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Keywords:

  • liquid chromatography–mass spectrometry;
  • organic anion transporting polypeptide 1B1 ;
  • pharmacokinetic;
  • pitavastatin;
  • polymorphism;
  • 388A>G

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. RESULTS
  6. Discussion
  7. Conclusion
  8. References

Purpose:  To investigate the contribution of the most frequent single nucleotide polymorphism (SNPs) of the organic anion transporting polypeptide 1B1 (OATP1B1) 388A>G to the pharmacokinetics of pitavastatin in Chinese healthy volunteers.

Methods:  Eighteen healthy volunteers participated in this study. Group 1 consisted of nine subjects who were of 388AA wild-type OATP1B1 genotype. Group 2 consisted of seven subjects with the 388GA genotype and two 388GG homozygotes. Two milligram of pitavastatin was administered orally to the volunteers. The plasma concentration of pitavastatin was measured for up to 48 h by liquid chromatography–mass spectrometry (LC–MS).

Results:  The pharmacokinetic parameters of pitavastatin were significantly different between the two genotyped groups. The concentration (Cmax) value was higher in the 388GA + 388GG group than that in the 388AA group (39·22 ± 8·45 vs. 22·90 ± 4·03 ng/mL, P = 0·006). The area under the curve to the last measurable concentration (AUC0–48) and area under the curve extrapolated to infinity (AUC0–∞) of pitavastatin were lower in the 388AA group than in the 388GA + 388GG group (100·42 ± 21·19 vs. 182·19 ± 86·46 ng h/mL, P = 0·024; 108·12 ± 24·94 vs. 199·64 ± 98·70ng h/mL, P = 0·026) respectively. The oral clearance (Cl/F) was lower in the 388GA + 388GG group than that in the 388AA group (12·46 ± 4·79 vs. 19·21 ± 3·74/h, P = 0·012). The elimination of half-life (t1/2) and peak concentration times (Tmax) values showed no difference between these groups.

Conclusions:  The OATP 388A>G polymorphism causes significant alterations in the pharmacokinetics of pitavastatin in healthy Chinese volunteers and this may well be clinically significant.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. RESULTS
  6. Discussion
  7. Conclusion
  8. References

Hyperlipidemia, plays a major role in the aetiology of coronary heart disease (CHD). A low plasma level of low-density lipoprotein (LDL)-cholesterol reduces CHD morbidity and mortality. The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins), considered first-line therapy for hyperlipidemia, inhibit the synthesis of mevalonate and lead to a reduction in plasma LDL-cholesterol.

Pitavastatin, a highly potent inhibitor of HMG-CoA, is sometimes called a ‘superstatins’ because of claimed, but insufficiently validated, more favourable clinical efficacy and safety profile (1–3). It is little metabolized by cytochrome P450 2C9 (CYP2C9) and lactonization is its main metabolic pathway. The lactone form can be easily converted to the parent drug (4–7). Pitavastatin is rapidly and efficiently taken up by the liver through the organic anion transporting polypeptide 1B1 (OATP1B1, OATP-C, gene SLCO1B1), which is a sodium-independent bile-acid transporter expressed at the sinusoidal membrane of human hepatocytes. The transporter plays an important role in the hepatocellular uptake of a variety of endogenous and foreign chemicals (8–10).

Although pitavastatin therapy has been shown to reduce substantially the level of LDL-cholesterol and the risk for cardiovascular disease in multiple patient subgroups, there is wide inter-individual variation in pitavastatin pharmacokinetics. Several single-nucleotide polymorphisms (SNPs) of OATP1B1 are known and two common single nucleotide polymorphisms, 388A>G and 521T>C, have been associated with a significant change in the transporter activity of OATP1B1 and to significantly alter the disposition of pitavastatin (11, 12). However, significant interethnic differences are known in the frequency distributions of these two polymorphisms. Recent studies reported that the 388A>G and 597C>T SNPs accounted for more than 70% of the variation in the three major haplotypes seen in a group of Chinese, Malays and Indians (13, 14).Meanwhile, the study showed that the frequencies of functional OATP1B1*15 alleles differed statistically between Chinese (8·2%) and Korean (14·0%) and Vietnamese (16·3%) (15). Furthermore, Xu et al. (16) reported that the frequencies of 388A>G and 521T>C variant alleles in the Chinese population were 73·4% and 14·0%, respectively, and their frequencies are similar to that in the Japanese, but significantly different from that seen in Caucasians and Blacks.

Over the past few decades, obesity and related hyperlipidemia have become serious public health problems in China. Pitavastatin, a third generation statin, shows potent effects on hyperlipidemia and has the potential to be an excellent addition to drugs available for this. However, OATP1B1 388A>G genetic polymorphism, affects the pharmacokinetics of pitavastatin in Chinese subjects is not known so far. This study aims to study this aspect in healthy Chinese volunteers.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. RESULTS
  6. Discussion
  7. Conclusion
  8. References

Subjects

Eighteen healthy volunteers were selected for this study and direct sequencing analysis was used to determine the OATP1B1 polymorphisms. Nine OATP1B1 388GG homozygotes and nine subjects with at least one 388A>G mutant allele (seven were 388AG heterozygotes and two were 388AA homozygotes) were recruited for the pitavastatin pharmacokinetic analysis. The study protocol was approved by the Ethics Committee of the school of Medicine, Nan Chang University, and written informed consents were obtained from all participants. Each subject was ascertained to be healthy by medical history, physical examination, routine blood and electrocardiographic tests. All of the subjects were non-smokers, abstained from drugs and did not take any coffee or alcohol for at least 1 week before entry into the study.

Study design

Each subject received a single oral dose of pitavastatin 2 mg with 150 ml water at 6:00 am after an overnight fast. Meals were allowed 4 h later after pitavastatin administration. Serial venous blood samples (5 mL each) were collected into EDTA containing tubes before and at 0·25, 0·50, 0·75, 1·0, 1·5, 2·0, 3·0, 4·0, 8·0, 12·0, 24·0, 36·0 and 48·0 h after dosing. Plasma samples were immediately stored at −20 °C after centrifugation.

OATP1B1 388G>A genotyping

Genomic DNA was extracted from peripheral blood by standard phenol–chloroform method. The polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method was used for genotyping as originally described by Xu et al. (16) and polymerase chain reaction (PCR) amplification was carried out by using the forward primer (5′-GCAAATAAAGGGGAATATTTCTC-3′) and reverse primer (5′-AGAGATGTAATTAAATGTATAC-3′) in a 25 μL of reaction system. The subjects were divided into two groups: a wild-type homozygote group (388AA) and a mutated group (388GA and 388GG).

Determination of pitavastatin in plasma

Plasma concentrations of pitavastatin were determined by the liquid chromatography–mass spectrometry with the Shimadzu LC/MS 2010EV system. A Shim-pack GVP-ODS C18 guard column (5·0 mm × 2·0 mm, Shimadzu, Kyoto, Japan) and a mobile phase consisting of 0·025% aqueous ammonia solution in 0·05mm ammonium acetate (A) and methanol (B) at a flow rate of 0·2 mL/min were applied. The gradient elution used was: 0·5 min to 7·0 min (B: 35–95%), 7·0 min to 8·5 min (B: 95%), 8·5 min to 9·0 min (B: 95–35%), and 9·0 min to 13·0 min (B: 35%) for column equilibration. Rosuvastatin was used as the internal standard. The analysis was performed in selective-ion monitoring mode at m/z 420·0 for pitavastatin and m/z 480·0 for rosuvastatin (Fig. 3). The lower limit of quantification for pitavastatin was 0·2 ng/mL. Typical chromatograms of pitavastatin and rosuvastatin (internal standard, IS) are shown in Fig. 4. The assay range used was 0·2–200 ng/mL. The correlation coefficient for pitavastatin calibration curves were higher than 0·99.

image

Figure 3.  The main pharmacokinetic parameters (Cmax, AUC0–48, AUC 0–∞, Cl/F) of a single dose of 2 mg pitavastatin with different OATP genotypes. Open triangles represent OATP 388AA genotype group (n = 9); solid triangles represent OATP 388GA + 388GG genotype group (n = 9).

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image

Figure 4.  The curve of mean time-concentration of pitavastatin in 15 healthy volunteers with different OATP genotypes after volunteers administrated a single oral dose of 2 mg pitavastatin. Open triangles represent OATP 388GA + 388GG genotype group (n = 9); solid triangles represent OATP 388AA genotype group (n = 9).Data are expressed as mean ± SE.

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Pharmacokinetic analysis

Individual pitavastatin plasma-concentration data were analysed using DAS 2·1 Microsoft. The maximum pitavastatin concentration (Cmax) and the corresponding peak times (Tmax) were determined from the respective observed plasma concentration-time data. The elimination rate constant (Ke) was obtained from the least squares fitted terminal log-linear portion of the plasma concentration-time profile. The elimination half-life (inline image) was calculated as 0·693/Ke.The area under the curve to the last measurable concentration (AUC0–t) was calculated by the linear trapezoidal rule. The area under the curve extrapolated to infinity (AUC0–∞) was calculated as AUC0–t + Ct/Ke, where Ct is the last measurable concentration.

Statistical analysis

The values were expressed as mean ± SD. Differences in pharmacokinetic data between the OATP1B1 388AA wild-type gene and the two mutated alleles 388AG and 388GG were evaluated statistically by independent-samples t-test, P < 0·05 was considered statistically significant. Statistical analysis was performed by the spss software for windows (version 10·0, SPSS Inc., Chicago, IL, USA).

RESULTS

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. RESULTS
  6. Discussion
  7. Conclusion
  8. References

OATP1B1 388G>A genotyping

The results of OATP1B1 388G>A genotyping, with band patterns of mutated alleles at 274 bp or 274 bp + 155 bp + 119 bp and band patterns of wild-type allele being 155 bp + 119 bp (Figs 1 and 2).

image

Figure 1.  Mutated group (388GA and 388GG). (a) was 388GA with three bands of 274 bp + 155 bp + 119 bp; (b) was 388GG with one band of 274 bp. DNA Marker with six bands of 100 bp + 200 bp + 500 bp + 750 bp + 1000 bp + 1500 bp were used to compare and analyse.

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image

Figure 2.  Wild type homozygote group (388AA) with two bands of 155 bp + 119 bp.

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Pharmacokinetic analysis

The main pharmacokinetic parameters of pitavastatin showed significant differences between the two genotyped groups. The mean Cmax, AUC0–48, and AUC0–∞ in the 388A>G mutated group were greater than those of the 388 AA group, where the mean Cl/F was lower in the former than in the latter (Fig. 3). The Cmax value was higher in the 388GA + 388GG group than that in the 388AA group (39·22 ± 8·45 vs. 22·90 ± 4·03 ng/mL, P = 0·006) and the AUC(0–48) and AUC(0–∞) were lower in the 388AA group than in the 388GA + 88GG group (100·42 ± 21·19 vs. 182·19 ± 86·46 ng h/mL, P = 0·024; 108·12 ± 24·94 vs. 199·64 ± 98·70 ng h/mL, P = 0·026) respectively (Table 1). The Cl/F value was lower in the 388GA + 388GG group than in the 388AA group (12·46 ± 4·79 vs. 19·21 ± 3·74 L/h, P = 0·012). There was no difference in the other Ka (per h), Ke (per h), Tmax (h), t1/2 (h) between the two groups. Figure 4 shows the time-concentration curve of pitavastatin according to genotypes. The AUC0–∞ of pitavastatin is higher in the OATP1B1 388GA + 388GG group than in the 388AA group, and the Cmax was also higher in the 388GA + 388GG group compared with the 388AA group.

Table 1.   Pharmacokinetic parameters of pitavastatin in subjects with OATP1B1 388GA or 388GG genotypes and wild-type homozygotes (388AA) after a single oral dose of 2 mg
Parameter2 mg
388GA + 388GG (n = 6) 388AA (n = 9) P
Ka (per h)2·79 ± 2·173·38 ± 3·500·691
Ke (per h)0·07 ± 0·040·05 ± 0·020·236
Tmax (h)0·72 ± 0·150·71 ± 0·190·876
Cmax (ng/mL)39·22 ± 8·4522·90 ± 4·030·006
AUC0–t (ng h/mL)182·19 ± 86·46100·42 ± 21·190·024
AUC0–∞ (ng h/mL)199·64 ± 98·70108·12 ± 24·940·026
t1/2 (h)13·00 ± 7·7813·08 ± 4·810·975
Cl/F (L/h)12·46 ± 4·7919·21 ± 3·740·012

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. RESULTS
  6. Discussion
  7. Conclusion
  8. References

Our study shows that the disposition of pitavastatin is significantly altered by the 388A>G genetic variation. The mean AUC0–48, AUC0–∞ and Cmax values in the OATP 388GA + 388GG mutant group were about 80%, 84% and 71% higher than corresponding values for the 388AA wild-type group. However, the oral elimination of pitavastatin (Cl/F) in the OATP 388GA + 388GG mutant group was about 35% lower than in the 388AA wild-type group.

Chung et al. (11) showed that OATP1B1 variant haplotypes have a significant effect on the pharmacokinetics of pitavastatin. They found dose-normalized AUC and Cmax of pitavastatin to be 1·4- and 18-fold higher, respectively, in subjects heterozygous for the OATP*15 allele vs. subjects without this allele. OATP SNPs in various ethnic populations have been reported with frequencies of functional SLCO1B1*15 alleles which differed even between Chinese (8·2%), Korean (14·0%) and Vietnamese (16·3%) populations (14, 17). Significant interethnic differences were observed in the genotype frequency distributions across the promoter SNP [g.−1118 7G>A (P ≤ 0·030)] as well as three coding region SNPs [c.388A>G (P≤ 0·001); c.571T>C (P ≤ 0·001); c.597C>T (P ≤ 0·001)] in healthy Chinese, Malay and Indian subjects (13). The frequencies of 388A>G and 521T>C variant alleles in the Chinese population were 73·4% and 14·0%, respectively, which are similar to those in the Japanese, but significantly different from those in Caucasians and Blacks (16).

Statins, mostly metabolized by members of CYPs, are potentially susceptible to the effect of genetic polymorphism of those enzymes. In vitro studies indicate that CYP2C9 metabolism is not an important mechanism for pitavastatin clearance (4, 18). However, other OATP1B1 SNPs may have an effect on the pharmacokinetics of some statins. Nishizato et al. (19) have reported that OATP1B1 512T > C mutation effected the pharmacokinetics of pravastatin and plasma concentrations were higher in those affected than in those who were not and total body clearance was also lower. The 521T > C genotype was not associated with altered pharmacokinetics of rosuvastatin in Chinese, Malay and Asian-Indian populations (20). The effect of OATP1B1 512T > C mutation on the pharmacokinetic of pitavastatin requires investigation. Although the metabolism of pitavastatin is complex, lactonization is the major pathway. Our study was conducted in a small group of healthy subjects at a single dosage of 2 mg of pitavastatin. Further studies should be carried out on the effects of OAT1B1 genetic polymorphism during chronic dosing of the drug.

Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. RESULTS
  6. Discussion
  7. Conclusion
  8. References

OATP1B1 388A>G polymorphism plays a significant role on the pharmacokinetics of pitavastatin in healthy Chinese volunteers and this may be one of interpretations for the inter difference in pitavastatin disposition.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. RESULTS
  6. Discussion
  7. Conclusion
  8. References
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