 to 9-cis-retinoic acid, a ligand for retinoic acid receptor and retinoid X receptor (Duester, 1996; Zhang et al., 2000). Estradiol, a main estrogen, is metabolized to 2-hydroxyestradiol by human CYP1A2 and CYP3A4 (Yamazaki et al., 1998). Agreeing with previous studies, our study shows that CYP1A2 inhibitor inhibited MLT, RAL, and E2 by 94, 83, and 44%, respectively, in human liver microsomes (Fig. 3). In contrast, dog CYP1A2 was found to not be responsible for MLT, RAL, and E2 activities. These species differences in CYP1A2 substrate specificity for endogenous substances permit us to speculate that the contribution of CYP1A2 in physiological homeostasis is different between humans and dogs. In fact, a CYP1A2-deficient phenotype in humans has not yet been reported (http:/www.imm.ki.se/CYPalleles), although CYP1A2-deficient dogs exist (Mise et al., 2004a; Tenmizu et al., 2004). However, the contribution of CYP1A2 to the metabolism of endogenous substances in vivo is not completely understood. For example, it has been reported that extrahepatic CYP1B1 has MLT activity (Ma et al., 2005) and that cytosolic aldehyde dehydrogenase and intestinal microsomal CYP2J4 has RAL activity (Duester, 1996; Zhang et al., 1998). To characterize the physiological role of CYP1A2, additional studies are required.
In general, pharmacokinetic data from animals along with in vitro metabolic data are used for the prediction of human in vivo pharmacokinetics. Also, in toxicological or pharmacological studies, the correlation between the systemic exposure of a compound and its toxicological or pharmacological effect is evaluated for the prediction of effects in humans. In these preclinical studies, the genetic homogeneity of the animals used will be very important for reliable prediction. The current study shows that POD, EROD, and TC activities in beagle dogs are significantly different between CYP1A2 genotypes (C/C, C/T, and T/T). The genotype frequency of T/T, CYP1A2-deficient dog, is more than 10% (Mise et al., 2004a; Tenmizu et al., 2004). Therefore, CYP1A2 genotypes in beagle dogs should be a great concern for obtaining reliable preclinical study data (Kamimura, 2006).
In this study, 11 P450 activities were quantitatively compared between dogs and humans. AN, MDZ, RAL, and E2 activities were lower in dogs than humans. These data are basic to understanding species differences in P450 function and useful for animal selection in preclinical studies.
CYP1A2, which activates polycyclic aromatic hydrocarbons and aromatic amines, is a key enzyme in chemical carcinogenesis in humans and rats (Guengerich, 1997). However, in dogs, the contribution of CYP1A2 in the carcinogen metabolism is unknown. Using our approach applied to the current study, it may be possible to determine the contribution of CYP1A2 to the carcinogen metabolism in dogs and to discuss its toxicological significance.
In conclusion, substrate specificity of CYP1A2 in beagle dogs and a species difference in the specificity between beagle dogs and humans were determined. Tacrine, ethoxyresorufin, and phenacetin are probe substrates for CYP1A2 in both dogs and humans. However, caffeine, melatonin, 9-cis-retinal, and estradiol, which are substrate for human CYP1A2, are not good substrates for dog CYP1A2. The finding that there are species differences in substrate specificity of CYP1A2 between humans and beagle dogs is an important issue and must be considered for preclinical studies using beagle dogs.
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ABBREVIATIONS: P450, cytochrome P450; ANF, -naphthoflavone; PCR, polymerase chain reaction; bp, base pair; LC/MS/MS, liquid chromatography/tandem mass spectrometry; CV, coefficient of variation; HPLC, high-performance liquid chromatography; TC, tacrine 1-hydroxylase; EROD, ethoxyresorufin O-deethylase; POD, phenacetin O-deethylase; AP, aminopyrine N-demethylase; AN, aniline hydroxylase; BF, bufuralol 1'-hydroxylase; MDZ, midazolam 1'-hydroxylase; 13X, caffeine 7-demethylase; 17X, caffeine 3-demethylase; 37X, caffeine 1-demethylase; 137U, caffeine 8-hydroxylase; MLT, melatonin 6-hydroxylase; RAL, 9-cis-retinal oxidase; E2, estradiol 2-hydroxylase.
Address correspondence to: Masashi Mise, Pharmacokinetics Research Laboratories, Dainippon Sumitomo Pharma Co., Ltd., 33-94, Enoki-cho, Suita, Osaka 564-0053, Japan. E-mail: masashi-mise{at}ds-pharma.co.jp
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