Volume 62, Issue 5 p. 567-572
Free Access

Effect of SLCO1B1 genetic polymorphism on the pharmacokinetics of nateglinide

Wei Zhang

Wei Zhang

Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan 410078, P. R. C.

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Yi-Jing He

Yi-Jing He

Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan 410078, P. R. C.

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Chun-Ting Han

Chun-Ting Han

Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan 410078, P. R. C.

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Zhao-Qian Liu

Zhao-Qian Liu

Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan 410078, P. R. C.

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Qing Li

Qing Li

Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan 410078, P. R. C.

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Lan Fan

Lan Fan

Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan 410078, P. R. C.

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Zhi-Rong Tan

Zhi-Rong Tan

Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan 410078, P. R. C.

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Wei-Xia Zhang

Wei-Xia Zhang

Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan 410078, P. R. C.

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Bang-Ning Yu

Bang-Ning Yu

Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan 410078, P. R. C.

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Dan Wang

Dan Wang

Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan 410078, P. R. C.

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Dong-Li Hu

Dong-Li Hu

Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan 410078, P. R. C.

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Hong-Hao Zhou

Hong-Hao Zhou

Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan 410078, P. R. C.

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First published: 23 June 2006
Citations: 84
Professor Hong-Hao Zhou, Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan 410078, China.
Tel: + 86 73 1448 7233
Fax: + 86 73 1480 5379
E-mail:[email protected]

Abstract

Aims

Nateglinide is a meglitinide analogue with antidiabetic action. A recent study showed that SLCO1B1 (which codes the OATP1B1 gene, also known as OATP-C, OATP2) is a major determinant which markedly affects the pharmacokinetics of repaglinide. Our objective was to assess the association between single nucleotide polymorphisms (SNPs) of SLCO1B1 and the pharmacokinetics of nateglinide.

Methods

Seventeen healthy volunteers with different SLCO1B1 genotypes (11 with 521TT, four with 521TC and two with 521CC) were enrolled in this study. Each was given a single oral dose of 90 mg nateglinide. Plasma concentrations of nateglinide were measured up to 8 h by HPLC.

Results

The Cmax and AUC(0,∞) of nateglinide were 83% (P = 0.002) and 82% (P = 0.001) higher in the SLCO1B1521TC subjects (n = 4), and 76% (P = 0.016) and 108% (P = 0.001) higher in the SLCO1B1521CC subjects (n = 2) than in the SLCO1B1521TT subjects (n = 11), respectively. The t1/2 of nateglinide in SLCO1B1521CC subjects was 78% longer than that in 521TT subjects (P = 0.036). The difference in tmax values among the three genotypic groups was not statistically significant.

Conclusions

Our results suggest that OATP1B1-mediated hepatic uptake of nateglinide may be the prior step for its metabolism and elimination. SLCO1B1521T > C SNP might play an important role in the pharmacokinetics of nateglinide.

Introduction

The worldwide prevalence of diabetes is predicted to increase from 124 million to more than 221 million by the year 2010. However, of these, approximately 97% will have type 2 diabetes [1]. Nateglinide is a novel phenylalanine derivative and is administered prior to meals and stimulates rapid, short-term insulin secretion in a dose-dependent manner, decreasing mealtime plasma glucose concentrations [2]. As with other meglitinides, nateglinide stimulates pancreatic β-cell secretion by acting on ATP-sensitive K+ channels and on voltage-sensitive Ca2+ channels [3, 4].

The oral bioavailability of nateglinide is about 73%, and it is rapidly absorbed and extensively metabolized primarily by cytochrome P450 2C9 (CYP2C9) in the liver and a smaller fraction by CYP3A4 and CYP2D6 [5]. A previous report showed that the CYP2C9*3 allele was associated with significantly reduced oral nateglinide clearance and pharmacokinetic parameters, which seemed to be unaffected by CYP2C9*2 and CYP2D6*4 or *5 carriers. However, the interindividual variability in the pharmacokinetics of nateglinide could not be fully explained by CYP2C9 genotype [6]. A recent study carried out by Niemi et al. identified that SLCO1B1 genetic polymorphism is a major determinant of repaglinide disposition in vivo, which explained most of the interindividual variability in the pharmacokinetics of repaglinide and elucidated an actively transporting process in its disposition prior to metabolism by CYP450s in hepatocytes [7].

Genetic polymorphisms of SLCO1B1, causing reduced transport activity both in vitro and in vivo, have recently gained increasing interest. Several studies in Japanese subjects, white German and Finnish subjects showed that SLCO1B1*5, SLCO1B1*15 (a haplotype that consists of SLCO1B1*1b and *5 SNPs) and *17 (a haplotype that consists of SLCO1B1*15 and −11187G > A SNPs) were associated with markedly increased pravastatin plasma concentrations compared with those homozygous for the wild-type *1a or *1b alleles [8–10]. Because the allelic frequency of the 521T > C (Val174Ala) SNP, which defines the *5, *15 or *17 haplotypes, is relatively high (about 14%) in Chinese subjects, these findings would suggest that a large number of substrates of OATP1B1 may have plasma concentrations of the drug higher than expected.

Nateglinide is structurally very similar to repaglinide, and both are meglitinide analogues. Our study aimed to determine the effect of the most common single nucleotide polymorphism (521T > C) in the SLCO1B1 gene on the pharmacokinetics of nateglinide.

Methods

Subjects

After genotyping a total of one hundred and eleven Chinese healthy male volunteers aged from 19 to 22 years, 17 subjects were recruited for nateglinide pharmacokinetic analysis including 11 homozygotes for 521TT and six carriers with at least one 521C mutant allele (four subjects were 521TC heterozygotes and two were 521CC homozygotes). The study protocol was approved by the Ethics Committee of Central South University, Xiangya School of Medicine, and written informed consent was obtained from each participant. Each of them was healthy as determined by medical history, physical examination, routine blood tests, and electrocardiography. All the subjects were nonsmokers, abstained from drugs for at least 2 weeks before entry into the study, and did not take any coffee or alcohol for 1 week before the study.

Study design

After an overnight fast, each subject received a single oral dose of 90 mg nateglinide (three 30 mg tablets, Hunan Medicine Co. Ltd, Dongting Pharmaceutical Factory, China) with 100 ml water. All subjects received a standardized breakfast of 250 ml milk and 90 g cake 1 h after administration of nateglinide. Four hours later, warm meals were allowed. Venous blood samples (5 ml) were collected into EDTA tubes before and at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, and 8 h after nateglinide intake. During the whole trial, a group of physicians and nurses stood by for emergency medical treatment. The plasma was separated by centrifugation and immediately stored in polypropylene tubes at −20°C.

ARMS–PCR for 521T > C genotyping

The four primers used in amplification refractory mutation system (ARMS-PCR) were as follows. P1: 5′-AAGTAGTTAAATTTGTAATAGAAATGC-3′, P2: 5′-GGGTCATACATGTGGATATAAGT-3′, P3: 5′-AAGC ATATTACCCATGAAC G-3′, and P4: 5′-GTAGACAA AGGGAAAGTGATCATA-3′. PCR amplification was performed in a Perkin- Elmer DNA Model PJ2000 Thermal Cycler (Foster city, CA) in a total volume of 25 µl containing 2.5 µl 10 × buffer (with MgCl2), 0.2 mmol l−1 of each dNTP, 40 pmol l−1 of each primer, 2 U LA Taq enzyme and approximately 200 ng genomic DNA as a template. The PCR conditions involved an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 48°C for 30 s and extension at 72°C for 30 s with a final extension at 72°C for 7 min. The PCR products were detected by means of 2.5% agarose gel electrophoresis and were detected by ethidium bromide staining. Wild-type 521TT yielded two fragments of 260 bp and 179 bp in length, while the variant 521CC homozygotes gave two fragments of 260 bp and 123 bp and the heterozygotes were three fragments of 260 bp, 123 bp and 179 bp. Two samples of each different genotype were sequenced to confirm the expected sequences of each genotype. (Figure 1)

Details are in the caption following the image


(a) ARMS-PCR analysis result for the SLCO1B1521T > C polymorphism. Lane 1, genotype with T/C at 521; Lanes 2 and 3, genotype with T/T at 521; Lane 4, genotype with C/C at 521; Lane 5, DNA marker. (b) Direct sequencing results for the SLCO1B1521T > C SNP. Panel I represents a trace for a sample with a SLCO1B1521TT genotype. Panel II represents a trace for a sample from subject with a SLCO1B1521TC genotype. Panel III represents a trace for a homozygous SLCO1B1521CC individual

Assay of nateglinide

The detection of nateglinide was performed on an Agilent 1100 HPLC system (California, USA) consisting of a HP1100 pump, an automatic sampler, and a Variable Wavelength Detector (VWD). The Chem Station software (Agilent) was used for data analysis and processing. Compounds was separated on a Hypersil BDS C18 column (4.6 mm × 200 mm, 5 µm particle size) with a Phenomenex SecurityGuardTM guard column (Phenomenex, US) and quantified by UV detection at 214 nm. The mobile phase was composed of 0.02 m ammonium acetate solution : acetonitrile (60 : 40 v : v), and was delivered at a flow rate of 1.0 ml min−1. The mobile phase was prepared daily and was filtered through a 0.45 µm cellulose acetate membrane. Repaglinide was used as an internal standard. Each sample was prepared by the following procedure: In a 1.5 ml plastic tube, 200 µl of plasma were mixed with 400 µl of internal standard (10 µg ml−1), and the mixture was vortexed for 2 min. After centrifugation for 10 min at 13000 rev min−1, the supernatants were transferred into an injection vial, and 20 µl was injected into the HPLC. The limit of quantification was 93.75 ng ml−1 for nateglinide. The interday coefficients of variation (CV) were 5.03% at 750 ng ml−1, 6.49% at 1.5 µg ml−1 and 3.75% at 12 µg ml−1 (n = 5) replicates.

Pharmacokinetics analysis

The pharmacokinetics of nateglinide were analyzed with a noncompartmental method using the WinNonlin 4.1 (Pharsight Corp., Moutain View, CA, USA). The maximum concentration (Cmax) and the time to Cmax (tmax) were directly observed from the concentration-time curve. The plasma nateglinide half-life (t1/2) and the area under the plasma concentration–time curve (AUC) were calculated with the following equations: t1/2 = ln2/λz, AUC (0,∞) = AUC(0,8 h) +Cp(8 h)/λz, where λz represents the slope of the linear terminal part of the plasma concentration vs. time curve after semilogarithmic transformation, Cp(8 h) is the plasma concentration at 8 h and AUC(0,8 h) the AUC for the 8 h period after nateglinide intake by the linear trapezoidal rule.

Statistical analysis

Statistical analyses were performed by the SPSS software for Windows (Ver 10.0, SPSS Inc., 202 Chicago, IL). Data are expressed as mean ± SE. The differences in pharmacokinetic data were analysed by anova test. A P value of less than 0.05 was considered significant.

Results

According to SLCO1B1 genotype, all the subjects were divided into three groups, 521TT homozygotes (n = 11), 521TC heterozygotes (n = 4) and 521CC homozygotes (n = 2). Cmax, AUC(0,8 h) and AUC(0,∞) of nateglinide were statistically different among all three genotypic groups (2, 3 and Table 1). The Cmax was 83% and 76% higher in SLCO1B1521TC and 521CC subjects than in SLCO1B1521TT subjects (P = 0.002 and P = 0.016, respectively). The AUC(0,∞) was 82% and 108% in SLCO1B1521TC and 521CC subjects than in SLCO1B1521TT subjects (P = 0.001 and P = 0.001, respectively). The t1/2 of nateglinide in SLCO1B1521CC subjects was 78% longer than that in 521TT subjects (P = 0.036). The differences of tmax values among the three genotypic groups were not statistically significant.

Details are in the caption following the image


The plasma concentration-time curve of nateglinide in 17 healthy volunteers with different SLCO1B1 genotypes after a single oral dose of 90 mg nateglinide. Solid circles represent SLCO1B1521TT genotype group (n = 11) (●), triangles represent SLCO1B1521TC genotype group (n = 4) (▴), open circles represent SLCO1B1521CC genotype group (n = 2) (○). Data are expressed as mean ± SEM

Details are in the caption following the image


Individual AUC of nateglinide from time 0 to infinity values in subjects with different SLCO1B1 genotypes after a single oral dose of 90 mg nateglinide. Horizontal rules show mean values for all genotype groups

Table 1.
Pharmacokinetic changes after a single 90 mg oral dose of nateglinide in relation to SLCO1B1521T > C single nucleotide polymorphism
SLCO1B1 genotypes C max (ng ml−1) (95% CI) t 1/2(h) (95% CI) AUC(0,8 h) (ng ml−1 h) (95% CI) AUC(0,∞) (ng ml−1 h) (95% CI)
521TT (n = 11) 2708 ± 244 (2164, 3253) 1.24 ± 0.12 (0.96, 1.51) 5350 ± 530 (4170, 6530) 5705 ± 523 (4539, 6871)
521TC (n = 4) 4944 ± 626 (2950, 6938) 1.65 ± 0.46 (0.19, 3.10) 9748 ± 896 (6895, 12601) 10322 ± 1108 (6795, 13849)
521CC (n = 2) 4772 ± 1007 (−8025, 17568) 2.21 ± 0.06* (1.7, 2.7) 10974 ± 928 (−819, 22768) 11842 ± 1132 (−2251, 26236)
P value 0.002 0.080 0.000 0.000
  • Data are expressed as mean ± SEM; CI confidence interval; C max observed peak plasma concentration; t 1/2 elimination half-life; AUC(0,8 h) area under the concentration vs. time curve to 8 h; AUC(0,∞) area under the concentration vs. time curve to infinity. * P = 0.036 compared with SLCO1B1521TT subjects.

Discussion

In this study, we have provided evidence for the first time that SLCO1B1 polymorphism may have a considerable impact on the pharmacokinetics of nateglinide. The plasma concentration of nateglinide was significantly higher in subjects carrying the SLCO1B1521C allele than in subjects with the reference genotype. The AUC(0,∞) values of nateglinide varied five-fold among all the individuals and the mean AUC(0,∞) of this drug was about 82% and 108% higher in the SLCO1B1521TC and 521CC groups than that in the SLCO1B1521TT group. Our data strongly suggest that the OATP1B1-mediated transportation is at least partly involved in the hepatic uptake of nateglinide.

Nateglinide is metabolized by oxidative biotransformation mainly via CYP2C9 (70% of metabolism), and to a minor extent by CYP3A4 and CYP2D6 (30% of metabolism) [5, 11]. Kirchheiner et al.[6] found that the area under the concentration-time curve from 0 to 12 h and the peak plasma concentration of nateglinide were almost doubled in CYP2C9*3/*3 homozygotes compared with CYP2C9*1/*1 wild type homozygotes, while the total oral clearance of this drug decreased by about 50%. We ignored the genotype of CYP2C9 among subjects in the present study, which is a shortcoming. However, the allelic frequencies of CYP2C9*2 and CYP2C9*3 in Chinese subjects are about 0 (n = 1016) and 3.3% (n = 866), respectively [12], which indicates that there should be no more than one CYP2C9*1/*3 heterozygote among our 17 subjects.

They observed no association between the CYP2D6 genetic polymorphisms and the pharmacokinetics of nateglinide [6]. However, the effect of CYP3A4 gene mutation on the kinetics of nateglinide still remains unknown, which is possibly because of the rare functional variation of this isoenzyme and its extremely low frequencies. In the present study, we observed a five-fold variation in AUC(0,∞) of nateglinide among individuals with different genotypes. Since the frequency of CYP2C9*3/*3 homozygotes is very low (less than 0.2%) in Chinese healthy volunteers, metabolic phase seems not to be the major determinant of the interindividual variability in the exposure to nateglinide.

OATP1B1, one of the key transporters located on the basolateral membrane of human hepatocytes, transports a large number of structurally divergent compounds and has been implicated as a determinant factor in hepatic drug uptake and elimination [13]. Thus, impaired OATP1B1 transport activity caused by functional polymorphism may alter the pharmacokinetic features of OATP1B1 substrate drugs. SLCO1B1521T > C (Val174Ala), which defines either the *5 or *15 haplotypes, is relatively common (10% to 16%) in both Caucasians and Asians, which resulted in increased plasma concentration and decreased nonrenal clearance of pravastatin [8–10]. 521T > C mutation was also found to be associated with a markedly higher concentration of repaglinide in a recent study [7].

Gemfibrozil, a fibric acid derivative, is a potent inhibitor for CYP2C8, CYP2C9 and OATP1B1 [14, 15]. Co-adminstration of gemfibrozil with repaglinide, the substrate for CYP2C8 and CYP3A4, greatly increases the AUC by 8.1-fold [16]. In vitro, gemfibrozil is a more potent inhibitor of CYP2C9 than CYP2C8. However, concomitant use of gemfibrozil and itraconazole only raised the AUC of nateglinide, which is a substrate for CYP2C9 and CYP3A4, by less than 47%[17]. The discrepancy in the inhibitory effects of these two drug–drug interactions could be explained by the fact that both repaglinide and nateglinide could be substrates for OATP1B1, and OATP1B1 is (maybe) the only basolateral membrane transporter involved in the uptake of repaglinide (but not for nateglinide). If this transporter was inhibited by gemfibrozil, repaglinide would accumulate in the blood circulation and could not be metabolized and eliminated. Another plausible explanation is that the glucuronide metabolite of gemfibrozil is a much more potent inhibitor of CYP2C8 than CYP2C9 in vivo[18].

In conclusion, the SLCO1B1521T > C polymorphism plays an important role in the interindividual variability of plasma concentrations of nateglinide. Further studies are needed to confirm whether these differences in nateglinide kinetics caused by SLCO1B1 genotypes could lead to a clinical blood glucose-lowering effect. Genotype assay of patients with respect to the SLCO1B1521T > C SNP may be helpful for patients who require treatment with nateglinide.

This work was supported by the National Natural Scientific Foundation of China grants F30130210, C30000211 and C30200346 and by the China Medical Board of New York grants 99–697 and 01–755.