PHARMACOGENETICS

Effects of CYP3A5, MDR1 and CACNA1C polymorphismson the oral disposition and response of nimodipinein a Chinese cohort

Ying Zhao & Desheng Zhai & Hui He & Tingting Li &Xijing Chen & Hui Ji

Received: 21 July 2008 /Accepted: 14 January 2009 /Published online: 11 February 2009# Springer-Verlag 2009

AbstractPurpose Our objective was to study the effects of poly-morphic the CYP3A5 (allele *1 and *3), MDR1 [singlenucleotide polymorphisms (SNPs) G2677T, C3435T] andCACNA1C (SNPs rs2239128, rs2239050, rs2238032)genes on nimodipine oral disposition and response inhealthy Chinese subjects.Methods Pharmacokinetics and pharmacodynamics datawere obtained from a bioequivalence study, and the same 20subjects were genotyped for CYP3A, MDR1 and CACNA1C.An additional 41 healthy Chinese subjects were recruited toobtain an indication of the distribution of CACNA1Cpolymorphisms in the Chinese population. Racial differencesin the frequency of CACNA1C alleles were assessed. Thephenotype differences between genotypes were analyzed.Results The allelic frequencies of rs2239050 andrs2238032 in our Chinese cohort were different from thosein a Caucasian population (p<0.01). Subjects with mutantalleles (*3/*3) of the CYP3A5 gene had a decreased oralclearance of nimodipine, with a higher lnCmax or

lnAUC0�1 compared with those subjects with the hetero-zygote (*1/*3) or wild type (*1/*1) gene. The CACNA1Crs2239128 C and rs2239050 G SNPs were associated witha stronger efficacy compared with their respective alleles,rs2239128 T and rs2239050 C. MDR1 polymorphismsshowed no significance in terms of nimodipine disposition.Conclusions The polymorphic CYP3A5 (allele *1 and *3)and CACNA1C genes have effects on nimodipine oraldisposition and response in healthy Chinese subjects. Thehomozygous variant of CYP3A5 (*3/*3) was associatedwith significantly increased nimodipine exposure. CAC-NA1C SNPs rs2239128 C and rs2239050 G were associ-ated with a stronger efficacy.

Keywords CACNA1C . Cytochrome P450 3A5 .

Nimodipine . Pharmacogenomics . Single nucleotidepolymorphism

AbbreviationsCACNA1C the calcium channel, voltage-dependent, L-type

alpha 1C subunitCYP3A5 cytochrome P450 3A5 geneDBP diastolic blood pressureSBP systolic blood pressureSNP single nucleotide polymorphism

Introduction

Nimodipine, a dihydropyridine calcium channel blocker,has been shown to be effective in preventing ischemiccomplications following subarachnoid hemorrhage [1, 2].Owing to its neuroprotective properties, nimodipine is also

Eur J Clin Pharmacol (2009) 65:579–584DOI 10.1007/s00228-009-0619-6

Y. Zhao :H. He : T. Li :X. Chen (*) :H. Ji (*)School of Pharmacy, China Pharmaceutical University,Nanjing 210009, Chinae-mail: chenxj@jlonline.come-mail: huijicpu@163.com

Y. ZhaoSchool of Pharmacy, Xinxiang Medical University,Xinxiang 453003, China

D. ZhaiSchool of Public Health, Centrol South University,Changsha 410078, China

D. ZhaiDepartment of Public Health, Xinxiang Medical University,Xinxiang 453003, China

used in other cerebrovascular disorders, such as ischemicstroke [3] and multi-infarct dementia [4].

Orally administered nimodipine is well absorbed in thegastrointestinal tract, but subject to extensive first-pass metab-olism that results in significant inter-individual variations [1].Nimodipine is extensively metabolized by the cytochromeP450 3A (CYP3A) subfamily, especially CYP3A4 andCYP3A5 [5]. Inter-racial variability in CYP3A5 expressionhas been reported to be determined by the frequency of themost prevalent CYP3A5 aberrant alleles (*3) [6].

The multidrug transporter MDR1 (P-glycoprotein, P-gp)is an adenosine triphosphate (ATP)-dependent drug effluxpump. It has been reported that polymorphism of MDR1 atloci 3435 is one mechanism that would explain inter-individual variability in intestinal P-gp expression [7].Empirical evidences suggests that dihydropyridine calciumchannel blockers act as substrates and inhibitors of P-gp [8–10]. Due to their similar tissue localization, substrateoverlap and co-inducibility, CYP3A and P-gp have beenpostulated to coordinately regulate drug disposition. Poly-morphic CYP3A5 and MDR1 may contribute to the inter-individual variability in oral nimodipine disposition.

While single nucleotide polymorphisms (SNPs) reported toimpact treatment response have been identified for manyblood-vascular system drug classes [i.e. β1 blockers, angio-tensin converting enzyme (ACE) inhibitors, angiotensin IIblockers], few SNPs have been identified to date for the classof calcium channel blockers [11]. In a recent retrospectivepharmacogenomic study carried out in 120 Caucasiansubjects, 62 SNPs were investigated in the gene CACNA1C(encoding the calcium channel, voltage-dependent, L-typealpha 1C subunit). The results showed that three SNPs(rs2239128, rs2239050, rs2238032) in CACNA1C hadsignificant associations with antihypertensive outcome [12].

In the study reported here, we investigated the effect ofgenetic polymorphisms of the CYP3A5 (A6986G), twoSNPs (C3435T and G2677T) in the MDR1 gene and threeSNPs (rs2239128, rs2239050, rs2238032) in the CACNA1Cgene on the disposition and response of nimodipine inhealthy Chinese male subjects.

Methods

Subjects

All of the pharmacokinetics data were obtained from abioequivalence study, which was given the approval of theHuman Ethics Committee of the Institute of Dermatonosis,Chinese Academy of Medical Sciences (Nanjing, China).Informed written consent was obtained from the subjects.Twenty healthy male Chinese volunteers (mean age23.7 years, mean body mass index 21.5) were entered intothe trial in which each participant received a single dose(60 mg nimodipine) of test (20 mg tablet; Tianjin, China)and reference product (Nimotop, 30 mg tablet; Bayer AG,Germany) in a blind, balance 2×2 cross-over designexperiment, separated by a 1-week washout period.

Since the focus of this study was not bioequivalenceissues of the two formulations but the effect of polymorphicCYP3A5, MDR1 and CACNA1C genes on oral nimodipinedisposition and response, only the pharmacokinetic data ofNimotop were used.

In terms of genetic information, all 20 subjects weregenotyped for CYP3A, MDR1 and CACNA1C. An addi-tional 41 healthy Chinese subjects, with written informedconsent, were recruited to obtain CACNA1C polymor-phisms in a Chinese cohort.

Genotyping of CYP3A5, MDR1 and CACNA1Cpolymorphisms

DNA was extracted from peripheral whole blood of eachsubject using a Tiangen DNA extraction kit (Tiangen). Thegenotypes of MDR1 (G2677T, G2677A and C3435T) andCYP3A5 (A6986G) were identified by special primersamplification–polymerase chain reaction (SPA-PCR) asdescribed previously [13, 14,].

The genotypes of CACNA1C (rs2239128, rs2239050,rs2238032) were identified by SPA–PCR using primers aslisted in Table 1. Oligonucleotide primers were designedbased on the published CACNA1C sequence (AC005866.4,

Table 1 Oligonucleotide sequences

Primers 5′ position Sequence 3′ position

rs2239128 C/T 9225 CAT gCA gTg AgA gCC Agg CA 9244CAT gCA gTg AgA gCC Agg Cg

9428 Cgg gCA TCT TCA Tgg gAg AC 9409rs2239050 C/G 93031 TgA CTT ACT gAT TCA gAA gAC 93051

TgA CTT ACT gAT TCA gAA gAg93241 TAT CCC Agg ACC CTA CAC 93224

rs2238032 G/T 21058 CCA gTT gCA CAT ACA TgA CA 21077CCA gTT gCA CAT ACA TgA CC

21267 CAT AgA Agg ATC AgC CCA Ag 21248

580 Eur J Clin Pharmacol (2009) 65:579–584

AC005293.1, AC006051.1). The lengths of the ampliconswere 204, 211 and 210 bp for rs2239128, rs2239050 andrs2238032, respectively.

All oligonucleotide primers were synthesized by inte-grated DNA technologies (Sangon, Shanghai, China). AllPCR reactions were carried out in a total volume of 25 μL,in a reaction mixture containing 0.25 mM dATP, dCTP,dGTP and dTTP, 200 nM both forward and reverse primers,1.0 U Taq DNA polymerase (Tiangen), 5% DMSO and 20–140 ng genomic DNA in 1× PCR buffer (pH 8.3, 10×solution containing 100 mM Tris-HCl, 500 mM KCl and15 mM MgCl2 and 0.01% gelatin) (Tiangen).

The amplification protocol consisted of one cycle of95°C with a 120-s hold followed by 27 cycles of 95°Cwith a 10-s hold, a specific annealing temperature of 60°Cwith 20-s hold and 72°C with a 20-s hold. Positivecontrols with definite specific genotype templates andnegative controls without DNA templates were run withevery assay to assess the overall specificity.

Pharmacokinetic and pharmacodynamic procedures

Blood was obtained before dosing and at 0.25, 0.5, 0.75, 1, 1.5,2, 2.5, 3, 4, 6, 8, 10 and 12 h after nimodipine administration. Aliquid chromatographic–electrospray ionization–mass spectro-metric (LC-ESI-MS) method was used to determine nimodi-pine in human plasma [15]. The limit of quantification was0.5 ng/mL. The method was validated within the linear range0.5–100 ng/mL. The intra- and inter-day precision (expressedas the coefficient of variation) and accuracy (expressed asrelative error) of the assay were lower than 10%.

Pharmacokinetic parameters, such as the maximumplasma concentration ©max), were obtained directly fromthe measured data; area under the plasma concentration–time curve from 0 to infinity (AUC0�1) and oral clearance(Cl/F) were computed using WinNolin 5.0.1 (Pharsight,Mountain View, CA)

Systolic blood pressure (SBP) and diastolic bloodpressure (DBP) were measured at 0, 2, 4, 6, 8, 10 and12 h after administration. During the first 0–12 h after

nimodipine administration, the temperature, pulse andelectrocardiogram were recorded.

Statistical analysis

Statistical analyses were performed using SPSS ver. 12.0(SPSS, Chicago, IL). Measurement data results areexpressed as the mean ± standard deviation. Racialdifferences in allele frequencies as well as deviationfrom Hardy–Weinberg equilibrium were assessed usingthe χ2 test. Analysis of variance (one-way ANOVA) withLSD/Dunnett for post hoc analysis was used to comparethe pharmacokinetics parameters between different geno-types. Repeated measure analysis of variance was used tocompare the decrease in blood pressure due to nimodipineadministration between different genotypes. A probability(p) of less than 0.05 was considered to be statisticallysignificant.

Results

Distribution of CACNA1C allelic genes frequencyand genotypic frequency in healthy Chinese subjects

The allelic distribution of the three CACNA1C SNPs didnot deviate from Hardy–Weinberg equilibrium in the studygroup. The allelic and genotypic frequency of CACNA1C inthe healthy Chinese subjects is shown in Table 2. As seenthe allelic frequency of rs2239128 in our Chinese cohortwas similar to that found in a Caucasian population [12],while the allelic frequencies of rs2239050 and rs2238032 inour Chinese cohort were different from those in the sameCaucasian population (p<0.01).

Phenotype and genotype interaction

Among the 20 subjects, ten were identified as CYP3A5 *1allele carriers (three CYP3A5*1/*1 and seven CYP3A5*1/*3 individuals) and ten as carrying the CYP3A5 homozy-

Table 2 Distribution of CACNA1C allelic genes and their frequency and genotypic frequency in 61 Chinese healthy subjects compared with thatin 120 Caucasian subjects [12]

Race SNP Allele Frequency Allele Frequency Genotype Frequency Genotype Frequency Genotype Frequency

Chinese rs2239128 C 0.68 T 0.32 CC 0.41 CT 0.54 TT 0.05Caucasian 0.69 0.31Chinese rs2239050 C 0.87** G 0.13** CC 0.75 CG 0.23 GG 0.02Caucasian 0.22 0.78Chinese rs2238032 G 0.05** T 0.95** GG 0 TT 0.90 GT 0.10Caucasian 0.22 0.78

**p<0.01 compared to Caucasian population [12]SNP, Single nucleotide polymorphism

Eur J Clin Pharmacol (2009) 65:579–584 581

gous variant (CYP3A5*3/*3). The allele distribution did notdeviate from Hardy–Weinberg equilibrium in the group.

Homozygous variantCYP3A5 (*3/*3) was associated withsignificantly increased nimodipine exposure. Compared to

CYP3A5 *1 allele carriers, carriers of the CYP3A5 homozy-gous variant showed significantly lower Nimodipine oralclearance [538.1±152.7 vs. 757.7±259.6 L/h, p=0.033, 95%CI (confidence interval) for differences −419.763, −19.544,power of test 0.588), and significantly higher lnCmax (3.99±0.35 vs. 3.53±0.33 μg/L, p=0.008, 95% CI for differences0.131, 0.775, power of test 0.798) and lnAUC0∼−∞ (4.72±0.30 vs. 4.39±0.33 μg h/L, p=0.031, 95% CI for differences0.033, 0.623, power of test 0.599) (Fig. 1).

Homozygotes (CC, n=4), heterozygotes (CT, n=14) andthe homozygous variant (TT, n=2) at MDR1 nucleotideposition 3435 were identified. Three homozygotes (GG), 13heterozygotes (GT) and four homozygous variants (TT) atMDR1 nucleotide position 2677 were identified. Both ofthese allele distributions did not deviate from Hardy–Weinberg equilibrium in the group.

Although some trends in the oral nimodipine exposure wereseen for the 2677 genotype, no difference was detected for oralclearance, lnCmax or lnAUC0�1 between the MDR1 3435and 2677 genotypes, possibly due to the small sample sizes.

The presence of the rs2239128 SNP was associated witha significant SBP decrease in subjects with the CC variantcompared with those with the CT variant (9.0±10.1 vs. 2.1±9.1 mmHg, p=0.038). The rs2239050 SNP was associatedwith a significant SBP decrease in subjects with the CGvariant compared with those with the CC variant (9.8±7.7 vs. 3.6±10.3 mmHg, p=0.049). However, no differencewas detected for the nimodipine effect on SBP betweendifferent rs2238032 genotypes (TT and TG) nor was adifference detected for the nimodipine effect on diastolicblood pressure (DBP) between different CACNA1C(rs2239128, rs2239050 and rs2238032) genotypes (Table 3).

Discussion

In this study, we investigated the effect of the SNPsCYP3A5 (A6986G) and MDR1 (C3435T and G2677T) on

*

0

500

1000

1500

*1/*1or*1/*3

*1/*1or*1/*3

*1/*1or*1/*3

CYP3A5 Genotype

Cl/F

(L/h

)

A

*

0

1

2

3

4

5

*3/*3

*3/*3

*3/*3

CYP3A5 Genotype

lnC

max

B

*

0

1

2

3

4

5

6

CYP3A5 Genotype

lnA

UC

C

Fig. 1 Effect of the CYP3A5 genotype on nimodipine oral clearance(Cl/F, a), maximum plasma concentration (lnCmax, b) and area underthe concentration–time curve (lnAUC0∼−∞, (c). *p<0.05

Table 3 Effects of CACN1AC genotype on the blood pressure decrease of nimodipine

SNP Genotype N SBP decrease (mmHg) 95% CI fordifferences

Powerof test

2 h 4 h 6 h 8 h 10 h 12 h total

rs2239128 TC 12 5.3±12.0 5.4±6.9 4.1±9.4 3.1±6.4 −1.8±8.5 −3.7±7.8 2.1±9.1* −13.366, −0.440 0.565CC 8 13.8±9.5 10.8±9.8 8.1±9.8 10.5±12.6 7.5±9.7 3.4±9.5 9.0±10.1

rs2239050 CG 4 13.0±5.9 8.3±5.6 8.8±8.8 16.5±9.3 9.5±8.2 2.5±3.0 9.8±7.7* 0.312, 14.541 0.303CC 16 7.6±12.6 7.4±9.1 4.9±9.8 3.50±8.2 0.1±9.5 −1.7±9.8 3.6±10.3

rs2238032 TT 18 8.5±11.7 7.2±8.7 5.9±10.0 6.1±9.2 1.8±10.2 −0.7±9.4 4.8±10.2 −12.372, 11.502 0.051TG 2 10.5±14.8 10.5±6.4 3.5±2.1 6.0±19.8 3.0±9.9 −2.0±2.8 5.3±9.5

* p<0.05CI, Confidence intervalData are expressed as mean ± standard deviation

582 Eur J Clin Pharmacol (2009) 65:579–584

nimodipine oral disposition. Although many researchershave tried to examine the role of CYP3A5 on drugmetabolism and disposition, the results are still controver-sial [14, 16–18]. Jin et al. reported that CYP3A5 expressersdisplay a greater steady-state oral clearance of verapamilthan non-expressers [19]; however, they obtained oppositeresults on the role of CYP3A5 on dihydropyridine calciumchannel blocker metabolism. Kim et al. found thatCYP3A5*3/*3 carriers exhibited lower plasma amlodipineconcentrations than CYP3A5*1 carriers [20]. The results ofour study suggest that the homozygous variant CYP3A5(*3/*3) was associated with significantly increased nimo-dipine exposure. Given the finding that CYP3A5 genotypeimpacts on the pharmacokinetics of nimodipine, it isworthwhile determining the impact of CYP3A5 genotypeon the effects of therapy as well as the adverse effects andthe potential drug–drug interactions. Nimodipine is one ofthe few drugs for which level-1 evidence is available for thetreatment of vasospasm from aneurysmal subarachnoidhemorrhage, as defined by the U.S. Preventative ServicesTask Force [21]. The most significant adverse effect is thereduction of blood pressure in some patients. Clinicalstudies have documented a decrease in blood pressure insmall groups of patients, including cases where the patientwithdrew from the study due to pronounced hypotensioninduced by nimodipine administration [1, 22]. A number ofclinically relevant drug interactions have been reported. Forexample, in epileptic patients taking the anticonvulsantscarbamazepine, phenobarbital and/or phenytoin, there was aapproximate sevenfold decrease in the AUC of nimodipine[23]. However, in vitro studies and further larger sampleinvestigations should be designed to confirm the role of theCYP3A5 genotype on nimodipine metabolism and disposi-tion as well as the pharmacodynamic effects.

There is some evidence suggesting that nimodipine acts asa substrate of P-gp [9, 24] and that the MDR1 (3435 and2677) genotype can influence the disposition of amlodipine,a dihydropyridine calcium channel blocker [25]. However,the results of our study show that MDR1 polymorphismsplayed no significant role in nimodipine disposition. Inaddition to the small sample size possibly limiting thedetection of any significance, it is also possible that the roleof MDR1 in disposition was outweighed by metabolicenzymes, since nimodipine, which is well and quicklyabsorbed, is subject to extensive first-pass metabolism [1].

We report here the distribution of CACNA1C allelicgenes, their frequency and genotypic frequency in healthyChinese subjects. Consistent with results reported in theliterature [12], the rs2239128 SNP in our study wasassociated with a significant decrease in the SBP in subjectswith the CC variant compared with those with the CTvariant, and the rs2239050 SNP was associated with asignificant decrease in SBP in subjects with the CG variant

compared with those with the CC variant (Table 3).However, contrary to the findings in the literature [12],we were unable to detect a difference for the nimodipineeffect on SBP between different rs2238032 genotypes (TTand TG), nor for the nimodipine effect on DBP betweendifferent CACNA1C (rs2239128, rs2239050 andrs2238032) genotypes. It is possible that the negativeresults in our study represent an artifact, which is alwayspossible when attempts are made to assess multiple locigenotype/phenotype correlations in a limited number ofhealthy subjects. Further large sample studies should bedesigned to evaluate the relationship between CACNA1Cpolymorphisms and antihypertensive outcome. The ques-tion of whether the racial difference in allelic genefrequency in CACNA1C between Chinese and Caucasianplays a role in racial differences in the pharmacodynamicsof calcium channel blockers remains unanswered. Thiswarrants further evaluation.

Acknowledgements The research was founded by National NaturalScience Foundation of China (No. 20 30472060)

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