CYP3A5 Genotype Did Not Impact on Nifedipine Disposition in Healthy Volunteers

CYP3A5 genotype did not impact on nifedipine disposition in healthy volunteers

T Fukuda1,4 ABSTRACT 1,4 CYP3A5 expression is regulated by single-nucleotide polymorphisms (SNPs). S Onishi The CYP3A5 genotype might contribute to a marked interindividual variation 1 S Fukuen in CYP3A-mediated metabolism of drugs. Nifedipine is a typical substrate of Y Ikenaga1 CYP3A4 and CYP3A5 in vitro. The aim of this study was to elucidate the M Ohno1 influence of the CYP3A5 genotype on nifedipine disposition in healthy K Hoshino1 subjects. A single capsule containing 10 mg of nifedipine was administered to 1 16 healthy male Japanese subjects (eight subjects: CYP3A5*1/*3; eight K Matsumoto subjects: CYP3A5*3/*3). Blood samples were collected to analyze the A Maihara2 pharmacokinetics of serum nifedipine and nitropyridine metabolite (M-I). K Momiyama2 The area under the plasma concentration–time curve (AUC), the peak plasma T Ito3 concentration (Cmax) and the terminal half-life (t1/2) of nifedipine, and the 1 ratio of the nifedipine AUC to M-I AUC showed large intragroup variations, Y Fujio but no significant differences between the two genotypes. Based on the J Azuma1 present findings, the functional relevance of CYP3A5 polymorphism should be re-evaluated in clinical trials. 1Clinical Evaluation of Medicines and The Pharmacogenomics Journal (2004) 4, 34–39. doi:10.1038/sj.tpj.6500218 Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, Japan; 2Japan Clinical Published online 2 December 2003 Laboratories, Inc., Bioassay Division, Japan; 3Osaka Pharmacology Research Clinic, Japan Keywords: CYP3A5; nifedipine; polymorphism; clinical; pharmacokinetics

Correspondence: J Azuma, Clinical Evaluation of Medicines and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. INTRODUCTION Tel: þ 81 6 6879 8258 Cytochrome P450 3A (CYP3A) is abundantly expressed in human liver and small Fax: þ 81 6 6879 8259 1,2 E-mail: [email protected] intestine, and contributes to the metabolism of 50% of prescribed drugs. The activities of CYP3A in the general population show interindividual variations in 4These two authors contributed equally to this CYP3A-mediated metabolism of drugs.2 Recently, single-nucleotide polymorph- work isms (SNPs) were identified in intron 3 (A–G: CYP3A5*3) and exon 7 (G–A: CYP3A5*6) of the CYP3A5 gene.3 In addition, CYP3A5*5 and CYP3A5*7 were reported as a defective allele of CYP3A5, which gave a substantial impact on CYP3A5 expression.4,5 These SNPs cause a frame-shift mutation or alternative splicing and protein truncation, and result in the absence of CYP3A5, suggesting that only people with at least one CYP3A5*1 allele express large amounts of CYP3A5 protein. Therefore, these findings suggest that polymorphic CYP3A5 expression might be one factor contributing to the marked interindividual variation observed in CYP3A-mediated metabolism of drugs. We previously reported the frequencies of CYP3A5-related SNPs in 200 healthy Japanese subjects.6 As a result, the allele frequency of CYP3A5*3 was Received: 18 February 2003 approximately 70%, but CYP3A5*6 was not detected in the Japanese population. Revised: 06 October 2003 Accepted: 14 October 2003 Accordingly, these findings suggested that about 40% of Japanese express Published online 2 December 2003 relatively high levels of metabolically active CYP3A5 protein. CYP3A5 polymorphism and nifedipne pharmacokinetics T Fukuda et al 35

Table 1 Enzyme kinetic analyses of the conversion of M-I Table 2 Characteristics of the subjects in the study using baculovirus-expressed human ¼ P450s Subject no. Age (years) Height (cm) Weight (kg) BMI (%) Km (mM) Vmax (pmol/ Vmax/Km (ml/ min/pmol min/pmol CYP3A5*1/ P450) P450) *3 (n ¼ 8) 1 23 174.2 65.0 97.3 CYP3A4 3.0 3.1 1.0 2 21 171.2 65.4 102.1 CYP3A5 6.5 3.3 0.5 3 22 169.0 57.5 92.6 4 23 177.7 58.9 84.2 9 22 185.3 64.9 84.5 10 21 180.1 72.3 100.3 To obtain clinical evidence of CYP3A5 polymorphism, we 11 23 177.7 62.6 89.5 focused on nifedipine, a typical substrate of CYP3A4 and 12 23 163.3 49.8 87.4 CYP3A5,7–9 because a large individual difference was observed in nifedipine disposition,10 which was thought to Mean 22.3 174.8 62.1 92.2 be regulated by a genetic background rather than environ- SD 0.9 6.9 6.7 7.0 ment.11 Consistently, using baculovirus-expressed human CYP3A5*3/ CYP3A5 and CYP3A4, we confirmed their contribution to *3 (n ¼ 8) the metabolism of nifedipine (Table 1). These findings 5 22 170.8 56.1 88.0 suggested that CYP3A5 contributes to the metabolism of 6 32 182.4 70.9 95.6 nifedipine with kinetics similar to CYP3A4, implying that 7 21 166.7 60.9 101.4 the interindividual differences in nifedipine disposition 8 20 179.5 66.1 92.4 might be explained in part by CYP3A5 polymorphism. 13 22 179.4 61.2 85.6 Thus, in the present study, we evaluated the influence of the 14 21 175.0 62.9 93.2 CYP3A5 genotype on nifedipine disposition in healthy 15 22 173.5 57.7 87.2 subjects to examine the polymorphic activities of CYP3A5 16 25 171.8 71.6 110.8 in vivo. Mean 23.1 174.9 63.4 94.3 SD 3.9 5.3 5.7 8.4 RESULTS The subjects were genotyped and divided into two groups, CYP3A5*1/*3 and CYP3A5*3/*3 (Table 2). No subject had the other CYP3A5 alleles, CYP3A5*5, CYP3A5*6 and CYP3A5*7. First, the plasma concentration profiles of nifedipine and M- significant differences in the pharmacodynamics between I were compared between *1/*3 and *3/*3 groups. Unexpect- the two genotypes (Figure 3 and Table 4). edly, the time profiles of both plasma nifedipine and M-I were not significantly different between the two genotypes (Figure 1). Moreover, plasma nifedipine and M-I showed a DISCUSSION large intragroup variation. Next, the typical pharmacoki- In the present study, we examined the effects of CYP3A5 netic parameters of nifedipine, such as the area under the genotype on nifedipine pharmacokinetics, and demon- plasma concentration–time curve from 0 to 12 h after strated that an interindividual variation of plasma nifedi- administration (AUC0–12 h), the peak plasma concentration pine concentration was not over-ridden by the CYP3A5 (Cmax), terminal half-life (t1/2) and clearance (CL/F) were genotype. The interindividual variation was not beyond our calculated (Table 3). The AUC0–12 h values showed large conception and was almost similar to that described in the intragroup variations without significant differences be- previous report following the administration of a 10-mg tween the two genotypes (218.8780.9 ng h/ml in capsule.12 The present finding suggests that CYP3A5 poly- CYP3A5*1/*3 subjects, 178.7792.8 ng h/ml in CYP3A5*3/*3 morphism is unlikely to be responsible for interindividual subjects; mean7SD). Furthermore, the ratio of the nifedi- variation in the plasma level of nifedipine because the pine AUC0–12 h to the M-I AUC(4.77 in CYP3A5*1/*3 remaining CYP3A5 alleles, CYP3A5*5, CYP3A5*6 and subjects, 3.62 in CYP3A5*3/*3 subjects; mean) also showed CYP3A5*7, were not found in the present subjects. large intragroup variations with no significant differences With respect to nifedipine metabolism, nifedipine dis- between the two genotypes (Figure 2). The differences in the position is slightly affected by the expression of intestinal

Cmax, t1/2 and CL/F of nifedipine between the two groups CYP3As because grapefruit juice influences nifedipine were not significant. disposition significantly but to a lesser extent than felodi- Finally, we measured systolic and diastolic blood pressure pine or nisoldipine,10,13,14 suggesting that nifedipine is and pulse rate to estimate the significance of CYP3A5 mainly metabolized not in the intestine but in the liver. In polymorphism in the pharmacodynamics of nifedipine. addition, it is hypothesized that P-glycoprotein (P-gp) is Consistent with the pharmacokinetics, there were no responsible for the large interindividual difference in

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CYP3A-mediated drug disposition, since P-gp exists in the toward the metabolism of nifedipine at relatively low similar tissue to CYP3A4. However, this hypothesis is not concentrations. the case with nifedipine disposition because nifedipine is We, however, observed a discrepancy between the in vitro not a substrate of P-gp.15,16 Therefore, nifedipine pharma- and in vivo contribution of CYP3A5 to nifedipine metabo- cokinetics must be crucially determined by the total liver lism in the present study. Interestingly, a similar result has CYP3A activities. been obtained in the case of midazolam, a typical CYP3A5 It was reported that nifedipine, as well as midazolam, were substrate. Namely, midazolam pharmacokinetics was also not only metabolized by CYP3A4 but also by CYP3A5 in hardly influenced in vivo by the genotype of CYP3A5,18 vitro.7–9,17 Prior to clinical study, we conducted kinetic study although midazolam is metabolized in vitro by CYP3A5 on the formation of M-I using recombinant microsomes rather than CYP3A4.3,17,19 Several possibilities can be (CYP3A4 and CYP3A5) because previous reports provided proposed to explain these discrepancies between the in vitro the oxidation activity at a single high concentration of and in vivo data. nifedipine. We confirmed the contribution of CYP3A5 It was previously reported that total CYP3A activity showed an interindividual variation,2 and the ratio of CYP3A4 to CYP3A5 might also vary interindividually in the liver.20 Recently, Westlind-Johnsson et al21 reported that CYP3A5 did not contribute to total CYP3A activity using

Figure 1 Plasma concentration–time curves of nifedipine (a) and Figure 2 Individual (dot) and mean (line) values with SD of the M-I (b) in the CYP3A5*1/*3 and CYP3A5*3/*3 subjects. Values ¼ AUC0–12 h of nifedipine (a) and the ratio of the nifedipine ¼ represent the means with SD (n ¼ 8). AUC0–12 h to the M-I ¼ AUC0–12 h (b) in the two genotypes.

Table 3 Pharmacokinetic parameters of nifedipine after oral administration to subject with CYP3A5*1/*3 and CYP3A5*3/*3 genotypes (mean7SD)

Genotype Cmax (ng/ml) t1/2 (h) AUC0–12 (ng h/ml) CL/F (ml/min)

CYP3A5*1/*3 (n ¼ 8) 116743.3 1.7770.66 219780.9 8777375 CYP3A5*3/*3 (n ¼ 8) 111753.8 1.8171.09 178792.8 12467837

The Pharmacogenomics Journal CYP3A5 polymorphism and nifedipne pharmacokinetics T Fukuda et al 37

liver samples. That is, they indicated that Kuehl et al3 might midazolam. Based on all these observations, we speculated have overestimated the level of CYP3A5 protein, possibly that the amount of CYP3A5 protein in the liver is much due to problems with the method of quantification. On the lower than CYP3A4, although we should consider the other hand, Williams et al19 reported that the Km value of limitations of our study design. This speculation might be nifedipine was much lower for CYP3A4 than CYP3A5 under demonstrated by using CYP3A5-specific substrates, although the detailed conditions with cytochrome b5. Cytochrome b5 no drugs metabolized specifically by CYP3A5 have been was suggested to be an essential component in CYP3A4- reported yet. With respect to mRNA expression, it was catalyzed nifedipine oxidation in human liver micro- reported that CYP3A genes exhibit a degree of tissue-specific somes.22 Although these data seem to support the present expression and CYP3A5 is predominantly expressed in the findings in vivo, the same was not consistent in the case of adrenal grand, prostate and kidney.23 Therefore, CYP3A5 polymorphism might have a physiological and pharmaco- logical effect, which is related to the extrahepatic tissues. This possibility remains to be verified in further studies. Interindividual differences in nifedipine pharmacokinetics remain to be elucidated genetically. Here, we propose that polymorphic regulation of CYP3A4 gene transcription, including the polymorphisms of the promoter activities and transcriptional factors, may have to be taken into account to explain the variation of nifedipine pharmacokinetics. How- ever, a polymorphism, which plays a significant role in the activity of CYP3A4, has not yet been identified in the 50- regulatory region of the CYP3A4 gene.24 Therefore, we have focused on human PXR (hPXR) as a factor effecting CYP3A expression and identified splicing variants of hPXR as a possible factor in interindividual variation caused in CYP3A activity.25 Interestingly, we have found that mRNA expression of wild-type hPXR is well correlated with mRNA expression of CYP3A4 in liver sample (unpublished data), which is consistent with the recent report.21 In summary, we revealed that nifedipine disposition in vivo is not affected by the CYP3A5*3 allele. Owing to a discrepancy between the in vitro and in vivo contribution of CYP3A5, the functional relevance of CYP3A5 in humans should be re-evaluated by clinical studies for each drug.

MATERIALS AND METHODS In vitro Screening for the Contributions of CYP3A4 and CYP3A5 Nifedipine and phenytoin were purchased from Wako Pure Chemicals Co. (Osaka, Japan) and oxidized nifedipine (ULTRAFINE) was obtained from Funakoshi (Tokyo, Japan). Microsomes from baculovirus-infected insect cells expres- sing human CYP3A4 and CYP3A5 with NADPH cytochrome P450 reductase (GENTEST) were obtained from Daiichi Figure 3 Blood pressure (a) and pulse rate (b) after nifedipine Pure Chemicals Co. (Tokyo, Japan). NADPH was purchased administration. Values represent the means with SD (n ¼ 8). from Oriental Yeast Co. (Tokyo, Japan). Other reagents and

Table 4 Systolic and diastolic blood pressures change rate (%) after administration of nifedipine

1 h after administration of nifedipine 2 h after administration of nifedipine

Systolic Diastolic Systolic Diastolic

CYP3A5*1/*3 (n ¼ 8) 3.077.2 9.3710.4 2.775.7 6.9711.3 CYP3A5*3/*3 (n ¼ 8) 2.372.8 6.975.2 3.873.5 8.573.7

Values were the mean7SD of blood pressure changes (%). Each blood pressure change (%) was calculated by the formula ((measured–baseline)/baseline Â 100).

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organic solvents were obtained from Nakarai Tesq Chemical were also created based on the published sequence. These Industries (Kyoto, Japan). PCR reactions were carried out in 25 ml of solution consisting All incubation, extraction and other handling of samples of 2.5 mlof10Â PCR buffer, 0.2 mM of each dNTP, 0.4 mMof were carried out in amber vials. A mixture (0.50 ml) each primer, 90 ng of genomic DNA as a template and 1 U of containing 10 pmol of P450 protein from baculovirus- AmpliTaq Gold (Perkin-Elmer, Branchburg, NJ, USA). After expressed human CYP3A4 and CYP3A5 and 1 mM NADPH initial denaturation at 951C for 10 min, the amplification for in 0.1 M potassium phosphate buffer (pH 7.4) was incubated the CYP3A5*3 or *6 alleles was performed using 37 cycles of with nifedipine (final concentration: 1, 2, 5, 10 and 40 mM) 941C for 30 s, 561C(*3)or581C(*6) for 30 s and 721C for 30 s, at 371C after 2 min of preincubation without NADPH. The followed by 721C for 5 min for final extension. After PCR incubation was continued with gentle shaking at 371C for amplification, 5 ml of each PCR product was digested for a 30 min and the reaction was stopped with ethyl acetate minimum of 2 h at 371C with 5 U of DdeI before (3 ml). A measure of 50 ml of methanol and 12.5 mlof20mM electrophoresis using a 3% agarose gel. phenytoin (internal standard) were added to each sample. CYP3A5*5 and CYP3A5*7 alleles were also determined The organic layers were transferred to other vials after according to the method of van Schaik et al26 with some centrifugation at 1500 g for 10 min and evaporated to modifications. dryness. The residues were dissolved in 200 ml of the mobile phase. A measure of 50 ml were analyzed by high-perfor- Study Design mance liquid chromatography (HPLC) with a reverse-phase A single oral dose of 10 mg of nifedipine (Adalats Bayer, column (Mightysil RP-18 GP250, 4.6 Â 250 mm2, Kanto Germany) with 200 ml of water was administered to the Chemical Industries Ltd, Kyoto, Japan). The column tem- subjects at 01000 after overnight fasting. Blood samples perature was set at 401C. The mobile phase was composed of (7 ml each) were collected before administration and at 0.5, 40% acetonitrile (pH 3.0 with perchloric acid) and was 1, 1.5, 2, 3, 4, 6, 8 and 12 h after administration. During this delivered at a constant flow rate of 1.2 ml/min. Nifedipine, study, adverse effects were assessed on an ongoing basis as M-I and phenytoin were detected by a UV detector (Nano- subjects offered information. Blood pressure and pulse rate space, SHISEIDO, Tokyo, Japan) with a wavelength at were measured before administration and at 1, 2, 3, 12 and 254 nm. In determining kinetic parameters, nifedipine 24 h after administration. A 12-lead electrocardiogram was concentration ranged from all points. recorded before administration and at 2.5 and 24 h after administration. Hematologic tests, blood chemistry and Subjects urinalysis were performed before administration and at 24 h Institutional Review Board approval of the study protocol after administration. was obtained. In all, 16 healthy male Japanese volunteers Grapefruit juice, St John’s Wort, alcohol, caffeine-containing participated in this study (Table 2). The subjects gave written beverages, tobacco and exercises were not allowed during informed consent to participate. the study. A total of 33 healthy volunteers was screened by the genotyping test to find the eight CYP3A5*1/*3 subjects and Assays eight CYP3A5*3/*3 subjects. The genotyping test of CYP3A5 Blood samples were collected in heparinized tubes and was conducted according to the previous study.6,26 All adequately protected from light and centrifuged to obtain subjects were healthy as assessed by medical history, serum. The samples were stored frozen at –201C until physical examination, hematologic tests, blood chemistry analyzed. Plasma concentrations of nifedipine and M-I were and urinalysis, and the results of a positive test for hepatitis measured by HPLC as described previously with minor B and C, human immunodeficiency virus and syphilis. modifications.27–29 Briefly, nifedipine and M-I were ex- Eight CYP3A5*1/*3 and eight CYP3A5*3/*3 subjects who tracted with dichloromethane/pentane (3 : 7, v/v) and showed normal results on routine laboratory tests described separation of the compounds was achieved using an Inertsil above and the negative results of virus tests were selected. ODS-3 column (3 mM particles, 4.0 Â 100 mm2, GL Sciences Inc., Tokyo, Japan). The compounds were detected at a Genotyping Test wavelength of 230 nm using a UV detector (SPD-10Avp, Genomic DNA was isolated from peripheral leukocytes using SHIMADZU, Tokyo, Japan). QIAGEN blood kit. The genotypes of each individual at the CYP3A5*3 and CYP3A5*6 alleles were determined using Pharmacokinetic Parameters

PCR-restriction fragment length polymorphism analysis The peak plasma concentration (Cmax) and the time to reach 6 according to the previous report. For the analysis of the the Cmax (Tmax) were obtained as measured values. The CYP3A5*3 allele, the forward (CYP3A5 6956Fm; 50-CTT TAA apparent first-order elimination rate constant (K) of nifedi- AGA GCT CTT TTG TCT CTC A-30) and reverse (CYP3A5 pine was determined by linear regression analysis of the 7155R; 50-CCA GGA AGC CAG ACT TTG AT-30) primers were slope of the terminal phase using the last three or four used (GenBank accession no. AC005020). For the analysis of points on the log plasma drug concentration–time curve. 0 the CYP3A5*6 allele, the forward (CYP3A5 14505F; 5 -GTG The terminal elimination half-life (t1/2) was calculated from 0 GGT TTC TTG CTG CAT GT-3 ) and reverse (CYP3A5 the relation t1/2 ¼ 0.693/K. The area under the plasma 14741R; 50-GCC CAC ATA CTT ATT GAG AG-30) primers concentration–time curve from 0 to 12 h after administra-

The Pharmacogenomics Journal CYP3A5 polymorphism and nifedipne pharmacokinetics T Fukuda et al 39

tion (AUC0–12 h) was calculated by the linear trapezoidal 9 Gillam EM, Wunsch RM, Ueng YF, Shimada T, Reilly PE, Kamataki T et al. method. The area under the drug concentration–time curve Expression of cytochrome P450 3A7 in Escherichia coli: effects of 50 modification and catalytic characterization of recombinant enzyme from time to infinity (AUCN) was determined by the linear expressed in bicistronic format with NADPH-cytochrome P450 trapezoidal method with extrapolation to infinity. Clearance reductase. Arch Biochem Biophys 1997; 346: 81–90. (CL/F) was calculated as dose/(AUCN). The plasma nifedi- 10 Azuma J, Yamamoto I, Watase T, Seto Y, Tanaka T, Kato M et al. Effects of grapefruit juice on pharmacokinetics of the calcium antagonists pine/M-I ratio was calculated from the AUC0–12 h value of nifedipine and M-I. nifedipine and nisoldipine. Jpn Pharmacol Ther (Japanese) 1996; 24: 267–276. 11 Ozdemir V, Kalowa W, Tang BK, Paterson AD, Walker SE, Endrenyi L et Data Analysis al. Evaluation of the genetic component of variability in CYP3A4 The pharmacokinetic parameters and the plasma nifedipine/ activity: a repeated drug administration method. Pharmacogenetics 2000; 10: 373–388. M-I ratios were first analyzed by one-way ANOVA. If the 12 Renwick AG, Robertson DR, Macklin B, Challenor V, Waller DG, George overall F ratio was significant, further comparison of the CF. The pharmacokinetics of oral nifedipine—a population study. Br J means was performed with the Student’s t-test. Statistical Clin Pharmacol 1988; 25: 701–708. analysis of the pharmacodynamic variables of systolic and 13 Bailey DG, Spence JD, Munoz C, Arnold JM. Interaction of citrus juices with felodipine and nifedipine. Lancet 1991; 337: 268–269. diastolic blood pressure, and pulse rate made under resting 14 Hashimoto K, Shirafuji T, Sekino H, Matsuoka O, Onnagawa O, conditions, were performed using ANOVA with repeated Okamoto T et al. 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Midazolam is metabolized by at least three different cytochrome P450 Grants and a Grant-in-Aid for Scientific Research from the Ministry enzymes. Br J Anaesth 1994; 73: 658–661. of Education, Culture, Sports, Science and Technology of Japan. We 18 Shih PS, Huang JD. Pharmacokinetics of midazolam and 10-hydro- thank Mr Ogura and Dr Kanada for giving the opportunity for the xymidazolam in Chinese with different CYP3A5 genotypes. Drug Metab clinical trial. We also thank Mr Seto and Mr Ikeda for their help. Dispos 2002; 30: 1491–1496. 19 Williams JA, Ring BJ, Cantrell VE, Jones DR, Eckstein J, Ruterbories K et al. Comparative metabolic capabilities of CYP3A4, CYP3A5, and CYP3A7. Drug Metab Dispos 2002; 30: 883–891. 20 Lin YS, Dowling AL, Quigley SD, Farin FM, Zhang J, Lamba J et al. DUALITY OF INTEREST Co-regulation of CYP3A4 and CYP3A5 and contribution to hepatic and intestinal midazolam metabolism. 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