【作者】 郭歆；

【导师】 李元建；

【作者基本信息】 中南大学 ， 药理学， 2009， 博士

【摘要】 第一章药物转运蛋白基因的遗传多态性对替米沙坦药动学的影响研究背景替米沙坦（telmisartan）是一种血管紧张素Ⅱ（AngiontensinⅡ,AngⅡ）1型（AT1）受体拮抗药,具有高选择性、不可逆地拮抗AT₁受体,而不抑制血管紧张素转换酶,不影响缓激肽的效应;也不与其它激素受体和离子通道结合或产生阻断作用,因而不影响包括心血管调节在内的其它受体系统。近年来对替米沙坦研究的范围还涉及有心血管（心力衰竭、心肌肥厚、心肌梗死等）、内分泌（糖尿病等）、肾病（糖尿病肾病、蛋白尿等）和神经系统（脑卒中）等众多领域,其已成为近年来心血管领域研究的热点,倍受临床关注。替米沙坦的体内药物浓度及降压疗效存在很大的个体差异,而导致这种差异的原因目前尚不明确。多种药物转运蛋白参与替米沙坦的吸收（Absorption）、分布（Distribution）、代谢（Metabolism）、排泄（Excretion）过程,且研究表明,多种编码药物转运蛋白的基因存在单核苷酸基因多态性,遗传因素所导致的药物转运蛋白活性的差异可能是导致替米沙坦血药浓度和药物反应性个体差异的原因之一。替米沙坦的体内过程主要受MDR1（多药耐药蛋白1,Multidrug resistance 1,又名P-gp）、MRP2（多药耐药相关蛋白2,Multidrug resistance-related protein 2）和OATP1B3（有机阴离子转运体1B3亚型,Organic anion transporters 1B3）药物转运蛋白的影响,而上述转运蛋白的基因多态性可影响各自底物的血药浓度及药代动力学。因此,本章我们从健康受试者及高血压患者两大人群出发,探讨上述三种转运蛋白的基因多态性是否影响替米沙坦在人体内的药代动力学特征及降压疗效。方法采用限制性片段长度内切酶（PCR-RFLP）的方法对60名中国健康男性志愿者的MDR1 C3435T、MRP2 G1249A、MRP2 C3972T、OATP1B3T334G和OATP1B3 G699A位点进行基因分型,从中挑选适合的基因型个体进行进一步的研究。基因型符合要求的受试者按照40 mg的剂量单次口服替米沙坦片,并于服药前（0 h）及服药后0.25 h、0.5 h、0.75 h、1 h、1.5 h、2 h、3 h、4 h、6 h、8 h、10 h、12 h、24 h、48 h分别采集肘静脉血5 mL,分离血浆,采用HPLC-MS（High performance liquid chromatogram-mass spectrometry,高效液相色谱—质谱检测器）法测定受试者体内不同时间点血浆中替米沙坦的药物浓度。采用DAS 2.0软件计算替米沙坦的药代动力学参数,并使用SPSS 13.0软件包比较基因型组间替米沙坦的药动学参数。选择符合1999年WHO/ISH诊断标准的原发性高血压病人,治疗前测定基础血压。按照每天一次,每次40 mg的给药剂量,连续服用替米沙坦片30天,测定治疗30天后的血压,采集第30天服药前血样,进行替米沙坦稳态血浆药物谷浓度的测定并对MDR1 C3435T、MRP2 C3972T、OATP1B3T334G和OATP1B3 G699A位点进行基因分型。采用HPLC-FLD法测定血浆中替米沙坦的浓度,使用SPSS 13.0软件包比较基因型组间替米沙坦的稳态谷浓度及疗效间的差异。结果MDR1 C3435T、MRP2 G1249A、MRP2 C3972T、OATP1B3 T334G和OATP1B3 G699A位点的等位基因发生频率与文献报道及HapMap中报道的结果基本一致,其中OATP1B3 T334G和OATP1B3 G699A位点间呈完全连锁不平衡。19位参加药动学研究的受试者,服药后替米沙坦的平均药动学参数T_max、C_max、t_1/2、AUC_0-48和AUC_0-∞分别为1.4±0.7 h（0.5～3.0 h）、312.7±279.7μg·L^-1(26.4～871.7μg·L^-1)、13.9±8.1 h（2.5～38.9 h）、1477.4±927.4μg·h·L^-1(184.8～3134.0μg·h·L^-1)和1662.3±1100.1μg·h·L^-1(194.7～3816.2μg·h·L^-1)。个体间替米沙坦的上述药物代谢动力学参数问都存在很大的个体差异。经方差分析及独立样本t检验,替米沙坦的药动学参数在MDR1C3435T、MRP2 G1249A、MRP2 C3972T和OATP1B3 T334G/G699A位点不同基因型个体间均无显著性差异。58位高血压患者治疗前后的血压分别为164/94 mmHg（SBP变化范围:220～130 mmHg;DBP变化范围:128～70 mmHg）和143/87 mmHg（SBP变化范围:194～110 mmHg;DBP变化范围:110～65 mmHg）,服用替米沙坦30天后,其血浆中药物稳态谷浓度为50.10±6.95μg·L^-1(波动范围:0.34μg·L^-1～258.42μg·L^-1),个体间的药物血药浓度同样存在很大的个体差异。经统计学分析,MDR1 C3435T、MRP2 C3972T、OATP1B3T334G/OATP1B3 G699A基因多态性对替米沙坦的稳态谷浓度变化没有影响。MRP2 C3972T基因多态性与治疗前后收缩压的变化值有关,基因型为3972TT的个体表现出更好的降压效果。结论MDR1 C3435T、MRP2 G1249A、MRP2 C3972T、OATP1B3 T334G/G699A位点的基因多态性不影响健康中国男性志愿者替米沙坦的药物代谢动力学。MDR1 C3435T、MRP2 C3972T、OATP1B3 T334G/OATP1B3G699A基因多态性与高血压患者替米沙坦的稳态谷浓度间无相关性,但MRP2 C3972T多态可以影响替米沙坦降低收缩压的作用程度。第二章SIRT1和DDAH1基因的遗传多态性对中国人替米沙坦降压疗效的影响研究背景高血压病是近半个世纪以来影响人类健康和生活质量、导致人类死亡的重要疾病之一。替米沙坦是AT₁受体阻断剂,临床上广泛应用于高血压病的治疗。研究发现在不同人种中,替米沙坦对轻、中度高血压治疗的有效率约为52%-56%,且其降压疗效存在很大的个体差异,大量的患者在使用替米沙坦单一药物进行治疗时需要加大剂量或联合使用其他降压药物。然而到目前为止,导致这种显著地药物反应性个体差异的原因仍尚不明确。随着对替米沙坦药理作用研究的不断深入,人们发现替米沙坦除具有降压作用外,还具有改善胰岛素抵抗和糖、脂代谢紊乱,及抗动脉粥样硬化的作用,上述作用可能与替米沙坦增加PPAR-γ（Peroxisome proliferatoractivated receptor-γ,过氧化物酶体增生物激活受体-γ）蛋白表达、上调DDAH（Dimethylarginine dimethylaminohydrolase,二甲基精氨酸二甲氨水解酶）表达和降低ADMA（Asymmetric dimethylarginine,非对称性二甲基精氨酸）浓度有关。SIRT1属于沉默信息调节因子2（Sir2）家族成员,该蛋白家族的活性依赖于烟酰胺腺嘌呤二核昔酸（NAD⁺）的去乙酰化酶,其在调节基因表达、细胞凋亡、代谢和老化过程中发挥重要作用。有研究发现AT1受体基因的近侧启动子区（-61bp到+25bp）包含SP1结合位点,SIRT1能够抑制SP1结合到AT1受体基因启动子上,从而下调AT1受体的表达,进而影响AngⅡ发挥缩血管效应,因此我们推测,DDAH1和SIRT1基因的遗传多态性可能在受体后水平影响替米沙坦的降压疗效。本研究从药物作用受体后信号通路蛋白出发,研究SIRT1和DDAH1基因的遗传多态性对替米沙坦降压疗效的影响,从而寻找影响替米沙坦降压疗效的SNP,为临床合理应用替米沙坦进行降压治疗提供指导。方法原发性高血压患者58例,应用题米沙坦治疗前测量基础血压,并抽取5 ml肘静脉血,分离血浆用于测定基础状态ADMA的浓度,血细胞用于DNA的提取。入组的病人按照每天一次、每次40 mg的给药剂量,连续服用替米沙坦30天。试验第31天服药前测量治疗后血压,并抽取肘静脉血7 ml,用于治疗后ADMA浓度的测定。采用PCR-RFLP法对DDAH1 C143611G、DDAH1 C144563T和SIRT1C-1138T位点进行基因分型;使用HPLC-MS法测定治疗前后血浆中ADMA的浓度。使用SPSS 13.0软件包选取适当的统计学方法对替米沙坦的降压疗效及治疗前后ADMA水平的变化情况进行基因型组间的比较。结果高血压患者中DDAH1 C143611G、DDAH1 C144563T和SIRT1C-1138T位点的等位基因频率及基因型与HapMap中报道的中国北京人群的基因分布特征一致。58例高血压患者完成了本项研究,治疗前患者血浆中ADMA的浓度为0.355±0.014μmol·L^-1(波动范围:0.632μmol·L^-1～0.106μmol·L^-1)。按照每天给药一次,每次40 mg的剂量,连续服用替米沙坦片30天后,其血浆中ADMA的浓度为0.455±0.020μmol·L^-1(波动范围:0.759μmol·L^-1～0.140μmol·L^-1),其中14例患者治疗后血浆ADMA的浓度降低,44例患者治疗后血浆ADMA的浓度升高,治疗前后血浆ADMA的浓度存在显著的差异（p＜0.001）。DDAH1 C143611G多态与替米沙坦的降压疗效无关,但DDAH1C144563T多态与替米沙坦治疗前后收缩压（SBP）和舒张压（DBP）的改变程度有关;SIRT1 C-1138T多态与舒张压的变化程度有关。结论DDAH1 C144563T及SIRT1 C-1138T多态可影响替米沙坦的降压疗效。更多还原

【Abstract】 Chapter 1 The polymorphisms of drug transporters on the effect of telmisartan pharmacokineticsBackgroundTelmisartan is an angiotensinⅡreceptor antagonist,can high selectively and irreversibly antagonize AT1 receptor,but don’t inhibit the angiotensin-converting enzyme or affect the effects of bradykinin.It also does not bind or block other hormone receptors or ion channels,therefore does not affect the other receptor systems including the cardiovascular condition system.In recent years,much evdences has shown that,,beside the effect on decrease the blood pressure,telmisartan also has the effects on other cardiovascular disease,endocrine diseases,nephropathy and diseases of the nervous system.The basis for the remarkable interindividual differences in the plasma concentration and response of telmisartan remains unknown.Several drug transporters are involved in the ADME process of telmisartan.Pharmacogenetics studies have shown that genes encoding drug transporters have a variety of single nucleotide polymorphisms（SNP）.Genetic factors that affect the activity of drug transporters may be one of the reasons for the individual differences in telmisartan plasma concentration and drug response.There are three main drug transporters impact the process of telmisartan in vivo,MDR1（P-gp）,MRP2 and OATP1B3.The polymorphisms of the genes can affect the pharmacokinetics of their own substrates.Therefore, in our researches,we investigated genetic polymorphisms of these three drug transporters on the effect of telmisartan pharmacokinetics in healthy volunteers and the steady-state concentration or treatment in hypertensive patients.MethodsGenotyping of the MDR1 C3435T,MRP2 G1249A,MRP2 C3972T, OATP1B3 T334G and OATP1B3 G699A polymorphism were performed by using method of polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP）.Sixty unrelated male volunteers were enrolled and screened for their MDR1 C3435T genotypes,and were selected for participation in the further study.The subjects,whose genotype fit the requirements,received a single oral dose of 40 mg telmisartan.Venous blood samples of 5 mL were drawn from forearm vein into EDTA tubes before（0 h） and at 0.25,0.5,0.75,1,1.5,2,3,4, 6,8,10,12,24,and 48h,respectively,after telmisartan administration.Plasma was separated,and determined by high performance liquid chromatogrammass spectrometry（HPLC-MS）.The pharmacokinetics of telmisartan was analyzed with DAS 2.0 software.Statistical analyses were performed by the SPSS 13.0 software for Windows. Essential hypertension patients were requested to measure the basis blood pressure before telmisartan therapy.All the patients were treated with telmisartan for 30 days,according to once a day,40 mg per time in every morning.Blood pressure was measured in the thirty-first days,and venous blood samples were collected before telmisartan administration.The plasma was used to determine the concentration of telmisartan and whole blood was used to analyze the genotypes of MDR1 C3435T,MRP2 C3972T,OATP1B3 T334G and OATP1B3 G699A,respectively.The plasma concentration of telmisartan was determined by high performance liquid chromatogram-fluorescence detector（HPLC-FLD）.Statistical analyses were performed by the SPSS 13.0 software for Windows.ResultsThe allelic frequencies and genotype distribution of MDR1 C3435T, MRP2 G1249A,MRP2 C3972T,OATP1B3 T334G and OATP1B3 G699A were not different from those reported in Chinese.OATP1B3 T334G and OATP1B3 G699Apolymorphisms are in complete linkage disequilibrium.Mean±SD and variation of T_max,C_max,t_1/2,AUC_0-48 and AUC_0-∞ of 40 mg orally administrated telmisartan were 1.4±0.7 h（0.5～3.0 h）,312.7±279.7μg·L^-1(26.4～871.7μg·L^-1),13.9±8.1 h（2.5～38.9 h）,1477.4±927.4μg·h·L^-1 (184.8～3134.0μg·h·L^-1) and 1662.3±1100.1μg·h·L^-1(194.7～3816.2μg·h·L^-1), respectively,in the overall 19 cases of subjects.There are significant individual differences in the pharmacokinetics of telmisartan.The blood pressure of 58 cases of essential hypertensive patients before and after telmisartan therapy were 164/94 mmHg（range:220～130 mmHg for SBP;128～70 mmHg for DBP） and 143/87 mmHg（range:194～110 mmHg for SBP;110～65 mmHg for DBP） respectively.There was also significant individual difference in the concentration of telmisartan.Statistical analysis showed that the polymorphism of MDR1 C3435T,MRP2 C3972T,OATP1B3 T334G/OATP1B3 G699A were not associated with plasma steady-state concentration of telmisartan.MRP2 C3972T polymorphism affected ASBP with telmisartan therapy,individuals with the 3972TT genotype showed significantly higher degree of decrease in SBP after telmisartan therapy.ConclusionWe found that the MDR1 C3435T、MRP2 G1249A、MRP2 C3972T、OATP1B3 T334G and OATP1B3 G699A polymorphism were not associated with the pharmacokinetics parameters of telmisartan in this study.The genetic polymorphisms of MDR1 C3435T,MRP2 C3972T,OATP1B3 T334G/OATP 1B3 G699A were not associated with telmisartan steady-state concentration, MRP2 C3972T polymorphism affectedΔSBP with telmisartan therapy. Chapter 2 Effect of genetic polymorphisms in SIRT1 and DDAH1 on antihypertensive efficacy of telmisartan in a Chinese populationBackgroundHypertension is an important disease which affecting human health and quality of life since the nearly half a century.Telmisartan is an angiotensinⅡreceptor antagonist;it is widely used in clinical treatment of hypertension. Evidence has shown that the response rate of telmisartan ranged from 52%to 56%in mild-to-moderate hypertension in patients with different ethnic backgrounds.A considerable percentage of patients also need dose titration during hypertension monotherapy.The basis for the remarkable interindividual difference in telmisartan response remains unknown.With the deep researches on the pharmacological effects of telmisartan, scientists found that telmisartan can increase the expression of PPAR-γ, reduce the expression of DDAH（Dimethylarginine dimethylaminohydrolase） and increase the concentration of the endogenous NO synthase inhibitor-ADMA （Asymmetric dimethylarginine）.Therefore,telmisartan can not only decrease the BP,but also improve insulin resistance and glucose-lipid metabolism disorders,and have the antiatherogenic role.SIRT1 is one of silent information regulator 2（SIR2）.Sirtuins require nicotinamide adenine dinucleotide（NAD⁺） as a cosubstrate for the deacetylation reaction,and they are highly preserved among numerous species and are associated with longevity,cell cycle regulation,apoptosis,DNA damage repair,and muscle differentiation.Deletion analysis of the AT₁R gene promoter revealed that suppression of AT₁R expression by resveratrol,a well known SIRT1 activator, is dependent on the most proximal promoter region（from -61 bp to -25 bp）, which contains Sp1 binding site（GC box）,the data suggested that resveratrol inhibits Sp1 binding to AT1R gene promoter and AT₁R gene transcription, induced AT₁R downregulation.We,therefore,hypothesized that the polymorphism of DDAH1 and SIRT1 maybe affect the treatment of telmisartan in the level of post-receptor.In this part of our researches,we focus on whether the DDAH1 and SIRT1 genetic polymorphisms may affect the antihypertensive efficacy of telmisartan,in order to find the SNPs which can impact the response of telmisartan,and provide guidance for the clinical application of telmisartan.MethodsEssential hypertension patients were requested to measure the basic blood pressure,and collected 5 mL blood samples.The plasma was used to detect the concentration of ADMA,and the blood cell was used to extract DNA. Patients were treated with telmisartan for 30 days,according to once a day,40 mg per time in the morning.Measure the blood pressure in the 31th days,and 7 mL blood samples were collected before telmisartan administration.The plasma was used to determine the concentration of ADMA. Genotype of the DDAH1 C143611G、DDAH1 C144563T and SIRT1 C-1138T polymorphism were performed by using method of polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP）.The plasma concentration of ADMA was determined by high performance liquid chromatogram-mass spectrometry（HPLC-MS）.Statistical analyses were performed by the SPSS 13.0 software for Windows.ResultsThe genotype and allele frequencies distribution characteristics of DDAH1 C143611G,DDAH1 C144563T and SIRT1 C-1138T were the same as the results reported in HapMap.58 cases of patients had finished the study.The mean concentration of ADMA was 0.355±0.014μmol·L^-1(range from 0.632μmol·L^-1 to 0.106μmol·L^-1) before treatment,and 0.455±0.020μmol·L^-1(range from 0.759μmol·L^-1 to 0.140μmol·L^-1) after 30-day therapy treatment.Fourteen EH cases showed decreased plasma concentration of ADMA after telmisartan treatment, while 44 EH cases showed increased plasma concentration of ADMA after telmisartan treatment.Significant differences in plasma concentration of before and after telmisartan treatment was observed（p＜0.001）.Significant difference inΔSBP andΔDBP was observed among individuals with different genotypes of DDAH1 C144563T polymorphism. Significant difference inΔDBP was also observed among individuals with different genotypes of SIRT1 C-1138T polymorphism.No association was observed between DDAH1 C143611G polymorphism and the hypotensive effect of telmisartan.ConclusionDDAH1 C144563T and SIRT1 C-1138T polymorphism may affect the anti-hypertensive efficacy of telmisartan in Chinese population.更多还原