Regular Article

ABCB1 Polymorphism and Gender Affect the Pharmacokineticsof Amlodipine in Chinese Patients with Essential Hypertension:

A Population Analysis

Xiao-cong ZUO1,2, Wen-li ZHANG1,2, Hong YUAN1, Jeffrey S. BARRETT3, Ye HUA1,2, Zhi-jun HUANG1,2,Hong-hao ZHOU4, Qi PEI1, Cheng-xian GUO1, Jiang-lin WANG1,2 and Guo-ping YANG1,4,*

1Center of Clinical Pharmacology and Pharmacy Department, The Third Xiangya Hospital,Central South University, Changsha, China

2School of Pharmaceutical Science, Central South University, Changsha, China3Laboratory for Applied PK/PD, Clinical Pharmacology & Therapeutics Division,

The Children’s Hospital of Philadelphia, Philadelphia, PA, USA4Institute of Clinical Pharmacology, Central South University, Changsha, China

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: The effects of genetic polymorphisms of ABCB1 C3435T, POR*28, CYP3A4*1G andCYP3A5*3 variants and gender relating to metabolism on the exposure and response of amlodipine inChinese hypertensive patients were determined. Population pharmacokinetic analyses were performedon data which were collected prospectively from 60 Chinese patients with mild to moderate essentialhypertension [age range 40­74 years, males (n = 31), females (n = 29)] receiving oral racemic amlodipinefor 4 weeks. Blood pressure was evaluated at the end of weeks 0 and 4. Blood samples were collectedin heparinized tubes at the following times: 0, 2, 6, and 24 h on about day 28 after administration ofamlodipine. A one-compartment model with first-order elimination and absorption best described theamlodipine pharmacokinetic data. ABCB1 3435 genetic polymorphism and gender affect the amlodipineoral clearance (CL/F). CL/F (L/h) = 28.8 © (1 + GNDR)¹0.531 © (ABCB1 C3435T) where GNDR = 0 and 1are for male and female, respectively. The CL/F value in a male patient with the ABCB1 3435CC or CTgenotype is 28.8 L/h. Lower CL/F and higher exposure occurs in female subjects with the ABCB1 3435CCor CT genotype who have greater decreases in blood pressure after treatment with amlodipine. The resultsmay help to improve the efficacy and tolerability of amlodipine in essential hypertensive patients.

Keywords: ABCB1 C3435T; gender; amlodipine; hypertension; population pharmacokinetics

Introduction

Amlodipine, a third-generation calcium antagonist, is mainlyused in the treatment of angina and hypertension diseases.1) Ithas been shown to suppress the calcium influx through the L-typecalcium channel in peripheral vascular and coronary smoothmuscle cells, thus leading to significant vasodilation in periph-eral and coronary vascular beds. Structural characteristics ofamlodipine offer unique pharmacokinetic properties amongcalcium antagonists. It has a relatively high bioavailability, ranging

from 65% to 90%, and higher tissue affinity, which resulted in arelatively long time to peak plasma concentration (Cmax) of 6–12 hand a longer plasma elimination half-life (t1/2) of 30–50 hcompared with other dihydropyridine calcium antagonists.

What’s more, 90% of an amlodipine oral dose is convertedto inactive metabolites in the liver. Amlodipine functions as asubstrate of cytochrome P450(CYP) 3A2,3) and P-glycoprotein(P-gp), product of the multidrug resistance 1 (MDR1, known as theABCB1) gene.4,5) In studies of healthy subjects, our previousstudy6) and Kim et al.7) showed that the CYP3A5*3 allele is a main

Received November 20, 2013; Accepted January 23, 2014J-STAGE Advance Published Date: February 11, 2014, doi:10.2133/dmpk.DMPK-13-RG-127*To whom correspondence should be addressed: Guo-ping YANG, Ph.D., Center of Clinical Pharmacology and Pharmacy Department, TheThird Xiangya Hospital of Central South University, Changsha 410013, China. Tel. ©86-731-88618326, Fax. ©86-731-88618326, E-mail:ygp9880@126.comThis study was funded by Natural Science Foundation of Hunan Province (No. 11JJ2054), National Science and Technology Major Projects forMajor New Drugs Innovation and Development (No. 2012ZX09303014-001), National Natural Science Foundation of China (No. 81102512,81373476), International Science & Technology Cooperation Program of China (No. 0S2014ZR0110) and Program for New Century ExcellentTalents in University (No. NCET-13-0605).

Drug Metab. Pharmacokinet. 29 (4): 305–311 (2014). Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)

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determinant of the apparent clearance (CL/F) of amlodipine. Thepolymorphic ABCB1 gene in humans may also explain the inter-individual variation in the disposition of amlodipine.8) AmongAfrican-American men and women with early hypertensive renaldisease, the CYP3A4 genotype appears to be associated with bloodpressure response to amlodipine.9) Genetic and environmentalfactors contribute towards the variability of drug response. Todetermine the multifactorial effects on the exposure and responseof amlodipine, we developed a population pharmacokinetic modelof amlodipine in Chinese patients with mild to moderate essentialhypertension and investigated the potential role of the CYP3A andABCB1 genetic polymorphisms in the variability of amlodipinedisposition through assessing pharmacokinetic parameters and theassociation of genotypes on the CYP3A and ABCB1 genes withblood pressure responses to amlodipine.

Materials and Methods

Patients: For all patients, informed consent in accordance withthe guidelines of the declaration of Helsinki was obtained for thestudy, which had been approved by the Third Xiangya Hospitalof Central South University Ethics Committee. The clinical trialincluded 60 Han Chinese men and women between the ages of40 and 75 years and diagnosed with mild to moderate essentialhypertension (mean seated systolic blood pressure (SBP) 140–180mmHg or diastolic blood pressure (DBP) 90–110mmHg), andtaking stable doses of amlodipine. Subjects were excluded if theyhad a history of secondary hypertension, severe hypertension, acutephase of cerebral stroke, congestive heart failure, unstable anginapectoris, acute myocardial infarction within the previous 6 months,arrhythmia, bilateral renal artery stenosis, hyperkalemia, cancer,severe hepatic or renal diseases, or were unable to provide informedconsent. Pregnant women, breastfeeding women, alcohol or drugaddicts and those with mental disabilities were also not enrolled.

Study design: After eligibility was confirmed and consentobtained, patients were scheduled for the trial. The study consistedof two phases: a 1-week placebo washout phase and a 4-weektreatment period with amlodipine 5mg once daily.

Subjects with antihypertensive drugs had the medicationdiscontinued just before entry into the placebo washout phase.Participants were entered into the treatment phase if their meanseated systolic blood pressure was between 140 and 180mmHgor diastolic blood pressure was between 90 and 110mmHg. Theyreceived amlodipine 5mg once daily during the treatment phase.To ensure the patients’ compliance with amlodipine treatment,patients with good compliance were reminded to take theirmedicine every day by our appointed community physicians.

On day 1 of the placebo washout phase, the following evalu-ation was performed for patients to meet all inclusion criteria:complete medical history and routine physical exam, bloodpressure and heart rate, 12-lead electrocardiograph (ECG), andclinical laboratory testing.

Patients were evaluated at the initiation and end of the washoutday, and the end of the treatment study. Their visits were scheduledin the morning, and all evaluations were performed at drug troughlevel (24 « 2 h after the previous day’s dose) and before the nextdose was taken. The following evaluations were performed at theend of weeks 0 and 4: demographic data, blood pressure, heart rate,concomitant drugs, and other related clinical items.

Sampling: On about day 28, patients were admitted to theResearch Center of Clinical Pharmacology for 24-h pharmaco-

kinetic and pharmacodynamic assessments. Five ml blood sampleswere collected in heparinized tubes at the following times: 0,2, 6, and 24 h after administration of amlodipine. Samples werecentrifuged at 3,000 rpm for 10min and the plasma was separatedand stored at ¹20°C until analysis. Double tubes of each samplewere kept.

Determination of amlodipine concentrations: Plasmaamlodipine concentrations were analyzed by LC-MS/MS consist-ing of API 4000 triple-quadruple tandem mass. Detection wasperformed by ESI+ in MRM mode. For details, see our publishedarticles.6) The lower limit of quantification was 0.081 ng/ml andstandard calibration curves showed good linearity within the rangeof 0.0810 ng/ml to 36.31 ng/ml (r2 = 0.9956). The intra-day andinter-day coefficients of variation of all assays were less than 15%.

Genotyping: DNA was extracted from peripheral wholeblood of each subject using a Promega DNA extraction kit(DNA Mini-Prep Kit, Promega, Madison, WI). The genotypes ofABCB1 C3435T (rs1045642), CYP3A4 G20230A (CYP3A4*1G,rs2242480) and CYP3A5 A6986G (CYP3A5*3, rs776746), andP450 oxidoreductase (POR) A503V (POR*28, rs1057868) wereidentified by polymerase chain reaction-restriction fragment lengthpolymorphism analysis as described previously10) and results wereconfirmed for randomly selected individuals for each genotypeby a developed and validated automated DNA sequencer (ABIPRISM 3730 Sequence Detection System; Applied Biosystems) inShanghai Sangon Biological Engineering Technology & ServicesCo., Ltd. (Shanghai, China). Hardy-Weinberg equilibrium wasassessed by using the Pearson goodness of fit (»2) test.

Pharmacodynamics: Blood pressure determinations weremade using the calibrated Electronic Blood Pressure Monitor(HEM-7012, Omron Co. Inc., Tokyo, Japan) and the patient’s samearm throughout the course of the study. Blood pressure takenon day 0 was recorded in both arms. Seated blood pressure wasobtained on the sampling day at 0 h.

Population pharmacokinetic model development: The datawere graphically presented using R software, version 2.14.0 (Rfoundation for statistical computing, http://www.r-project.org). Thenonlinear mixed-effects modeling software program NONMEM(version VII, level 2.0; ICON Development Solutions, Ellicott City,MD) was used to determine amlodipine’s population pharmaco-kinetic using first-order conditional estimation method with thesubroutines ADVAN2 TRANS2. Maximum likelihood estimateswere carried out for CL/F and apparent volume of distribution(V/F). Various compartmental models and combinations of inter-individual and residual error models were assessed in describingthe plasma concentration-time data for amlodipine.Subject covariate analysis

Factors screened for effects on these parameters included thefollowing: gender (GNDR), age, height, body weight, body massindex, history of smoking, drinking and family hypertension,POR, CYP3A4, CYP3A5 and ABCB1 genotypes, alanine amino-transferase, aspartate aminotransferase, total protein (TP), totalbilirubin, direct bilirubin, blood urea nitrogen, serum creatinine,uric acid, hematocrit, and co-administration with drugs such asinsulin, metformin, acarbose and aspirin.

Continuous covariates were plotted against each subject numberto identify correlations between any two covariates. Box plots ofcontinuous covariates, sorted by categorical covariates, were alsocreated to identify the potential relationships between any twocovariates. Covariates with visual relationships were separately

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examined further by NONMEM for statistical significance. Thoseidentified as potentially influencing pharmacokinetic parameterswere then incorporated stepwise into the model.

Parameter-covariate relationships were included in a full tenta-tive pharmacokinetic model if the covariate contributed at leasta 3.84 change in the objective function (¡ = 0.05 for 1 degreeof freedom, »2). Covariates were then excluded from the modelusing a simple backward elimination method if the covariaterelationship did not contribute at least a 7.88 change in theobjective function (¡ = 0.005 for 1 degree of freedom, »2.Model assessment and validation

Graphic plots for validation of the population pharmacokineticmodel were constructed as follows: population predicted (PRED)and individual predicted (IPRED) values versus observed concen-trations (DV), and PRED versus conditional weighted residuals(CWRES). The accuracy and robustness of the final model selectedwas checked using the nonparametric bootstrap method and thestandardized visual predictive check (SVPC) method. Internalvalidation was undertaken using bootstrapping with the Perl speaksNONMEM (PsN) program (version 3.5.3).

SVPC displays observations and predictions on a standardizedscale. SVPC is better than VPC when there are multiple categori-cal covariate effects on pharmacokinetic parameters. The SVPCshows the percentile of each observation of each participant in themarginal distribution of model-simulated endpoints (Pi,j) as afunction of time using that participant’s own design template.11)

In this study, 1,000 data sets were simulated using the final modelbased on the original data set by setting NSUBPROBLEMS =1,000 in NONMEM to implement the SVPC.

Statistical analysis: SPSS for Windows (version 18.0;SPSS Inc., Chicago, IL) was used to analyze data described asmean « SD. The allele and genotype frequencies of the CYP3Aand ABCB1 polymorphisms were assessed for deviation from theHardy–Weinberg equilibrium using the »2 test. Pearson’s product-moment correlation was used to examine the relationship betweenthe area under the concentration–time curve (AUC) of amlodipineand the difference of seated systolic blood pressure from its base-line value. p values of less than 0.05 were considered statisticallysignificant.

Results

Patient demographics: Data were collected from 60 adultpatients with mild to moderate essential hypertension (31 men,29 women). Patient characteristics are presented in Table 1. AllSNP distributions of the POR*28, CYP3A5*3, CYP3A4*1G,ABCB1 C3435T were in Hardy–Weinberg equilibrium (p > 0.05).Mutant alleles and genotype frequencies are described in Table 2.

Amlodipine concentrations: A total of 228 plasma concen-trations of amlodipine are available for population modeling(Fig. 1). On the sampling day, the mean plasma concentrationsof amlodipine at 0, 2, 6, and 24 h were 7.32 « 3.34 ng/ml,8.95 « 4.00 ng/ml, 10.91 « 4.61 ng/ml and 7.51 « 3.21 ng/ml.Drug concentrations distributed in the absorption phase, distribu-tion phase and elimination phase.

Population pharmacokinetic analysis:Model development

Multiple structural models were explored to determine themodel that best fit the amlodipine concentration data. One-compartment and two-compartment models with zero-order andfirst-order absorption models with and without absorption lag timewere compared. We found that amlodipine pharmacokinetic datawere adequately shown by a one-compartment model with first-order elimination and absorption. The parameters estimated werethe absorption rate constant (Ka), oral clearance (CL/F), andapparent volume of distribution (V/F). Exponential and propor-tional plus additive error models were used to appropriatelydescribe the inter-subject and residual variabilities, respectively.Covariate analysis

In the forward model building step, adding GNDR, ABCB1C3435T genotypes and TP to CL/F decreased the OFV by morethan 3.84 (p < 0.05) at each addition. Adding ABCB1 C3435Tto V/F also decreased the OFV significantly. GNDR on CL/F andABCB1 C3435T on V/F of amlodipine appeared to be the mostimportant influential covariates.

In the final backward elimination step, a significant increase inOFV by more than 7.88 (p < 0.005) was observed on eliminationof GNDR and ABCB1 C3435T from the model. However, 0 fell inthe 95% confidence interval of the typical value of the effect ofABCB1 C3435T on V/F; therefore we removed it from the finalmodel. The covariate modeling process is shown in Table 3. CL/F

Table 1. Demographic, biological, and genetic data for patients

Characteristics Valuea Male Female

Number of patients 60 31 29Number of amlodipine concentrations 228 120 108Age (years) 59 (40, 74) 61 (40, 74) 58 (41, 71)Weight (kg) 64 (45, 100) 73.5 (52, 100) 62 (45, 77)Height (m) 1.63 (1.48, 1.71) 1.67 (1.59, 1.71) 1.57 (1.48, 1.71)BMI (kg/m2) 24.9 (16.5, 34.6) 25.4 (19.7, 34.6) 24.6 (16.5, 29.3)Systolic blood pressure (mmHg) 151 (133, 177) 151 (133, 177) 151 (133, 171)Diastolic blood pressure (mmHg) 91 (71, 107) 91 (72, 107) 91 (71, 105)Hemoglobin 130 (99, 167) 138 (99, 167) 125 (100, 150)Aspartate aminotransferase (U/L) 23 (15, 64) 23 (18, 63) 25 (15, 64)Alanine aminotransferase (U/L) 23.5 (7, 88) 26 (7, 88) 23 (8, 81)Total bilirubin (mmol/L) 12.2 (5.0, 31.7) 12.0 (5.0, 31.7) 12.2 (5.5, 22.4)Blood urea nitrogen (mmol/L) 5.01 (2.28, 13.85) 5.06 (2.70, 13.85) 4.54 (2.28, 8.02)Serum creatinine (mmol/L) 69 (25, 330) 69 (25, 330) 56.5 (33, 148)Number of patients with family hypertension history (male/female) 18/23Number of patients with smoking history (male/female) 12/0Number of patients with drinking history (male/female) 12/0

aValues are expressed as median (range) unless specified otherwise.

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was affected by gender and ABCB1 C3435T genotype, illustratedin the following equation:

CL=Fi ¼ 28:8� ð1þ GNDRiÞ�0:531 � ðABCB1 C3435TÞiwhere ABCB1 3435TT is 1.503 for individuals with lowerenzymatic activity of ABCB1. The parameters for the finalcovariate model for the CL/F of amlodipine are shown in Table 4.

The CL/F of amlodipine in male or subjects with ABCB1 3435TTgenotype is greater than that in female or ones with ABCB13435CC or CT genotype. The model estimates that a female sub-ject with ABCB1 3435TT genotype would have a CL/F of 30.0L/h. The residual error model indicated that an additive term inaddition to the proportional error term was applicable to the study.Model validation

Figure 2 shows the goodness-of-fit plots for the finalamlodipine 1-compartment model. The individual- and popula-tion-predicted plasma amlodipine concentrations matched theobserved plasma amlodipine concentrations, demonstrating thatthe model adequately described the data. The CWRES showed arandom distribution around zero and 95% of them were within anacceptable range (–2 to 2).

The final model was subjected to bootstrap analysis. The meanparameter estimates and percentile-based bootstrap 95% CIsobtained from 1,000 runs are presented in Table 4. Minimizationand the covariance step were successful in 100.0% and 87.3%of bootstrap runs, and the mean parameter estimates were notstatistically different from the estimates obtained using the originaldataset. The model is also stable because the variability estimatesobtained from bootstrap analysis are close to those obtained fromthe original dataset.

Figure 3 shows a SVPC of the pharmacokinetic model foramlodipine after oral administration to adult patients with mild

Fig. 1. Observed plasma concentrations of amlodipine versus time

Table 3. Population pharmacokinetics modeling process

No. Model description OFV ¦OFV

Model building (df = 1, p = 0.05, 3.84)1 1-Compartment base model 624.82 Add GNDR on CL/F in model 1 613.0 11.83 Add TP on CL/F in model 2 608.9 4.14 Add ABCB1 on CL/F in model 3 604.3 4.65 Add ABCB1 on V/F in model 4 591.5 12.8Model elimination (df = 1, p = 0.005, 7.88)6 Remove GNDR on CL/F in model 5 601.7 ¹10.27 Remove ABCB1 on CL/F in model 5 599.9 ¹8.48 Remove TP on CL/F in model 5 594.7 ¹3.29 Remove ABCB1 on V/F in model 5 604.3 ¹12.8Final model 607.2

OFV, objective function value; GNDR, gender; TP, total protein; ABCB1, thegene encoding P-glycoprotein.

Table 4. Population pharmacokinetic parameters of amlodipine in 60 adultpatients with mild to moderate essential hypertension and bootstrapvalidation

ParametersFinal model Bootstrap

Estimate RSE (%) Median 95% CI

CL/F (L/h) 28.8 7.1 28.9 (25.7, 32.6)V/F (L) 858 23.6 846 (98.4, 1,174)Ka (h¹1) 0.229 27.1 0.225 (0.031, 0.332)GNDR CL/F (L/h) ¹0.531 26.4 ¹0.551 (¹0.764, ¹0.283)ABCB1 CL/F (L/h) 1.503 30.2 1.594 (1.063, 3.066)Inter-individual variabilityCL/F (%) 36.1 12.7 35.6 (28.8, 43.9)Residual variabilityCV (%) 18.5 9.0 18.2 (15.4, 20.8)SD (µg/L) 0.615 30.7 0.622 (0.27, 0.85)

CL/F, apparent clearance; V/F, apparent distribution volume; GNDR, gender;ABCB1, the gene encoding P-glycoprotein; CV, proportional residual variance;SD, additive residual variance; RSE, relative standard error; CI, confidenceinterval.

Table 2. Frequency of genotype POR*28, CYP3A4*1G, CYP3A5*3 and ABCB1 C3435T in 60 essential hypertensive patients

Name GenotypesFrequency ofgenotype (%)(n = 60)

No. ofmales

No. offemales

AlleleFrequencyof allele(%)

Frequency of allelein males

(%)

Frequency of allelein females

(%)

POR*28(C>T)

CC 48.3 14 15 C 69.2 66.1 72.4CT 41.7 13 12 T 30.8 33.9 27.6TT 10.0 4 2

CYP3A4*1G(G>A)

GG 53.3 17 15 G 71.7 74.2 69.0GA 36.7 12 10 A 28.3 25.8 31.0AA 10.0 2 4

CYP3A5*3(A>G)

AA 11.7 4 3 A 31.7 32.2 31.0AG 40.0 12 12 G 68.3 67.8 69.0GG 48.3 15 14

ABCB1 C3435T(C>T)

CC 50.0 15 15 C 70.0 69.4 70.7CT 40.0 13 11 T 30.0 30.6 29.3TT 10.0 3 3

POR, P450 oxidoreductase; CYP, cytochrome P450; ABCB1, the gene encoding P-glycoprotein.

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to moderate essential hypertension. The open circles representcalculated Pi,j for each observation versus time, obtained from1,000 simulated datasets.

Exposure-response analysis for SBP: Figure 4 shows therelationship between changes in systolic blood pressure on aboutday 28 from its baseline and the AUC of amlodipine. Exploratoryplots indicated that a linear model was preferable for modeling thedependence of SBP on amlodipine exposure. The coefficient ofcorrelation between them was 0.534 (p < 0.05).

Discussion

A 1-compartment linear model with first-order absorption

Fig. 3. Visual predictive check for final covariate model, SVPC plots foramlodipine pharmacokinetic modelOpen circles, calculated Pi,j for each observation versus time; dashed line,model-predicted 5th, 50th, and 95th percentiles of model-predicted Pi,j (frombottom to top). Pi,j, percentile of each observation of each participant in themarginal distribution of model-simulated endpoints.

Fig. 4. Exploratory plots of blood pressure response as a function of AUC

a

b

c

Fig. 2. Goodness-of-fit plots for the final model(a) Population-predicted amlodipine plasma concentration (PRED) versusobserved amlodipine plasma concentration (DV); (b) individual-predictedamlodipine plasma concentration versus DV; (c) conditional weighted residualsversus PRED.

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and elimination was found to be the best structural model foramlodipine in the present study. This is in agreement with findingsin previous studies.12,13) Gender and ABCB1 genotype significantlyaffected the CL/F of amlodipine. Lower CL/F and higher expo-sure occurs in female subjects with the ABCB1 3435CC or CTgenotype. The population values of CL/F for a subject in this studywere 28.8 L/h and 19.9 L/h for males and females, respectively,which is consistent with previous reports that hypertensive childand adolescent subjects have CL/F values of 23.7 L/h in males and17.6 L/h in females12) and, is also similar to 23.0 L/h and 20.8 L/hcalculated by lean body weight in males and females with meanages greater than 70 years, respectively.14)

The frequencies of ABCB1 3435CC, CT and TT are 0.5, 0.4and 0.1, which is similar to the report on Chinese hypertensivepatients.15) Higher CL/F of amlodipine was observed in subjectscarrying ABCB1 3435TT in the present study. This is consistentwith the report that 3435TT genotypes increased the amlodipineconsumption.8) That is to say, subjects with the ABCB1 3435TTgenotype need higher dose of amlodipine to obtain the sameantihypertensive effect as the ABCB1 3435CC and CT genotypegroups.

It has been reported that P-gp was widely distributed in severalhuman tissues, including liver, intestine, pancreas and kidney.16)

The polymorphism in exon 26 (C3435T) of MDR-1 is related tothe decreased protein expression and function, and affects theabsorption and tissue concentrations of the substrate of MDR-1.17)

The expression level of P-gp was higher in ABCB1 3435CCor 3435CT subjects than in 3435TT ones. Low P-gp activity inthe liver leading to higher absorption of amlodipine in intra-hepatocellular is suggested to result in increased hepatic CYP3Ametabolism for co-substrates of CYP3A and P-gp.18) This mayexplain our results that the CL/F of amlodipine in subjects with theABCB1 3435TT genotype is higher than that in subjects with theABCB1 3435CC or 3435CT genotype. Our findings showed thatthe oral clearance of amlodipine was 1.50-fold higher in participantscarrying the ABCB1 3435TT genotype than in individuals with theABCB1 3435CT or 3435CC genotype, which is similar to previousreports.8,19) However, the effect of ABCB1 genetic variants onpharmacokinetics of other P-gp substrates remains controver-sial.20,21) ABCB1 haplotype-based analysis of the populationpharmacokinetics of amlodipine derived from G2677T/C3435Tstill need to be further investigated in a large scale population.

Females possessing the 2677TT-3435TT haplotype have higherCYP3A4 mRNA expression levels (median, 10.7; range, 5.92–15.2amol/µg total RNA) than those with 2677GG-3435CC (median,3.03; range 1.38–4.68) or 2677GT-3435CT (median, 4.31; range,0.07–9.42) (p = 0.022).22) In addition, previous surveys usingmdr1a (¹/¹), mdr1b (¹/¹) and mdr1a/1b (¹/¹) mice housed inAmsterdam have identified that the mice lacking P-gp are capableof inducing CYP3A expression in hepatocytes.23) The mostextensively studied CYP3A5 SNP, designated as CYP3A5*1,expresses a high level of functional CYP3A5 protein in subjectswith CYP3A5*1/*1 or CYP3A5*1/*3 genotypes and very low orundetectable levels of functional CYP3A5 protein in ones withCYP3A5*3/*3 genotypes. Zhao et al.24) showed that CYP3A5*3/*3 was linked with a significant increase in Cmax and AUC0¹¨ ofnimodipine. Although our previous study6) in healthy volunteersindicates that the CYP3A5*1 allele is an important factor in thedisposition of amlodipine in a single-dose study, which is inagreement with Kim’s report,25) the CYP3A5 genotype was not

brought into the final model.The CYP3A4*1G mutant allele, occurring at a frequency of

0.30 in Chinese renal transplant recipients,10) might be relatedto the higher CYP3A4 enzyme activity,10,26) while CYP3A4*1Gexpression seemed to increase the effect of atorvastatin, probablyresulting from a lower enzymatic activity of the CYP3A4*1Gallele.27) POR is the only flavoprotein that donates electrons tomicrosomal P450 enzymes. POR*28 C>T polymorphism wasassociated with CYP3A activity.28,29) Our results did not show anysignificant effect of POR*28, CYP3A4*1G or CYP3A5*3 variantson the disposition of amlodipine in essential hypertensive patients.The major reason is that a study involving a larger populationsample is needed to take into account genetic linkage analysisbetween CYP3A4*1G and CYP3A5*3 on the biotransformation ofamlodipine.

This study shows that gender is an important factor in thedisposition of amlodipine. The CL/F of amlodipine in the hyper-tensive female patients is less than that in male patients, whichis line with previous statements.12–14) Thus, amlodipine concen-trations were higher in women than men and the decrease in BPwith amlodipine was greater within the female group, similar tothe literature.30,31) These findings may be partly explained by theresearch done by Wolbold et al.32) Although CYP3A4 is dif-ferentially expressed between the two genders, with womenexpressing about twice the amount of the enzyme on the basisof microsomal protein content and faster clearance of CYP3Asubstrates in women, treatment with newer calcium antagonistssuch as amlodipine, felodipine and nitrendipine was associatedwith an average reduction of CYP3A4 expression by 45%. There-fore, it is likely that female patients receiving amlodipine or otherlong-acting calcium antagonists would have a significantly lowerCYP3A4 expression than males. In addition, the gender distri-bution and body weight are significantly correlated and thecorrelation coefficient is ¹0.51, which is consistent with Rohatagiet al.’s report.13) Thus, the effect of gender on the pharmacokineticsof amlodipine may be partly explained by the weight differencebetween males and females. This needs to be clarified by a large-scale population study.

Our investigation suggested a positive relationship between thedifference in SBP from pre- and post-dose of amlodipine and thesteady-state AUC of amlodipine. The influencing factors of theABCB1 C3435T variant and gender on the pharmacokinetics ofamlodipine would have similar effects on the pharmacodynamicsin hypertensive patients. This suggests that a greater BP responsein female than male patients may be explained by the lower CL/Fand higher drug exposure of amlodipine in the female group,similarly to the effect of the ABCB1 C3435T allele on drugexposure and BP response described above. Because only thebaseline and the terminal point value of SBP and DBP wererecorded, an exposure-response model could not be obtained dueto the lack of convergence. Further studies need to investigatethe exact prediction of change in SBP or DBP treatment withamlodipine.

In conclusion, this is the first study to extensively investigate theeffect of ABCB1 C3435T, POR*28, CYP3A4*1G and CYP3A5*3variants and gender on the disposition of amlodipine in essentialhypertensive patients. The study results suggest that ABCB1C3435T polymorphism and gender are determinant factors ofamlodipine pharmacokinetic variability. Lower CL/F and higherexposure occurs in female subjects with the ABCB1 3435CC or

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CT genotype who have greater decreases in blood pressure aftertreatment with amlodipine. Thus, a higher dosage needs to beadministered to males with the ABCB1 3435TT genotype toachieve a better antihypertensive effect. The results may be goodfor amlodipine dose optimization and help to improve the efficacyand tolerability of amlodipine in essential hypertensive patients.

References

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