OATP1B1及CYP3A4基因多态性对阿托伐他汀转运代谢影响的分子机制研究
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摘要
背景：
阿托伐他汀（Atorvastatin, ATV）是新一代β-羟基-β-甲基戊二酰辅酶A（HMG-CoA）还原酶抑制剂，可竞争性抑制胆固醇的生物合成，显著降低血脂水平。虽然ATV已广泛用于高胆固醇血症的治疗，但其调脂疗效呈现明显的个体差异仍给临床应用带来困扰。有研究表明，ATV与其他他汀类不同之处在于，其既是摄入型转运体OATP1B1的底物，又主要经CYP3A4代谢。故OATP1B1及CYP3A4的基因多态很可能影响ATV的转运与代谢，从而导致ATV疗效和副作用个体差异的出现。因此，系统研究与ATV转运、代谢相关OATP1B1及CYP3A4的基因多态性及其相互间的关联意义重大。
目的：
本课题旨在通过构建OATP1B1~*1a、~*5和~*15重组质粒模型，研究ATV在不同OATP1B1基因型个体中药物体外转运的差异；通过野生型及不同位点突变型CYP3A4重组酶系，研究不同CYP3A4基因型代谢ATV的差异；通过统计分析研究所获数据，探讨ATV在不同基因型个体中转运和代谢差异的分子机制及二者的关联性；为ATV在临床的合理应用提供理论与实验依据。
方法：
1、建立HPLC及LC-MS检测ATV生物样本的方法，摸索ATV的重组蛋白酶样品及HEK293T细胞样品处理方法。
2、构建稳定表达目的基因的细胞模型。将携带OATP1B1~*15-GFP融合基因的慢病毒（OATP1B1~*15质粒构建、慢病毒包装、滴度测定由上海吉凯基因完成）感染HEK293T细胞，建立稳定表达OATP1B1~*15的HEK293T细胞模型，结合本实验室已成功表达OATP1B1~*1a、OATP1B1~*5的HEK293T细胞模型，采用上述三种细胞模型比较ATV在不同基因型OATP1B1个体中摄取转运过程的差异。
3、ATV在表达OATP1B1~*1a、~*5及~*15的HEK293T细胞中吸收转运实验。实验分为空白对照组、OATP1B1~*1a、~*5及~*15三种质粒组，考察摄取缓冲液PH对摄取过程的影响；考察不同时间点（5、10、20、40、60min）对摄取过程的影响；考察底物浓度（5、10、20、50、100、150μM）对摄取过程的影响。采用LC-MS法测定细胞样品中ATV的量，比较ATV在三种不同基因型质粒模型中的摄取动力学参数（K_m、V_(max)、CL_(int)值），分析两个位点突变对ATV转运过程的影响。
4、ATV在野生型及不同位点突变型CYP3A4重组酶系中体外酶促动力学分析。实验分为空白对照组和CYP3A4~*1（wild type）、CYP3A4~*3（M445T）、CYP3A4~*5（P218R）、CYP3A4~*16（T185S）、CYP3A4~*18（L293P）五种重组酶组，每组分别加入系列浓度的ATV10μl，孵育体系中重组酶终浓度为1mg/ml，温孵60min后，加入100μl冰乙腈终止反应后处理样品，采用RP-HPLC法测定孵育体系中ATV及其代谢产物的量，比较ATV在CYP3A4野生型与突变型重组酶系中的酶促动力学参数（K_m、V_(max)、CL_(int)值），分析CYP3A4不同位点突变对ATV代谢的影响。
5、数据处理与统计分析。所有数据均以平均数±标准差（mean±SD）表示，通过米曼氏方程模型、Lineweaver-Burk作图法计算ATV在不同OATP1B1质粒模型中摄取动力学参数，以及ATV在野生型和突变体重组酶系药物代谢动力学参数。采用SPSS12.0软件进行统计分析，组间差异采用t检验，统计结果以P>0.05表示差异无统计学意义，P<0.05表示差异有统计学意义，P<0.01表示显著性差异。
结果：
1、建立的LC-MS检测方法完全能够符合ATV生物样本分析测试的要求，建立的HPLC检测方法符合ATV及其代谢产物分析测试的要求，两种检测方法均具有较高的灵敏度和特异性；成功建立ATV的重组蛋白酶样品及HEK293T细胞样品处理方法，为本课题实施奠定了可靠基础。
2、获得OATP1B1~*15慢病毒融合质粒，其病毒滴度为2x109TU/ml，感染HEK293T后，通过荧光检测，观察到GFP的表达，荧光表达随着时间延长及感染复数(Multiplicity of infection, MOI)值增加而增强、增多，最佳MOI值为50，此时Lenti-OATP1B1~*15感染HEK293T的感染率可达80%；通过Western Blot检测感染的293T样品，可以观察到95kDa附近有特征带表达，其大小和OATP1B1~*15融合蛋白（104kDa）相吻合。表明稳定感染Lenti-OATP1B1~*15的HEK293T细胞模型构建成功。
3、OATP1B1~*1a-HEK293T对ATV的摄取动力学参数K_m与V_(max)及CL_(int)分别为21.17±3.96uM、9.13±1.42pmol·min~(-1)·mg~(-1)protein、0.43；OATP1B1~*5-HEK293T对ATV的摄取动力学参数K_m、V_(max)及CL_(int)分别为13.95±2.32μM、3.88±0.49pmol·min~(-1)·mg~(-1)protein、0.28；OATP1B1~*15-HEK293T对阿托伐他汀摄取的动力学参数K_m与V_(max)及CL_(int)分别为19.37±2.71μM、7.98±1.03pmol·min~(-1)·mg~(-1)protein、0.41。用SPSS12.0进行统计分析，OATP1B1~*5(521T>C)基因型个体对ATV的转运活性低于野生型OATP1B1~*1a(P<0.05)，差异有统计学意义；而OATP1B1~*15(388A>G521T>C)基因型个体与野生型OATP1B1~*1a个体对ATV转运活性差异无统计学意义(P>0.05)。
4、野生型CYP3A4~*1重组酶酶促动力学参数K_m、V_(max)及CL_(int)值分别为88.65±19.32μM、16.39±4.10pmol/min/pmol P450、0.18；突变型CYP3A4~*3酶促动力学参数K_m、V_(max)及CL_(int)值分别为92.58±9.33μM、23.86±5.02pmol/min/pmolP450、0.26，其活性略大于野生型；突变型CYP3A4~*5酶促动力学参数K_m、V_(max)及CL_(int)值分别为109.38±8.21μM、44.84±6.73pmol/min/pmol P450、0.41，其活性显著大于野生型。突变型CYP3A4~*16酶促动力学参数K_m、V_(max)及CL_(int)值分别为156.87±20.87μM、15.67±6.03pmol/min/pmol P450、0.10，其活性显著低于野生型；突变型CYP3A4~*18酶促动力学参数K_m、V_(max)及CL_(int)值分别为100.37±18.09μM、18.48±5.63pmol/min/pmol P450、0.18，其代谢ATV的活性与野生型相近。
结论：
本文从分子学水平探明OATP1B1多态性对ATV转运影响的机制及CYP3A4多态性对ATV代谢影响的机制。
1、结果提示，OATP1B1521位点可能是ATV转运的分子作用点，也是影响OATP1B1对ATV转运能力的关键位点。该位点发生突变会导致转运入肝至靶点的ATV量减少，难以达到相应的降脂疗效，故临床上应关注患者OATP1B1521位点的突变情况，指导ATV临床合理用药。
2、结果提示，CYP3A4445、CYP3A4218、CYP3A4185位点可能是ATV代谢的分子作用点，也是影响CYP3A4对ATV代谢活性的关键位点。这些位点发生突变会导致氨基酸发生变化，从而影响对ATV的代谢活性，故ATV在临床用药时也应关注患者CYP3A4不同位点的突变情况。
3、如服用ATV的患者转运体基因型为OATP1B1~*5，则转运入肝脏的ATV减少，同时该患者为CYP3A4~*3/~*5突变型，则对ATV代谢能力提高，会引起作用于靶点的药物浓度降低，不但导致治疗失败，还可能会引起外周毒副作用，则需通过增加给药剂量或换用其他的降脂药，达到相应的降血脂疗效。故在临床应用中结合OATP1B1及CYP3A4突变情况指导ATV用药，将更具合理性。
Background:
Atorvastatin is a new generation of β-hydroxy-β-methylglutaryl coenzyme A(HMG-CoA) reductase inhibitor, its own biological activity after into the bodywithout metabolism, competitive inhibition of cholesterol biosynthesis, are widelyused in clinical treatment of hypercholesterolemia and elevated serum cholesterol ofmixed hyperlipidemia. ATV transported into hepatic by the organic aniontransporting polypeptide1B1(OATP1B1), To inhibit HMG-CoA reductase combinedwith medroxyprogesterone valerate competitively, reduce the formation of cholesterolto reach the effect of lipid-lowering, ATV mainly metabolismed by CYP3A4, andthen excreted in the form of metabolite finally.
Objectives:
The aim of this project is build OATP1B1~*1a,~*5and~*15recombinant plasmidmodel to study transport differences and its molecular mechanism of atorvastatin indifferent OATP1B1genotype mutant in vitro study. To study the metabolism producedifferences and its molecular mechanism of atorvastatin in wild-type and differentpoints mutant CYP3A4recombinant enzyme. Analysis the experimental data ofatorvastatin in vitro studies, and analysis statins in toxic side effects or ineffectivetreatment caused by differences in transport and metabolism processes that may occurin different genotypes. To provide a theoretical basis for in vivo experiments and theindividual drugs in clinical.
Methods:
1、Create HPLC and LC-MS detection method to detect biologic samples ofATV, and explore cell sample and recombinant enzyme sample preparation method.
2、To infect HEK293T cells with OATP1B1~*15-GFP fusion gene lentivirus (Thejob of OATP1B1~*15plasmid construct, the packaging of lentiviral, titer determ-ination completed by the company of Kaiji gene, Shanghai), To detect the target gene expression of OATP1B1~*15by observing fluorescence and Western blot, Toestablish stable expression of OATP1B1~*15-HEK293T cells model, our laboratoryhas successfully expressed OATP1B1~*1a, OATP1B1~*5HEK293T cell-model, usingthese three cell-models investigated ATV uptake in different genotypes OATP1B1individual transporter process differences.
3、The transport experiment of ATV in OATP1B1~*1a,~*5and~*15-HEK293Tcells. The experiment was divided into blank control group, the OATP1B1~*1a,~*5and~*15three kinds of plasmid group, To investigate the impact of uptake buffer pHon the uptake process; To investigate uptake process at different time points (5,10,20,40,60min). To investigate the impact of substrate concentration (5,10,20,50,100,150μM) to the intake process. To determination the amount of ATV in cell samplesby LC-MS method ATV uptake in the three different genotypes plasmid modelkinetic parameters (K_m, V_(max)and CL_(int)), analysis the effect of two site mutations onATV transit process.
4、The enzyme kinetics study of ATV in wild-type and different point mutantCYP3A4recombinant enzymes in vitro. The experiment was divided into blankcontrol group, CYP3A4~*1, CYP3A4~*3, CYP3A4~*5, CYP3A4~*16, and CYP3A4~*18five kinds of recombinase group, each group were added series of concentrations ofthe ATV10μL, in the incubation system final concentration of the recombinantenzyme is1mg/ml, after incubated60minutes, add100μL ice acetonitrile toterminate reaction, The amount of ATV and its metabolites in the incubation systemdeterminated by RP-HPLC method, To compare the enzymatic kinetic parameters(K_m, V_(max), and CL_(int)) of ATV in wild type and mutant CYP3A4recombinant enzymes,analysis the impact of different sites mutations of CYP3A4to metabolism of ATV.
5、Data processing and statistical analysis: All data are demonstrated bymean±standard deviation (Mean±SD), calculate the uptake kinetics parametersenzymes kinetic parameters of ATV in different OATP1B1plasmid model throughMichaelis-Menton equation model, and Lineweaver-Burk plot. Using SPSS12.0software for data processing, the difference between the groups with the design of thet-test, the statistical results is P>0.05as no significant difference, the statistical resultsis P<0.05as significant difference. the statistical results is P<0.01indicated that there as a very significant difference.
Results:
1、 The established LC-MS detection method is developed to meet theatorvastatin cell sample analysis and testing requirements, with high sensitivity andspecificity. The established HPLC method developed to meet the requirements of theanalysis and testing of atorvastatin and its metabolites with high sensitivity andspecificity.
2、 Recombinant lenti-viral titer of Lenti-OATP1B1~*15is2x109TU/ml;Lenti-OATP1B1~*15after the infection of HEK293T fluorescent protein with timeand MOI (Multiplicity of infection, MOI) value increased with the increase;50is thebest MOI value. Lenti-OATP1B1~*15transfected HEK293T transfection rate is80%when MOI is50. Detected by Western Blot transfected into293T samples observed95kDa near the bar characteristics with its size and OATP1B1double mutant fusionprotein (~76kDa+28kDa=104kDa) is consistent. That OATP1B1double mutant wassuccessfully expressed.
3、OATP1B1~*1a and OATP1B1~*5gene atorvastatin intake are quite different,OATP1B1~*5gene reduce the transport capacity of atorvastatin. Compared withOATP1B1~*15, OATP1B1~*5transport activity lower. The uptake kinetic parametersK_m、V_(max)and CL_(int)of OATP1B1~*1a-HEK293T are21.17±3.96μM,9.13±1.42pmol·min~(-1)·mg~(-1)protein,0.43. OATP1B1~*5-HEK293T are13.95±2.32μM3.88±0.49pmol·min~(-1)·mg~(-1)protein,0.28. OATP1B1~*5-HEK293T atorvastatin uptake kineticparameters K_m, V_(max)and CL_(int)are19.37±2.71μM7.98±1.03pmol·min~(-1)·mg~(-1)protein,0.41, respectively. Statistical analysis by SPSS12.0, Series of concentrations of ATVtransported in OATP1B1~*1a and OATP1B1~*5, OATP1B1~*15and OATP1B1~*5arestatistically significant.
4、Results of research are CYP3A4~*1enzyme kinetics parameters of the K_mandV_(max)and CL_(int)values were88.65±19.32μM,16.39±4.10pmol/min/pmol P450,0.18;Enzymatic kinetic parameters K_m, V_(max)and CL_(int)values of CYP3A4~*5are109.38±8.21μM,44.84±6.73pmol/min/pmol P450,0.41, its activity significantly greater thanthe wild type; enzyme kinetics parameters K_m, V_(max)and CL_(int)values of CYP3A4~*3 are92.58±9.33μM23.86±5.02pmol/min/pmol P450,0.26, its activity is slightly largerthan the wild type. CYP3A4~*16enzymatic kinetic parameters of the K_m, V_(max)andCL_(int)values are156.87±20.87μM,15.67±6.03pmol/min/pmol P450,0.10, its activitysignificantly lower than the wild type. CYP3A4~*18enzyme kinetics parameters ofthe K_m, V_(max)and CL_(int)values are100.37±18.09μM,18.48±5.63pmol/min/pmol P450,0.18, on behalf similar activity of wild-type.
Conclusions：
In this subject, Proven mechanism of OATP1B1polymorphisms on ATVaffected by the transit mechanism and CYP3A4polymorphisms on ATV metabolismfrom the molecular level can be the innovation of this article.
1、Our present results suggest that the OATP1B1521site might be the molecularaction site of transport ATV, also be the key affect point of transport capacity of ATV.The sites mutate will lead to the reduction of ATV transport into hepatic targets, it isdifficult to achieve the appropriate lipid-lowering efficacy, it is necessary for us topay close attention to the OATP1B1521point mutation of patients in clinically,guiding ATV clinical rational drug use.
2、Our present results suggest that CYP3A4445, CYP3A4218, and CYP3A4185sites may be the molecular role of point of ATV metabolism, also affect CYP3A4on the the ATV metabolic activity of key sites. These mutation site causes amino acidchange, which affects the metabolic activity of ATV, so the ATV in the clinical use ofdrugs should also be concerned about the different sites in patients with CYP3A4mutations.
3、The results suggest that, such as patients taking ATV with transportergenotype of OATP1B1~*5, ATV transported into the liver to reduce the patient for theCYP3A4~*3/~*5mutant, ATV metabolic capacity to improve, give rise to the role oftarget The drug concentration decreased, not only lead to treatment failure, but alsocause peripheral side effects, you will need by increasing the dose or switching toother lipid-lowering drugs, lipid-lowering efficacy. Therefore, in clinical applicationsshould be based on the mutation of OATP1B1and CYP3A4to guide the rational useof the ATV.
阿托伐他汀（Atorvastatin, ATV）是新一代β-羟基-β-甲基戊二酰辅酶A（HMG-CoA）还原酶抑制剂，可竞争性抑制胆固醇的生物合成，显著降低血脂水平。虽然ATV已广泛用于高胆固醇血症的治疗，但其调脂疗效呈现明显的个体差异仍给临床应用带来困扰。有研究表明，ATV与其他他汀类不同之处在于，其既是摄入型转运体OATP1B1的底物，又主要经CYP3A4代谢。故OATP1B1及CYP3A4的基因多态很可能影响ATV的转运与代谢，从而导致ATV疗效和副作用个体差异的出现。因此，系统研究与ATV转运、代谢相关OATP1B1及CYP3A4的基因多态性及其相互间的关联意义重大。
目的：
本课题旨在通过构建OATP1B1~*1a、~*5和~*15重组质粒模型，研究ATV在不同OATP1B1基因型个体中药物体外转运的差异；通过野生型及不同位点突变型CYP3A4重组酶系，研究不同CYP3A4基因型代谢ATV的差异；通过统计分析研究所获数据，探讨ATV在不同基因型个体中转运和代谢差异的分子机制及二者的关联性；为ATV在临床的合理应用提供理论与实验依据。
方法：
1、建立HPLC及LC-MS检测ATV生物样本的方法，摸索ATV的重组蛋白酶样品及HEK293T细胞样品处理方法。
2、构建稳定表达目的基因的细胞模型。将携带OATP1B1~*15-GFP融合基因的慢病毒（OATP1B1~*15质粒构建、慢病毒包装、滴度测定由上海吉凯基因完成）感染HEK293T细胞，建立稳定表达OATP1B1~*15的HEK293T细胞模型，结合本实验室已成功表达OATP1B1~*1a、OATP1B1~*5的HEK293T细胞模型，采用上述三种细胞模型比较ATV在不同基因型OATP1B1个体中摄取转运过程的差异。
3、ATV在表达OATP1B1~*1a、~*5及~*15的HEK293T细胞中吸收转运实验。实验分为空白对照组、OATP1B1~*1a、~*5及~*15三种质粒组，考察摄取缓冲液PH对摄取过程的影响；考察不同时间点（5、10、20、40、60min）对摄取过程的影响；考察底物浓度（5、10、20、50、100、150μM）对摄取过程的影响。采用LC-MS法测定细胞样品中ATV的量，比较ATV在三种不同基因型质粒模型中的摄取动力学参数（K_m、V_(max)、CL_(int)值），分析两个位点突变对ATV转运过程的影响。
4、ATV在野生型及不同位点突变型CYP3A4重组酶系中体外酶促动力学分析。实验分为空白对照组和CYP3A4~*1（wild type）、CYP3A4~*3（M445T）、CYP3A4~*5（P218R）、CYP3A4~*16（T185S）、CYP3A4~*18（L293P）五种重组酶组，每组分别加入系列浓度的ATV10μl，孵育体系中重组酶终浓度为1mg/ml，温孵60min后，加入100μl冰乙腈终止反应后处理样品，采用RP-HPLC法测定孵育体系中ATV及其代谢产物的量，比较ATV在CYP3A4野生型与突变型重组酶系中的酶促动力学参数（K_m、V_(max)、CL_(int)值），分析CYP3A4不同位点突变对ATV代谢的影响。
5、数据处理与统计分析。所有数据均以平均数±标准差（mean±SD）表示，通过米曼氏方程模型、Lineweaver-Burk作图法计算ATV在不同OATP1B1质粒模型中摄取动力学参数，以及ATV在野生型和突变体重组酶系药物代谢动力学参数。采用SPSS12.0软件进行统计分析，组间差异采用t检验，统计结果以P>0.05表示差异无统计学意义，P<0.05表示差异有统计学意义，P<0.01表示显著性差异。
结果：
1、建立的LC-MS检测方法完全能够符合ATV生物样本分析测试的要求，建立的HPLC检测方法符合ATV及其代谢产物分析测试的要求，两种检测方法均具有较高的灵敏度和特异性；成功建立ATV的重组蛋白酶样品及HEK293T细胞样品处理方法，为本课题实施奠定了可靠基础。
2、获得OATP1B1~*15慢病毒融合质粒，其病毒滴度为2x109TU/ml，感染HEK293T后，通过荧光检测，观察到GFP的表达，荧光表达随着时间延长及感染复数(Multiplicity of infection, MOI)值增加而增强、增多，最佳MOI值为50，此时Lenti-OATP1B1~*15感染HEK293T的感染率可达80%；通过Western Blot检测感染的293T样品，可以观察到95kDa附近有特征带表达，其大小和OATP1B1~*15融合蛋白（104kDa）相吻合。表明稳定感染Lenti-OATP1B1~*15的HEK293T细胞模型构建成功。
3、OATP1B1~*1a-HEK293T对ATV的摄取动力学参数K_m与V_(max)及CL_(int)分别为21.17±3.96uM、9.13±1.42pmol·min~(-1)·mg~(-1)protein、0.43；OATP1B1~*5-HEK293T对ATV的摄取动力学参数K_m、V_(max)及CL_(int)分别为13.95±2.32μM、3.88±0.49pmol·min~(-1)·mg~(-1)protein、0.28；OATP1B1~*15-HEK293T对阿托伐他汀摄取的动力学参数K_m与V_(max)及CL_(int)分别为19.37±2.71μM、7.98±1.03pmol·min~(-1)·mg~(-1)protein、0.41。用SPSS12.0进行统计分析，OATP1B1~*5(521T>C)基因型个体对ATV的转运活性低于野生型OATP1B1~*1a(P<0.05)，差异有统计学意义；而OATP1B1~*15(388A>G521T>C)基因型个体与野生型OATP1B1~*1a个体对ATV转运活性差异无统计学意义(P>0.05)。
4、野生型CYP3A4~*1重组酶酶促动力学参数K_m、V_(max)及CL_(int)值分别为88.65±19.32μM、16.39±4.10pmol/min/pmol P450、0.18；突变型CYP3A4~*3酶促动力学参数K_m、V_(max)及CL_(int)值分别为92.58±9.33μM、23.86±5.02pmol/min/pmolP450、0.26，其活性略大于野生型；突变型CYP3A4~*5酶促动力学参数K_m、V_(max)及CL_(int)值分别为109.38±8.21μM、44.84±6.73pmol/min/pmol P450、0.41，其活性显著大于野生型。突变型CYP3A4~*16酶促动力学参数K_m、V_(max)及CL_(int)值分别为156.87±20.87μM、15.67±6.03pmol/min/pmol P450、0.10，其活性显著低于野生型；突变型CYP3A4~*18酶促动力学参数K_m、V_(max)及CL_(int)值分别为100.37±18.09μM、18.48±5.63pmol/min/pmol P450、0.18，其代谢ATV的活性与野生型相近。
结论：
本文从分子学水平探明OATP1B1多态性对ATV转运影响的机制及CYP3A4多态性对ATV代谢影响的机制。
1、结果提示，OATP1B1521位点可能是ATV转运的分子作用点，也是影响OATP1B1对ATV转运能力的关键位点。该位点发生突变会导致转运入肝至靶点的ATV量减少，难以达到相应的降脂疗效，故临床上应关注患者OATP1B1521位点的突变情况，指导ATV临床合理用药。
2、结果提示，CYP3A4445、CYP3A4218、CYP3A4185位点可能是ATV代谢的分子作用点，也是影响CYP3A4对ATV代谢活性的关键位点。这些位点发生突变会导致氨基酸发生变化，从而影响对ATV的代谢活性，故ATV在临床用药时也应关注患者CYP3A4不同位点的突变情况。
3、如服用ATV的患者转运体基因型为OATP1B1~*5，则转运入肝脏的ATV减少，同时该患者为CYP3A4~*3/~*5突变型，则对ATV代谢能力提高，会引起作用于靶点的药物浓度降低，不但导致治疗失败，还可能会引起外周毒副作用，则需通过增加给药剂量或换用其他的降脂药，达到相应的降血脂疗效。故在临床应用中结合OATP1B1及CYP3A4突变情况指导ATV用药，将更具合理性。
Background:
Atorvastatin is a new generation of β-hydroxy-β-methylglutaryl coenzyme A(HMG-CoA) reductase inhibitor, its own biological activity after into the bodywithout metabolism, competitive inhibition of cholesterol biosynthesis, are widelyused in clinical treatment of hypercholesterolemia and elevated serum cholesterol ofmixed hyperlipidemia. ATV transported into hepatic by the organic aniontransporting polypeptide1B1(OATP1B1), To inhibit HMG-CoA reductase combinedwith medroxyprogesterone valerate competitively, reduce the formation of cholesterolto reach the effect of lipid-lowering, ATV mainly metabolismed by CYP3A4, andthen excreted in the form of metabolite finally.
Objectives:
The aim of this project is build OATP1B1~*1a,~*5and~*15recombinant plasmidmodel to study transport differences and its molecular mechanism of atorvastatin indifferent OATP1B1genotype mutant in vitro study. To study the metabolism producedifferences and its molecular mechanism of atorvastatin in wild-type and differentpoints mutant CYP3A4recombinant enzyme. Analysis the experimental data ofatorvastatin in vitro studies, and analysis statins in toxic side effects or ineffectivetreatment caused by differences in transport and metabolism processes that may occurin different genotypes. To provide a theoretical basis for in vivo experiments and theindividual drugs in clinical.
Methods:
1、Create HPLC and LC-MS detection method to detect biologic samples ofATV, and explore cell sample and recombinant enzyme sample preparation method.
2、To infect HEK293T cells with OATP1B1~*15-GFP fusion gene lentivirus (Thejob of OATP1B1~*15plasmid construct, the packaging of lentiviral, titer determ-ination completed by the company of Kaiji gene, Shanghai), To detect the target gene expression of OATP1B1~*15by observing fluorescence and Western blot, Toestablish stable expression of OATP1B1~*15-HEK293T cells model, our laboratoryhas successfully expressed OATP1B1~*1a, OATP1B1~*5HEK293T cell-model, usingthese three cell-models investigated ATV uptake in different genotypes OATP1B1individual transporter process differences.
3、The transport experiment of ATV in OATP1B1~*1a,~*5and~*15-HEK293Tcells. The experiment was divided into blank control group, the OATP1B1~*1a,~*5and~*15three kinds of plasmid group, To investigate the impact of uptake buffer pHon the uptake process; To investigate uptake process at different time points (5,10,20,40,60min). To investigate the impact of substrate concentration (5,10,20,50,100,150μM) to the intake process. To determination the amount of ATV in cell samplesby LC-MS method ATV uptake in the three different genotypes plasmid modelkinetic parameters (K_m, V_(max)and CL_(int)), analysis the effect of two site mutations onATV transit process.
4、The enzyme kinetics study of ATV in wild-type and different point mutantCYP3A4recombinant enzymes in vitro. The experiment was divided into blankcontrol group, CYP3A4~*1, CYP3A4~*3, CYP3A4~*5, CYP3A4~*16, and CYP3A4~*18five kinds of recombinase group, each group were added series of concentrations ofthe ATV10μL, in the incubation system final concentration of the recombinantenzyme is1mg/ml, after incubated60minutes, add100μL ice acetonitrile toterminate reaction, The amount of ATV and its metabolites in the incubation systemdeterminated by RP-HPLC method, To compare the enzymatic kinetic parameters(K_m, V_(max), and CL_(int)) of ATV in wild type and mutant CYP3A4recombinant enzymes,analysis the impact of different sites mutations of CYP3A4to metabolism of ATV.
5、Data processing and statistical analysis: All data are demonstrated bymean±standard deviation (Mean±SD), calculate the uptake kinetics parametersenzymes kinetic parameters of ATV in different OATP1B1plasmid model throughMichaelis-Menton equation model, and Lineweaver-Burk plot. Using SPSS12.0software for data processing, the difference between the groups with the design of thet-test, the statistical results is P>0.05as no significant difference, the statistical resultsis P<0.05as significant difference. the statistical results is P<0.01indicated that there as a very significant difference.
Results:
1、 The established LC-MS detection method is developed to meet theatorvastatin cell sample analysis and testing requirements, with high sensitivity andspecificity. The established HPLC method developed to meet the requirements of theanalysis and testing of atorvastatin and its metabolites with high sensitivity andspecificity.
2、 Recombinant lenti-viral titer of Lenti-OATP1B1~*15is2x109TU/ml;Lenti-OATP1B1~*15after the infection of HEK293T fluorescent protein with timeand MOI (Multiplicity of infection, MOI) value increased with the increase;50is thebest MOI value. Lenti-OATP1B1~*15transfected HEK293T transfection rate is80%when MOI is50. Detected by Western Blot transfected into293T samples observed95kDa near the bar characteristics with its size and OATP1B1double mutant fusionprotein (~76kDa+28kDa=104kDa) is consistent. That OATP1B1double mutant wassuccessfully expressed.
3、OATP1B1~*1a and OATP1B1~*5gene atorvastatin intake are quite different,OATP1B1~*5gene reduce the transport capacity of atorvastatin. Compared withOATP1B1~*15, OATP1B1~*5transport activity lower. The uptake kinetic parametersK_m、V_(max)and CL_(int)of OATP1B1~*1a-HEK293T are21.17±3.96μM,9.13±1.42pmol·min~(-1)·mg~(-1)protein,0.43. OATP1B1~*5-HEK293T are13.95±2.32μM3.88±0.49pmol·min~(-1)·mg~(-1)protein,0.28. OATP1B1~*5-HEK293T atorvastatin uptake kineticparameters K_m, V_(max)and CL_(int)are19.37±2.71μM7.98±1.03pmol·min~(-1)·mg~(-1)protein,0.41, respectively. Statistical analysis by SPSS12.0, Series of concentrations of ATVtransported in OATP1B1~*1a and OATP1B1~*5, OATP1B1~*15and OATP1B1~*5arestatistically significant.
4、Results of research are CYP3A4~*1enzyme kinetics parameters of the K_mandV_(max)and CL_(int)values were88.65±19.32μM,16.39±4.10pmol/min/pmol P450,0.18;Enzymatic kinetic parameters K_m, V_(max)and CL_(int)values of CYP3A4~*5are109.38±8.21μM,44.84±6.73pmol/min/pmol P450,0.41, its activity significantly greater thanthe wild type; enzyme kinetics parameters K_m, V_(max)and CL_(int)values of CYP3A4~*3 are92.58±9.33μM23.86±5.02pmol/min/pmol P450,0.26, its activity is slightly largerthan the wild type. CYP3A4~*16enzymatic kinetic parameters of the K_m, V_(max)andCL_(int)values are156.87±20.87μM,15.67±6.03pmol/min/pmol P450,0.10, its activitysignificantly lower than the wild type. CYP3A4~*18enzyme kinetics parameters ofthe K_m, V_(max)and CL_(int)values are100.37±18.09μM,18.48±5.63pmol/min/pmol P450,0.18, on behalf similar activity of wild-type.
Conclusions：
In this subject, Proven mechanism of OATP1B1polymorphisms on ATVaffected by the transit mechanism and CYP3A4polymorphisms on ATV metabolismfrom the molecular level can be the innovation of this article.
1、Our present results suggest that the OATP1B1521site might be the molecularaction site of transport ATV, also be the key affect point of transport capacity of ATV.The sites mutate will lead to the reduction of ATV transport into hepatic targets, it isdifficult to achieve the appropriate lipid-lowering efficacy, it is necessary for us topay close attention to the OATP1B1521point mutation of patients in clinically,guiding ATV clinical rational drug use.
2、Our present results suggest that CYP3A4445, CYP3A4218, and CYP3A4185sites may be the molecular role of point of ATV metabolism, also affect CYP3A4on the the ATV metabolic activity of key sites. These mutation site causes amino acidchange, which affects the metabolic activity of ATV, so the ATV in the clinical use ofdrugs should also be concerned about the different sites in patients with CYP3A4mutations.
3、The results suggest that, such as patients taking ATV with transportergenotype of OATP1B1~*5, ATV transported into the liver to reduce the patient for theCYP3A4~*3/~*5mutant, ATV metabolic capacity to improve, give rise to the role oftarget The drug concentration decreased, not only lead to treatment failure, but alsocause peripheral side effects, you will need by increasing the dose or switching toother lipid-lowering drugs, lipid-lowering efficacy. Therefore, in clinical applicationsshould be based on the mutation of OATP1B1and CYP3A4to guide the rational useof the ATV.
引文
[1] King A. Risk factors: Everything in moderation-binge drinking increases CVD risk [J]. NatRev Cardiol,2011,8(2):62.
[2] Han SH, Kang EW, Yoon SJ, et al. Combined vascular effects of HMG-CoA reductaseinhibitor and angiotensin receptor blocker in non-diabetic patients undergoing peritonealdialysis [J]. Nephrol Dial Transplant,2011, Epub ahead of print.
[3] Cziraky MJ, Willey VJ, McKenney JM, et al. Statin safety: an assessment using anadministrative claims database [J]. Am J Cardiol,2006,97(8A):61C-68C.
[4] Simran D S, Martin A K, David W J. Clinical Implications of Pharmacogenetic Variation onthe Effects of Statins [J]. Drug Saf,2011,34(1):1-19.
[5]王震宇,禹同生,王健,等.阿托伐他汀的药理作用及临床应用进展[J].中国新药杂志,2010,19(18):1684-1687.
[6] Silva M, Matthews M L, Jarvis C, et al. Meta-analysis of drug-induced adverse eventsassociated with intensive-dose statin therapy [J]. Clin Ther,2007,29:253–260.
[7] Lins RL, Matthys KE, Verpooten GA, et al. Pharmacokinetics of atorvastatin and itsmetabolites after single and multiple dosing in hypercholesterolaemic haemodialysispatients[J]. Nephrology and Dial Transplant,2003,18:967–976.
[8] Willrich MAV, Hirata MH, Genvigir FDV, et al. CYP3A5*3A allele is associated withreduced lowering-lipid response to atorvastatin in individuals with hypercholesterolemia [J].Clin Chim Acta,2008,398(1-2):15-20.
[9] Ho RH, Choi L, Lee W, et al. Effect of drug transporter genotypes on pravastatin dispositionin European and African-American participants [J]. Pharmacogenet Genomics,2007,17(8):647-656.
[10] Hagenbuch B, Meier PJ. Organic anion transporting polypeptides of the OATP/SLC21family: phylogenetic classification as OATP/SLCO super-family, new nomenclature andmolecular/functional properties [J]. P flugers Arch,2004,447(5):653-665.
[11] Niemi M. Role of OATP transporters in the disposition of drugs [J]. Pharmacogenomics,2007,8(7):787-802.
[12] Niemi M, Schaeffeler E, Lang T, et al. High plasma pravastatin concentrations areassociated with single nucleotide polymorphisms and haplotypes of organic aniontransporting polypeptide-C (OATP-C,SLCO1B1)[J]. Pharmacogenetics,2004,14(7):429-440.
[13] Pasanen MK, Fredrikson H, Neuvonen PJ, et al. Different effects of SLCO1B1polymo-rphism on the pharmacokinetics of atorvastatin and rosuvastatin [J]. Clin Pharmacol Ther,2007,82(6):726-733.
[14] Ieiri I, Suwannakul S, Maeda K, et al. SLCO1B1(OATP1B1, an up take transporter) andABCG2(BCRP, an efflux transporter) variant alleles and pharmacokinetics of pitavastatin inhealthy volunteers [J]. Clin Pharm acol Ther,2007,25:541-547.
[15] Deng JW, Song IS, Shin HJ, et al. The effect of SLCO1B1*15on the disposition ofpravastatin and pitavastatin is substrate dependent: the contribution of transporting activitychanges by SLCO1B1*15[J]. Pharmacogenet Genomics,2008;18(5):424-433.
[16] Eiselt R, Domanski T L, Zibat A, et al. Identification and functional characterization of eightCYP3A4protein variants [J]. Pharmacogenetics,2001,11:447-458.
[17] Dai D, Tang J, Rose R, et al. Identification of variants of CYP3A4and characterization oftheir abilities to metabolize testosterone and chlorpyrifos [J]. J Pharmacl Exp Ther,2001,299:825-831.
[18] Matsumura K, Saito T, Takahashi Y, et al. Identifieation of a novel polymrphic enhancer ofthe human CYP3A4gene [J]. Mol Pharmacol,2004,65:326-334.
[19] Nicolas JM, Espie P, Molimard M. Gender and interindividual variability inpharmacokinetics [J]. Drug. Metab. Rev.2009,41:408-421.
[20] Kouji K, Margaret E B. CYP3A4Genotypes and Plasma Lipoprotein Levels Before andAfter Treatment With Atorvastatin in Primary Hypercholesterolemia [J]. The AmericanJournal of Cardiology,2004,93:104-107.
[21] Wei Zhang, Yan-Zi Chang, Quan-Cheng Kan, et al. CYP3A4*1G genetic polymorphisminfluences CYP3A activity and response to fentanyl in Chinese gynecologic patients [J]. EurJ Clin Pharmacol,2010,66:61-66.
[22] Konig J, Cui Y, Nies A T, Keppler D. A novel humanorganic anion transportingpolypepti-de localized to the basolateralhepatocyte membrane [J]. Am. J Physio Gastrointest.Liver Physiol,2000,278: G156–G164.
[23] Kim R. Genetic variations and frequeneies of major haplotypes in SLCOIB1encoding thetransporter OATPIB1in Japanese subjects: SLC0lB1*17is more prevalent than*15[J]. DrugMetab Pharmacokinet,2007,22:456-461.
[24] Annick Seithel, Sonja Eberl, Katrin Singer, et al. The Influence of Macrolide Antibiotics onthe Uptake of OrganicAnions and Drugs Mediated by OATP1B1and OATP1B3[J]. DrugMetabolism and Disposition,2007,35:779–786
[25] Niemi M, Backman JT, Kajosaari LI, et al. Polymorphic organic anion transportingpolypep-tde1B1is a major determinant of repaglinide pharmacokinetics [J]. Clin PharmacolTher,2005,77(6):468-478
[26] Gui C, Obaidat A, Chaguturu R. Development of a cell-based high-throughput assay toscreen for inhibitors of organic anion transporting polypeptides1B1and1B3[J]. Curr ChemGenomics.2010,1(4):1-8
[27] Tirona RG, Leake BF, Merino G, et al. Polymorphic in OATP-C; identification of multiplealle-ic Variants associated with altered transport activity among European-and African-Americans [J]. J Biol Chem,2001,276(38):35669-35675.
[28] Nozawa T, Nakajima M, Tamai I, et al. Genetic polymorphisms of human organic anion tran-sporters OATP-C and OATP-B; allele frequencies in the Japanese population and fucionalAnalysis [J]. J Phatmacol Exp Ther,2002,302(2):804-813.
[29] Nishizato Y, Ieier I, Suzuki H, et al. Polymorphisms of OATP-C and OATP3genes;consepuences for pravastatin pharmacokinetics [J]. Chin pharmacol Ther,2003,73(6):554-565.
[30]杨彪，梅晰凡，刘福强.HEK293细胞复苏培养及冻存[J].辽宁医学院学报,2007,28(6):1-3.
[31]沈大棱,吴超群.细胞生物学[M].复旦大学出版社,2007,263-267.
[32] Chung YK, Heo HJ, Kim EK，et al．Inhibitory effect of ursolic acid purified from Origanummajorana L on the acetylcholinesterase [J]. Mol Cells,2001,11(2):137-143.
[33] Maurizio Federico, Lentivirus Gene Engineering Protocols (Methods in Molecular Biology)[J]. Humana Press,2003,1:30.
[34] Chalfie M, Tu Y, Euskirehen G, et al.Green fuoreseent protein as a marker for geneexpression [J]. Science,1994,263(14):802-805.
[35] Maeda K, Sugiyama Y. Impact of genetic polymorphisms of transporters on thepharmacokinetic, pharmacodynamic and toxicological properties of anionic drugs [J]. DrugMetab.Pharmacokinet.2008,23:223-235.
[36] Ieiri I, Higuchi S, Sugiyama Y. Genetic polymorphisms of uptake (OATP1B1,1B3) andefflux (MRP2, BCRP) transporters: implications for inter-individual differences in thepharmacokinetics and pharmacodynamics of statins and other clinically relevant drugs [J].Expert Opin Drug Metab Toxicol.2009,5(7):703-729.
[37] Richard HH, Rommel GT, BrendaF LE, et al. Drug and Bile Acid Transporters inRosuvastatin Hepatic Uptake: Function Expression and Pharmacogenetics [J]. Gastroenter-ology,2006,130:1793–1806.
[38]张伟,贺毅憬,周宏灏.有机阴离子转运多肽1B1的遗传药理学进展[J].中国临床药理学与治疗学.2008,13(7):721-727.
[39] Neuvonen PJ. Drug interactions with HMG-CoA reductase inhibitors (statins): theimportance of CYP enzymes, transporters and pharmacogenetics [J]. Curr Opin InvestigDrugs,2010,11(3):323-332.
[40] Toda T, Eliasson E, Yama D, et al. Roles of different CYP enzymes in the formation ofspecific fluvastatin metabolites by human liver microsomes [J]. Basic Clin PharmacolToxicol.2009,105(5):327-332.
[41] Niemi M, Cascorbi I, Sugiy Y, et al. Glyburide and glimepiride pharmacokinetics in subjectswith different CYP2C9genotypes [J]. Clin Pharmacol. Ther2002,72:326-332.
[42] Bolego C, Baetta R, Bellosta S, et al. Safety considerations for statins [J]. Curr Opin Lipidol,2002,13(6):637-644.
[43] Kajinami K, Brousseau ME, Ordovas JM, et al. CYP3A4genotypes and plasma lipoproteinlevels before and after treatment with atorvastatin in primary hypercholesterolemia [J]. Am JCardiol,2004,93(1):104-107.
[44] Daly AK. Significance of the minor cytochrome P4503A isoforms [J]. Clin Pharmacokinet,2006,45(1):13-31.
[45] Floyd MD, Gervasini G, Masica AL, et al. Genotype-phenotype associations for commonCYP3A4and CYP3A5variants in the basal and induced metabolism of midazolam inEuropean and African-American men and women [J]. Pharmacogenetics,2003,13:595-606.
[46] Alessio Provenzani, Monica Notarbartolo, Manuela Labbozzetta, et al. The effect ofCYP3A4and ABCB1single nucleotide polymorphisms on tacrolimus dose requirements inCaucasian liver transplant patients [J]. Ann Transplant,2009,14(1):23-31.
[47] Fujino H, Saito T, Tsunenari Y, et al. Metabolic properties of the acid and lactone forms ofHMG-CoA reductase inhibitors [J]. Xenobiotica,2004,34(11):961-971.
[48] Kajinami K, Akao H, Polisecki E, et al. Pharmacogenomics of statin responsiveness [J]. AmJ Cardiol,2005,96(9):65-70.
[49]王晓雯,曾苏.人细胞色素P4503A4突变体CYP3A4.3, CYP3A4.4, CYP3A4.5和CYP3A4.18重组酶的异源表达与活性[J].中国药理学与毒理学杂志,2009,23(6):456-463.
[50] Fiegenbaum M, Da Silveira FR, Van Der Sand CR, et al. The role of common variants ofABCB1, CYP3A4, and CYP3A5genes in lipid-lowering efficacy and safety of simvastatintreatment [J]. Clin Pharmacol Ther2005,78(5):551-558.
[51] Rashid S, Curtis DE, Garuti R, et al. Decreased plasma cholesterol and hypersensitivity tostatins in mice lacking Pcsk [J]. Proc Nat Acad Sci U S A,2005,102(15):5374-5379.
[52] Venrooij FV, Stolk RP, Banga J-D, et al. Common cholesteryl ester transfer protein genepolymorphisms and the effect of atorvastatin therapy in type2diabetes [J]. Diabetes Care,2003,26(4):1216-1223.
[53]张莉莉,吕安林,贾国良等.载脂蛋白E基因多态性对他汀类药物调脂消斑的影响[J].第四军医大学学报,2004,25(11):1029-1031.
[54] Pedro-Botet J, Schaefer EJ, Bakker-Arkema RG, et al. Apolipoprotein E genotype affectsplasma lipid response to atorvastatin in a gender specific manner [J]. Atherosclerosis,2001,158(1):183-193.
[55] Neuvonen P J. Drug interactions with HMG-CoA reductase inhibitors (statins): theimportance of CYP enzymes, transporters and pharmacogenetics [J]. Curr Opin InvestigDrugs,2010,11(3):323-332.
[56] Cuiping Chen, Rouchelle J.M, Scott D.C. et al. Differential Interation of3-Hydroxy-3-Methylglutarl-CoA Reductase Inhibtors With ABCB1, ABCC2, and OATP1B1[J]. DRUGMETABOLISM AND DISPOSITION,2005,33:537–546.
[1] Watanabe RM. Drugs, diabetes and pharmacogenomics: the road to personalized therapy [J].Pharmacogenomics,2011,12(5):699-701.
[2] Neuvonen P J. Drug interactions with HMG-CoA reductase inhibitors (statins): the importanceof CYP enzymes, transporters and pharmacogenetics [J]. Curr Opin Investig Drugs,2010,11(3):323-332.
[3] Toda T, Eliasson E, Puist H, et al. Roles of different CYP enzymes in the formation of specificfluvastatin metabolites by human liver microsomes [J]. Basic Clin Pharmacol Toxicol,2009,105(5):327-332
[4] Niemi M, Cascorbi I, Turnquist H, et al. Glyburide and glimepiride pharmacokinetics insubjects with different CYP2C9genotypes [J]. Clin Pharmacol Ther.2002,72:326-332.
[5] Julia K, Dirk K, Tuist H, et al. Influence of CYP2C9polymorphisms on the pharmacokineticsand cholesterol-lowering activity of (-)-3S,5R-fluvastatin and (＋)-3R,5S-fluvastatin inhealthy volunteers [J]. Clin Pharmacol Ther.2003,74:186-194.
[6] Kouji K, Margaret E B, Turnt D, et al. CYP3A4Genotypes and Plasma Lipoprotein LevelsBefore and After Treatment With Atorvastatin in Primary Hypercholesterolemia [J]. TheAmerican Journal of Cardiology,2004,93:104-107.
[7] Hagenbuch B, Meier PJ. Organic anion transporting polypeptides of the OATP/SLC21family:phylogenetic classification as OATP/SLCO superfamily, new nomenclature and molecular/functional properties [J]. Pflugers Arch,2004,447(5):653-665.
[8] Niemi M. Role of OATP transporters in the disposition of drugs [J]. Pharmacogenomics,2007,8(7):787-802.
[9] Nozawa T, Nakajima M, Tamai I, et al. Genetic polymorphisms of human organic aniontransporters OATP-C(SLC-1A6) and OATP-B(SLC-1A9): allele frequencies in the Japanesepopulation and functional analysis [J]. J Pharm Acol Exp Ther,2002,302(2):804-813.
[10] Niemi M, Schaeffeler E, Lang T, et al. High plasma pravastatin concentrations are associatedwith single nucleotide polymorphisms and haplotypes of organic anion transportingpolypeptide-C (OATP-C, SLCO1B1)[J].Pharmacogenetics,2004,14(7):429-440.
[11] Kim EY, Cho DY, Shin HJ, et al. Duplex pyrosequencing assay of the388A> Gand521T> CSLCO1B1polymorphisms in three Asian populations [J]. Clin Chim Acta,2008,388(1P2):68-72.
[12] Masaru H, Kazuya M, Yoshihisa S, et al. Contribution of OATP2(OATP1B1) and OATP8(OATP1B3) tothe hepatic up take of pitavastatin in humans [J]. J Pharmacol Exp,2004,311:139-146.
[13] Chung, J.Y, Cho DY, Ieiri I, et al. Effect of OATP1B1(SLCO1B1) variant alleles on thepharmacokinetics of pitavastatin in healthy volunteers [J]. Clin Pharmacol,2005,78:342–350.
[14] Ieiri I, Suwannakul S, Maeda K, et al. SLCO1B1(OATP1B1, an up take transporter) andABCG2(BCRP, an efflux transporter) variant alleles and pharmacokinetics of pitavastatin inhealthy volunteers [J]. Clin Pharm acol Ther,2007,25:541-547.
[15] Deng JW, Song IS, Tu H, et al. The effect of SLCO1B1*15on the disposition of pravastatinand pitavastatin is substrate dependent: the contribution of transporting activity changes bySLCO1B1*1[J]. Pharmacogenet Genomics.2008,18(5):424-433.
[16] Pasanen MK, Fredrikson H, Neuvonen PJ, et al. Different Effects of SLCO1B1Polymorp-hism on the Pharmacokinetics of Atorvastatin and Rosuvastatin [J].Clinical Pharmacology&Therapeutics,2007,82:726–733.
[17] JH Choi, MG Lee, J-Y Cho, et al. Influence of OATP1B1Genotype on the Pharmacokineticsof Rosuvastatin in Koreans [J]. Clinical Pharmacology Therapeutics,2008,83(2):251-257.
[18] Nishizato Y, Ieiri I, Suzuki H, et al. Polymorphisms of OATP-C(SLC-1A6) and OAT3(SLC-2A8) genes:consequences for p ravastatin pharmacokinetics [J]. Clin Pharmacol Ther,2003,73(6):554-565.
[19] Niemi M, Pasanen MK, Teiri Y, et al. SLCO1B1polymorphism and sex affect thepharmacokinetics of pravastatin but not fluvastatin [J]. Clin Pharmacol Ther,2006,80:356–366.
[20] Ho RH, Choi L, Lee W, et al. Effect of drug transporter genotypes on p ravastatin dispositionin European-and African-American participants [J]. Pharmacogenet Genomics,2007,17(8):647-656.
[21] Zhang W, Chen B L, Ozdemir V, et al. SLCO1B1521T→C functional genetic polymorphismand lipid-lowering efficacy of multiple-dose pravastatin in Chinese coronary heart diseasepatients [J]. Br J Clin Pharmacol,2007,64(3):346-352.
[22]温爱萍,徐小薇,严晓伟.有机阴离子转运多肽1B1遗传多态性对普伐他汀药动学的影响[J].中国药学杂志,2008,43(22):1735-1739.
[23] Choi MK, Shin HJ. Differential effect of genetic variants of Na(+)-taurocholateco-transporting polypeptide (NTCP) and organic anion-transporting polypeptide1B1(OATP1B1) on the uptake of HMG-CoA reductase inhibitors [J]. Xenobiotica,2011,41(1):24-34.
[24] Min Kim, Turnquist H, Jackson J, et al. The multidrug resistance transporter ABCG2(breastcancer resistance protein effluxes Hoechst33342and is overexpressed in hematopoietic stemcells [J]. Clin Can Res,2002,8(1):22-28.
[25] Sang SL, Hye E J, Joo MY, et al. Identification and Functional Assessment of BCRPPolymorphisms in a Korean Population [J]. The American Society for Pharmacology andExperimental Therapeutics,2007,35:623-632.
[26] Zamber CP, Lamba JK, Yasuda K, et al. Natural allelic variants of breast cancer resistanceprotein (BCRP) and their relationship to BCRP expression in human intestine [J].Pharmacogenetics,2003,13:19-28.
[27] Zhang Wei, Yu Bang-Ning, Li Xi, et al. Role of BCRP421C>A polymorphism onrosuvastatin pharmacokinetics in healthy Chinese males [J]. Clinica Chimica Acta,2006,373:99-103.
[28] Keskitalo JE, Pasanen MK, Jin LR, et al. Different effects of the ABCG2c.421C>A SNP onthe pharmacokinetics of fluvastatin, pravastatin and simvastatin [J]. Phar pharmacogenomics,2009,10(10):1617-1624.
[29] Cudaback E, Li X, Montine KS, et al. Apolipoprotein E isoform-dependent microgliamigration [J]. J Bio Chem,2011, Epub ahead of print.
[30] Fu YH, Lv RJ, Jin LR, et al. Association of apolipoprotein E polymorphisms with temporallobe epilepsy in a Chinese Han population [J]. Epilepsy Res,2010,91(2-3):253-259.
[31]李虎,赵水平.载脂蛋白E及胆固醇酯转运蛋白基因多态性与冠心病的相关性研究[J].临床荟萃,2009,24(24):2127-2130.
[32] Ballantyne CM, Herd JA, Stein EA, et al. Apolipoprotein E genotypes and response ofplasma lipids and progressionregression of coronary atherosclerosis to lipid-lowering drugtherapy [J]. J Am Coll Cardiol,2000,36(5):1572-1578.
[32] Pedro-Botet J, Schaefer EJ, Bakker-Arkema RG, et al. Apolipoprotein E genotype affectsplasma lipid response to atorvastatin in a gender specific manner [J]. Atherosclerosis,2001,158(1):183-193.
[33] Huang W, Ye X, Li X, et al. The inhibition activity of chemical constituents in hawthorn fruitand their synergistic action to HMG-CoA reductase [J]. Zhong guo Zhong Yao Za Zhi,2010,35(18):2428-2431.
[34]童煜. HMG-CoA还原酶基因多态与冠心病的相关性研究[D].四川:四川大学遗传学,2003.
[35] Chasman DI, Posada D, Subrahmanyan L, et al. Pharmacogenetic study of statin therapy andcholesterol reduction [J]. JAMA,2004,291(23):2821-2827.
[36] Zee A-HK, Olaf H, Stricker BH, et al. Genetic polymorphisms: importance for response toHMG-CoA reductase inhibitors [J]. Atherosclerosis,2002,163(2):213-222.
[37] Agellon L, Quinet E, Gilletle D, et al. Organization of the human cholestery estentransrerproein gene [J]. Biochemistry,2009,29:1372-1376.
[38]张震宇,王颍,谢玉才.胆固醇酯转移蛋白与氟伐他汀降脂疗效的关系[J],临床心血管杂志.2008,24(8):590-592.
[39] Schmitz G, Langmann T. Pharmacogenomicsof cholesterol lowering therapy [J]. VascPharmacol2006,44(2):75-89.
[40] Grooth DE, Kim YR, Song J, et al. Genetic variations of cholesterol ester transfer proteingene in Koreans [J]. Hum Biol,2001,73(6):815-821.
[41]朱燕林,严晓伟,鄢盛凯.胆固醇酯转移蛋白TaqB基因多态性与辛伐他汀调脂疗效的相关性[J].中国临床药理学杂志,2004,152:367-375.
[42] Kathiresan S, Manning A K, Demissie S, et al. A genome-wide association study for bloodlipid phenotypes in the Framingham Heart Study [J]. BMC Med Genet,2007,8(1): S1-17.
[2] Han SH, Kang EW, Yoon SJ, et al. Combined vascular effects of HMG-CoA reductaseinhibitor and angiotensin receptor blocker in non-diabetic patients undergoing peritonealdialysis [J]. Nephrol Dial Transplant,2011, Epub ahead of print.
[3] Cziraky MJ, Willey VJ, McKenney JM, et al. Statin safety: an assessment using anadministrative claims database [J]. Am J Cardiol,2006,97(8A):61C-68C.
[4] Simran D S, Martin A K, David W J. Clinical Implications of Pharmacogenetic Variation onthe Effects of Statins [J]. Drug Saf,2011,34(1):1-19.
[5]王震宇,禹同生,王健,等.阿托伐他汀的药理作用及临床应用进展[J].中国新药杂志,2010,19(18):1684-1687.
[6] Silva M, Matthews M L, Jarvis C, et al. Meta-analysis of drug-induced adverse eventsassociated with intensive-dose statin therapy [J]. Clin Ther,2007,29:253–260.
[7] Lins RL, Matthys KE, Verpooten GA, et al. Pharmacokinetics of atorvastatin and itsmetabolites after single and multiple dosing in hypercholesterolaemic haemodialysispatients[J]. Nephrology and Dial Transplant,2003,18:967–976.
[8] Willrich MAV, Hirata MH, Genvigir FDV, et al. CYP3A5*3A allele is associated withreduced lowering-lipid response to atorvastatin in individuals with hypercholesterolemia [J].Clin Chim Acta,2008,398(1-2):15-20.
[9] Ho RH, Choi L, Lee W, et al. Effect of drug transporter genotypes on pravastatin dispositionin European and African-American participants [J]. Pharmacogenet Genomics,2007,17(8):647-656.
[10] Hagenbuch B, Meier PJ. Organic anion transporting polypeptides of the OATP/SLC21family: phylogenetic classification as OATP/SLCO super-family, new nomenclature andmolecular/functional properties [J]. P flugers Arch,2004,447(5):653-665.
[11] Niemi M. Role of OATP transporters in the disposition of drugs [J]. Pharmacogenomics,2007,8(7):787-802.
[12] Niemi M, Schaeffeler E, Lang T, et al. High plasma pravastatin concentrations areassociated with single nucleotide polymorphisms and haplotypes of organic aniontransporting polypeptide-C (OATP-C,SLCO1B1)[J]. Pharmacogenetics,2004,14(7):429-440.
[13] Pasanen MK, Fredrikson H, Neuvonen PJ, et al. Different effects of SLCO1B1polymo-rphism on the pharmacokinetics of atorvastatin and rosuvastatin [J]. Clin Pharmacol Ther,2007,82(6):726-733.
[14] Ieiri I, Suwannakul S, Maeda K, et al. SLCO1B1(OATP1B1, an up take transporter) andABCG2(BCRP, an efflux transporter) variant alleles and pharmacokinetics of pitavastatin inhealthy volunteers [J]. Clin Pharm acol Ther,2007,25:541-547.
[15] Deng JW, Song IS, Shin HJ, et al. The effect of SLCO1B1*15on the disposition ofpravastatin and pitavastatin is substrate dependent: the contribution of transporting activitychanges by SLCO1B1*15[J]. Pharmacogenet Genomics,2008;18(5):424-433.
[16] Eiselt R, Domanski T L, Zibat A, et al. Identification and functional characterization of eightCYP3A4protein variants [J]. Pharmacogenetics,2001,11:447-458.
[17] Dai D, Tang J, Rose R, et al. Identification of variants of CYP3A4and characterization oftheir abilities to metabolize testosterone and chlorpyrifos [J]. J Pharmacl Exp Ther,2001,299:825-831.
[18] Matsumura K, Saito T, Takahashi Y, et al. Identifieation of a novel polymrphic enhancer ofthe human CYP3A4gene [J]. Mol Pharmacol,2004,65:326-334.
[19] Nicolas JM, Espie P, Molimard M. Gender and interindividual variability inpharmacokinetics [J]. Drug. Metab. Rev.2009,41:408-421.
[20] Kouji K, Margaret E B. CYP3A4Genotypes and Plasma Lipoprotein Levels Before andAfter Treatment With Atorvastatin in Primary Hypercholesterolemia [J]. The AmericanJournal of Cardiology,2004,93:104-107.
[21] Wei Zhang, Yan-Zi Chang, Quan-Cheng Kan, et al. CYP3A4*1G genetic polymorphisminfluences CYP3A activity and response to fentanyl in Chinese gynecologic patients [J]. EurJ Clin Pharmacol,2010,66:61-66.
[22] Konig J, Cui Y, Nies A T, Keppler D. A novel humanorganic anion transportingpolypepti-de localized to the basolateralhepatocyte membrane [J]. Am. J Physio Gastrointest.Liver Physiol,2000,278: G156–G164.
[23] Kim R. Genetic variations and frequeneies of major haplotypes in SLCOIB1encoding thetransporter OATPIB1in Japanese subjects: SLC0lB1*17is more prevalent than*15[J]. DrugMetab Pharmacokinet,2007,22:456-461.
[24] Annick Seithel, Sonja Eberl, Katrin Singer, et al. The Influence of Macrolide Antibiotics onthe Uptake of OrganicAnions and Drugs Mediated by OATP1B1and OATP1B3[J]. DrugMetabolism and Disposition,2007,35:779–786
[25] Niemi M, Backman JT, Kajosaari LI, et al. Polymorphic organic anion transportingpolypep-tde1B1is a major determinant of repaglinide pharmacokinetics [J]. Clin PharmacolTher,2005,77(6):468-478
[26] Gui C, Obaidat A, Chaguturu R. Development of a cell-based high-throughput assay toscreen for inhibitors of organic anion transporting polypeptides1B1and1B3[J]. Curr ChemGenomics.2010,1(4):1-8
[27] Tirona RG, Leake BF, Merino G, et al. Polymorphic in OATP-C; identification of multiplealle-ic Variants associated with altered transport activity among European-and African-Americans [J]. J Biol Chem,2001,276(38):35669-35675.
[28] Nozawa T, Nakajima M, Tamai I, et al. Genetic polymorphisms of human organic anion tran-sporters OATP-C and OATP-B; allele frequencies in the Japanese population and fucionalAnalysis [J]. J Phatmacol Exp Ther,2002,302(2):804-813.
[29] Nishizato Y, Ieier I, Suzuki H, et al. Polymorphisms of OATP-C and OATP3genes;consepuences for pravastatin pharmacokinetics [J]. Chin pharmacol Ther,2003,73(6):554-565.
[30]杨彪，梅晰凡，刘福强.HEK293细胞复苏培养及冻存[J].辽宁医学院学报,2007,28(6):1-3.
[31]沈大棱,吴超群.细胞生物学[M].复旦大学出版社,2007,263-267.
[32] Chung YK, Heo HJ, Kim EK，et al．Inhibitory effect of ursolic acid purified from Origanummajorana L on the acetylcholinesterase [J]. Mol Cells,2001,11(2):137-143.
[33] Maurizio Federico, Lentivirus Gene Engineering Protocols (Methods in Molecular Biology)[J]. Humana Press,2003,1:30.
[34] Chalfie M, Tu Y, Euskirehen G, et al.Green fuoreseent protein as a marker for geneexpression [J]. Science,1994,263(14):802-805.
[35] Maeda K, Sugiyama Y. Impact of genetic polymorphisms of transporters on thepharmacokinetic, pharmacodynamic and toxicological properties of anionic drugs [J]. DrugMetab.Pharmacokinet.2008,23:223-235.
[36] Ieiri I, Higuchi S, Sugiyama Y. Genetic polymorphisms of uptake (OATP1B1,1B3) andefflux (MRP2, BCRP) transporters: implications for inter-individual differences in thepharmacokinetics and pharmacodynamics of statins and other clinically relevant drugs [J].Expert Opin Drug Metab Toxicol.2009,5(7):703-729.
[37] Richard HH, Rommel GT, BrendaF LE, et al. Drug and Bile Acid Transporters inRosuvastatin Hepatic Uptake: Function Expression and Pharmacogenetics [J]. Gastroenter-ology,2006,130:1793–1806.
[38]张伟,贺毅憬,周宏灏.有机阴离子转运多肽1B1的遗传药理学进展[J].中国临床药理学与治疗学.2008,13(7):721-727.
[39] Neuvonen PJ. Drug interactions with HMG-CoA reductase inhibitors (statins): theimportance of CYP enzymes, transporters and pharmacogenetics [J]. Curr Opin InvestigDrugs,2010,11(3):323-332.
[40] Toda T, Eliasson E, Yama D, et al. Roles of different CYP enzymes in the formation ofspecific fluvastatin metabolites by human liver microsomes [J]. Basic Clin PharmacolToxicol.2009,105(5):327-332.
[41] Niemi M, Cascorbi I, Sugiy Y, et al. Glyburide and glimepiride pharmacokinetics in subjectswith different CYP2C9genotypes [J]. Clin Pharmacol. Ther2002,72:326-332.
[42] Bolego C, Baetta R, Bellosta S, et al. Safety considerations for statins [J]. Curr Opin Lipidol,2002,13(6):637-644.
[43] Kajinami K, Brousseau ME, Ordovas JM, et al. CYP3A4genotypes and plasma lipoproteinlevels before and after treatment with atorvastatin in primary hypercholesterolemia [J]. Am JCardiol,2004,93(1):104-107.
[44] Daly AK. Significance of the minor cytochrome P4503A isoforms [J]. Clin Pharmacokinet,2006,45(1):13-31.
[45] Floyd MD, Gervasini G, Masica AL, et al. Genotype-phenotype associations for commonCYP3A4and CYP3A5variants in the basal and induced metabolism of midazolam inEuropean and African-American men and women [J]. Pharmacogenetics,2003,13:595-606.
[46] Alessio Provenzani, Monica Notarbartolo, Manuela Labbozzetta, et al. The effect ofCYP3A4and ABCB1single nucleotide polymorphisms on tacrolimus dose requirements inCaucasian liver transplant patients [J]. Ann Transplant,2009,14(1):23-31.
[47] Fujino H, Saito T, Tsunenari Y, et al. Metabolic properties of the acid and lactone forms ofHMG-CoA reductase inhibitors [J]. Xenobiotica,2004,34(11):961-971.
[48] Kajinami K, Akao H, Polisecki E, et al. Pharmacogenomics of statin responsiveness [J]. AmJ Cardiol,2005,96(9):65-70.
[49]王晓雯,曾苏.人细胞色素P4503A4突变体CYP3A4.3, CYP3A4.4, CYP3A4.5和CYP3A4.18重组酶的异源表达与活性[J].中国药理学与毒理学杂志,2009,23(6):456-463.
[50] Fiegenbaum M, Da Silveira FR, Van Der Sand CR, et al. The role of common variants ofABCB1, CYP3A4, and CYP3A5genes in lipid-lowering efficacy and safety of simvastatintreatment [J]. Clin Pharmacol Ther2005,78(5):551-558.
[51] Rashid S, Curtis DE, Garuti R, et al. Decreased plasma cholesterol and hypersensitivity tostatins in mice lacking Pcsk [J]. Proc Nat Acad Sci U S A,2005,102(15):5374-5379.
[52] Venrooij FV, Stolk RP, Banga J-D, et al. Common cholesteryl ester transfer protein genepolymorphisms and the effect of atorvastatin therapy in type2diabetes [J]. Diabetes Care,2003,26(4):1216-1223.
[53]张莉莉,吕安林,贾国良等.载脂蛋白E基因多态性对他汀类药物调脂消斑的影响[J].第四军医大学学报,2004,25(11):1029-1031.
[54] Pedro-Botet J, Schaefer EJ, Bakker-Arkema RG, et al. Apolipoprotein E genotype affectsplasma lipid response to atorvastatin in a gender specific manner [J]. Atherosclerosis,2001,158(1):183-193.
[55] Neuvonen P J. Drug interactions with HMG-CoA reductase inhibitors (statins): theimportance of CYP enzymes, transporters and pharmacogenetics [J]. Curr Opin InvestigDrugs,2010,11(3):323-332.
[56] Cuiping Chen, Rouchelle J.M, Scott D.C. et al. Differential Interation of3-Hydroxy-3-Methylglutarl-CoA Reductase Inhibtors With ABCB1, ABCC2, and OATP1B1[J]. DRUGMETABOLISM AND DISPOSITION,2005,33:537–546.
[1] Watanabe RM. Drugs, diabetes and pharmacogenomics: the road to personalized therapy [J].Pharmacogenomics,2011,12(5):699-701.
[2] Neuvonen P J. Drug interactions with HMG-CoA reductase inhibitors (statins): the importanceof CYP enzymes, transporters and pharmacogenetics [J]. Curr Opin Investig Drugs,2010,11(3):323-332.
[3] Toda T, Eliasson E, Puist H, et al. Roles of different CYP enzymes in the formation of specificfluvastatin metabolites by human liver microsomes [J]. Basic Clin Pharmacol Toxicol,2009,105(5):327-332
[4] Niemi M, Cascorbi I, Turnquist H, et al. Glyburide and glimepiride pharmacokinetics insubjects with different CYP2C9genotypes [J]. Clin Pharmacol Ther.2002,72:326-332.
[5] Julia K, Dirk K, Tuist H, et al. Influence of CYP2C9polymorphisms on the pharmacokineticsand cholesterol-lowering activity of (-)-3S,5R-fluvastatin and (＋)-3R,5S-fluvastatin inhealthy volunteers [J]. Clin Pharmacol Ther.2003,74:186-194.
[6] Kouji K, Margaret E B, Turnt D, et al. CYP3A4Genotypes and Plasma Lipoprotein LevelsBefore and After Treatment With Atorvastatin in Primary Hypercholesterolemia [J]. TheAmerican Journal of Cardiology,2004,93:104-107.
[7] Hagenbuch B, Meier PJ. Organic anion transporting polypeptides of the OATP/SLC21family:phylogenetic classification as OATP/SLCO superfamily, new nomenclature and molecular/functional properties [J]. Pflugers Arch,2004,447(5):653-665.
[8] Niemi M. Role of OATP transporters in the disposition of drugs [J]. Pharmacogenomics,2007,8(7):787-802.
[9] Nozawa T, Nakajima M, Tamai I, et al. Genetic polymorphisms of human organic aniontransporters OATP-C(SLC-1A6) and OATP-B(SLC-1A9): allele frequencies in the Japanesepopulation and functional analysis [J]. J Pharm Acol Exp Ther,2002,302(2):804-813.
[10] Niemi M, Schaeffeler E, Lang T, et al. High plasma pravastatin concentrations are associatedwith single nucleotide polymorphisms and haplotypes of organic anion transportingpolypeptide-C (OATP-C, SLCO1B1)[J].Pharmacogenetics,2004,14(7):429-440.
[11] Kim EY, Cho DY, Shin HJ, et al. Duplex pyrosequencing assay of the388A> Gand521T> CSLCO1B1polymorphisms in three Asian populations [J]. Clin Chim Acta,2008,388(1P2):68-72.
[12] Masaru H, Kazuya M, Yoshihisa S, et al. Contribution of OATP2(OATP1B1) and OATP8(OATP1B3) tothe hepatic up take of pitavastatin in humans [J]. J Pharmacol Exp,2004,311:139-146.
[13] Chung, J.Y, Cho DY, Ieiri I, et al. Effect of OATP1B1(SLCO1B1) variant alleles on thepharmacokinetics of pitavastatin in healthy volunteers [J]. Clin Pharmacol,2005,78:342–350.
[14] Ieiri I, Suwannakul S, Maeda K, et al. SLCO1B1(OATP1B1, an up take transporter) andABCG2(BCRP, an efflux transporter) variant alleles and pharmacokinetics of pitavastatin inhealthy volunteers [J]. Clin Pharm acol Ther,2007,25:541-547.
[15] Deng JW, Song IS, Tu H, et al. The effect of SLCO1B1*15on the disposition of pravastatinand pitavastatin is substrate dependent: the contribution of transporting activity changes bySLCO1B1*1[J]. Pharmacogenet Genomics.2008,18(5):424-433.
[16] Pasanen MK, Fredrikson H, Neuvonen PJ, et al. Different Effects of SLCO1B1Polymorp-hism on the Pharmacokinetics of Atorvastatin and Rosuvastatin [J].Clinical Pharmacology&Therapeutics,2007,82:726–733.
[17] JH Choi, MG Lee, J-Y Cho, et al. Influence of OATP1B1Genotype on the Pharmacokineticsof Rosuvastatin in Koreans [J]. Clinical Pharmacology Therapeutics,2008,83(2):251-257.
[18] Nishizato Y, Ieiri I, Suzuki H, et al. Polymorphisms of OATP-C(SLC-1A6) and OAT3(SLC-2A8) genes:consequences for p ravastatin pharmacokinetics [J]. Clin Pharmacol Ther,2003,73(6):554-565.
[19] Niemi M, Pasanen MK, Teiri Y, et al. SLCO1B1polymorphism and sex affect thepharmacokinetics of pravastatin but not fluvastatin [J]. Clin Pharmacol Ther,2006,80:356–366.
[20] Ho RH, Choi L, Lee W, et al. Effect of drug transporter genotypes on p ravastatin dispositionin European-and African-American participants [J]. Pharmacogenet Genomics,2007,17(8):647-656.
[21] Zhang W, Chen B L, Ozdemir V, et al. SLCO1B1521T→C functional genetic polymorphismand lipid-lowering efficacy of multiple-dose pravastatin in Chinese coronary heart diseasepatients [J]. Br J Clin Pharmacol,2007,64(3):346-352.
[22]温爱萍,徐小薇,严晓伟.有机阴离子转运多肽1B1遗传多态性对普伐他汀药动学的影响[J].中国药学杂志,2008,43(22):1735-1739.
[23] Choi MK, Shin HJ. Differential effect of genetic variants of Na(+)-taurocholateco-transporting polypeptide (NTCP) and organic anion-transporting polypeptide1B1(OATP1B1) on the uptake of HMG-CoA reductase inhibitors [J]. Xenobiotica,2011,41(1):24-34.
[24] Min Kim, Turnquist H, Jackson J, et al. The multidrug resistance transporter ABCG2(breastcancer resistance protein effluxes Hoechst33342and is overexpressed in hematopoietic stemcells [J]. Clin Can Res,2002,8(1):22-28.
[25] Sang SL, Hye E J, Joo MY, et al. Identification and Functional Assessment of BCRPPolymorphisms in a Korean Population [J]. The American Society for Pharmacology andExperimental Therapeutics,2007,35:623-632.
[26] Zamber CP, Lamba JK, Yasuda K, et al. Natural allelic variants of breast cancer resistanceprotein (BCRP) and their relationship to BCRP expression in human intestine [J].Pharmacogenetics,2003,13:19-28.
[27] Zhang Wei, Yu Bang-Ning, Li Xi, et al. Role of BCRP421C>A polymorphism onrosuvastatin pharmacokinetics in healthy Chinese males [J]. Clinica Chimica Acta,2006,373:99-103.
[28] Keskitalo JE, Pasanen MK, Jin LR, et al. Different effects of the ABCG2c.421C>A SNP onthe pharmacokinetics of fluvastatin, pravastatin and simvastatin [J]. Phar pharmacogenomics,2009,10(10):1617-1624.
[29] Cudaback E, Li X, Montine KS, et al. Apolipoprotein E isoform-dependent microgliamigration [J]. J Bio Chem,2011, Epub ahead of print.
[30] Fu YH, Lv RJ, Jin LR, et al. Association of apolipoprotein E polymorphisms with temporallobe epilepsy in a Chinese Han population [J]. Epilepsy Res,2010,91(2-3):253-259.
[31]李虎,赵水平.载脂蛋白E及胆固醇酯转运蛋白基因多态性与冠心病的相关性研究[J].临床荟萃,2009,24(24):2127-2130.
[32] Ballantyne CM, Herd JA, Stein EA, et al. Apolipoprotein E genotypes and response ofplasma lipids and progressionregression of coronary atherosclerosis to lipid-lowering drugtherapy [J]. J Am Coll Cardiol,2000,36(5):1572-1578.
[32] Pedro-Botet J, Schaefer EJ, Bakker-Arkema RG, et al. Apolipoprotein E genotype affectsplasma lipid response to atorvastatin in a gender specific manner [J]. Atherosclerosis,2001,158(1):183-193.
[33] Huang W, Ye X, Li X, et al. The inhibition activity of chemical constituents in hawthorn fruitand their synergistic action to HMG-CoA reductase [J]. Zhong guo Zhong Yao Za Zhi,2010,35(18):2428-2431.
[34]童煜. HMG-CoA还原酶基因多态与冠心病的相关性研究[D].四川:四川大学遗传学,2003.
[35] Chasman DI, Posada D, Subrahmanyan L, et al. Pharmacogenetic study of statin therapy andcholesterol reduction [J]. JAMA,2004,291(23):2821-2827.
[36] Zee A-HK, Olaf H, Stricker BH, et al. Genetic polymorphisms: importance for response toHMG-CoA reductase inhibitors [J]. Atherosclerosis,2002,163(2):213-222.
[37] Agellon L, Quinet E, Gilletle D, et al. Organization of the human cholestery estentransrerproein gene [J]. Biochemistry,2009,29:1372-1376.
[38]张震宇,王颍,谢玉才.胆固醇酯转移蛋白与氟伐他汀降脂疗效的关系[J],临床心血管杂志.2008,24(8):590-592.
[39] Schmitz G, Langmann T. Pharmacogenomicsof cholesterol lowering therapy [J]. VascPharmacol2006,44(2):75-89.
[40] Grooth DE, Kim YR, Song J, et al. Genetic variations of cholesterol ester transfer proteingene in Koreans [J]. Hum Biol,2001,73(6):815-821.
[41]朱燕林,严晓伟,鄢盛凯.胆固醇酯转移蛋白TaqB基因多态性与辛伐他汀调脂疗效的相关性[J].中国临床药理学杂志,2004,152:367-375.
[42] Kathiresan S, Manning A K, Demissie S, et al. A genome-wide association study for bloodlipid phenotypes in the Framingham Heart Study [J]. BMC Med Genet,2007,8(1): S1-17.