摘要
目的
1.研究POR常见多态基因A503V的多态性对CYP3A体内活性的影响；
2.研究POR A503V基因多态性对中国汉族健康志愿者体内氨氯地平药代动力学的影响；
3.研究中国汉族健康志愿者血浆中miR-34a的水平与氨氯地平体内药代动力学参数间的相关性,探讨微小RNA在人体氨氯地平药代动力学个体差异中的作用及可能机制；
4.在中国汉族高血压患者中研究POR、CYP3A4、CYP3A5和MDR1四种基因的遗传多态性对氨氯地平药代动力学及药物效应的影响；
5.应用定量药理学方法,研究遗传因素和环境因素对氨氯地平干预中国汉族高血压患者的药代及药效动力学的影响,建立PK/PD数学模型。
方法
1.POR常见多态基因A503V的多态性对CYP3A体内活性的影响研究
采用PCR产物直接测序法在中国汉族男性健康志愿者中对POR常见多态基因A503V进行分型,并根据基因分型结果随机筛选22名健康受试入组者。随机将受试者分成两组,每组11人。临床试验分为三个周期,每周期间洗脱期7天。两组受试者于前两周期内分别静脉注射咪达唑仑注射液5mg或空腹口服咪达唑仑7.5mg,于第三周期统一单次口服苯磺酸氨氯地平片5mg(为第二部分研究内容),并在给药前后采集系列血样标本。所有药物在空腹12小时后晨起给予。采用HPLC-MS/MS法测定血浆中咪达唑仑(MID)和1-羟咪达唑仑(1-OH MID)的浓度,应用DAS2.0软件对药代动力学参数进行计算。以1-羟咪达唑仑与咪达唑仑血药浓度-时间曲线下面积比值(AUC1-OH-MID/AUCMID)和咪达唑仑1h的代谢率(MR)作为反应体内CYP3A活性的指标。采用SPSS13.0进行统计学分析,各组之间主要药代动力学参数的差异采用单因素方差分析或配对-t检验进行比较。
2.健康中国汉族男性志愿者中POR A503V基因多态性对氨氯地平体内的药代动力学的影响
临床试验同第1部分。采用HPLC-MS/MS法测定氨氯地平的血药浓度并应用DAS2.0软件对药代动力学参数进行计算。采用SPSS13.0进行统计学分析,并进行配对t检验或单因素方差分析比较PORA503V各基因型CC组、CT组及TT组之间主要药代动力学参数的差异。
3.中国汉族男性健康志愿者血浆中miR-34a表达水平与氨氯地平体内药代动力学的相关性研究
利用第2部分氨氯地平的血药浓度数据和血浆样本,通过real time定量PCR方法测定服药前(Oh)血浆中miR-34a人体内的表达量。采用SPSS13.0进行统计学分析,用独立样本t检验和非参数相关性分析方法来评价miR-34a人体内表达量与氨氯地平药代动力学的相关性。
4.中国汉族高血压患者中POR、CYP3A4、CYP3A5和MDR1基因多态性对氨氯地平药代动力学及降压效应的影响
筛选中国汉族轻中度原发性高血压患者,采用测序方法对PORA503V、CYP3A4*1G、CYP3A5*3、MDR1C3435T四种基因进行分型。所有患者经过一周安慰剂洗脱后,符合试验方案并愿意继续参与试验的受试者连续口服苯磺酸氨氯地平4周,每天1次,每次5mg。在第4周最后一次给药前(第28天)及给药后2h、6h、24h各采集外周静脉血5m1,并测量记录治疗前后的血压。采集血、尿生物标本进行实验室检查,同时观察和记录不良反应发生情况。利用HPLC-MS/MS法测定氨氯地平的血药浓度。通过SPSS13.0对所得数据进行分析,经单因素方差分析或配对-t检验比较PORA503V、CYP3A4、CYP3A5、MDR1各基因型之间主要药代动力学和药效之间的差异。
5.氨氯地平干预中国汉族高血压患者的遗传及环境因素的PK/PD定量药理学研究
采用第4部分的临床试验数据建立数据库。使用NONMEM7.2软件并基于FOCEI方法对氨氯地平的数据进行群体药动学(PPK)建模和群体药效学(PPD)建模。
结果
1.POR常见多态基因A503V的多态性对CYP3A体内活性的影响研究
22位受试者入组参与了临床试验,其中TT基因型型7名、CT基因型型8名, CC基因型型7名,三种基因型个体在年龄、身高、体重方面均无显著性差异。静脉注射咪达唑仑后,TT基因型组1-羟基咪达唑仑的AUCo_t明显高于CC基因型组(p=0.026),TT组1-羟基咪达唑仑Cmax显著高于CC组(p=0.001)和CT组(p=0.002)；口服咪达唑仑后三基因组间的主要药代动力学参数无统计学意义。以1-OH-MID AUC0-8/MID AUC0-t为评价指标时,静脉注射和口服咪达唑仑后,TT基因型组的CYP3A酶的活性较其他基因型组相比较有升高的趋势,但差异未达到统计学意义。而以咪达唑仑1h代谢率(MR)为评价指标时,静脉注射咪达唑仑后,三组间咪达唑仑MR存在显著统计学差异(ANOVA,p=0.032)；TT基因型组与MR显著高于CT基因型组(P=0.010)和CC基因型组(P=0.031)；口服咪达唑仑后,各基因型间MR无统计学差异。
2.中国汉族健康男性志愿者中POR A503V多态性对氨氯地平体内药代动力的影响
22位受试者口服5mg苯磺酸氨氯地平片后,POR A503V CC基因型组、CT基因型组及TT基因型组的药代动力学参数Tmax分别为(5.43±3.21)h.(4.88±1.55)h和(4.57±1.51)h；AUCo-96分别为(154.76士15.58)ng/mL/h.(126.41±37.88)ng/mL/h和(141.49±31.46)ng/mL/h；Cmax分别为(4.87±0.99)ng/mL.(3.93±1.08)ng/mL和(5.47±0.91)ng/mL；t1/2分别为(38.94士15.81)h、(41.24士17.35)h和(33.66士13.19)h.对三组间氨氯地平的药代动力学参数进行单因素分析显示,三组间Cmax的差异有统计学意义(P=0.025),TT基因型组Cmax显著高于CC基因型组(p=0.008),三组间其余药代动参数均无统计学差异。
3.中国汉族健康男性志愿者血浆中miR-34a水平与氨氯地平体内药代动力学间的相关性研究
通过提取血浆中miRNA,成功完成21位受试者血浆miR-34a人表达水平的半定量测定(1位志愿者血浆RNA提取和检测失败)。根据血浆miR-34a的水平分为血浆miR-34a高水平组(高于中位数,n=11)与低水平组(低于中位数,n=10)的氨氯地平药代动力学参数Cmax分别为(4.62±1.02)ng/mL和(4.65±1.32)ng/mL, AUC0-96分别为(151.61±28.67) ng/mL/h和(129.75±32.48) ng/mL/h,两组间经独立样本t检验无统计学差异。分别对氨氯地平Cmax和AUC0-96与miR-34a表达水平进行非参数相关性分析,相关系数r分别为-0.215、-0.385,呈现负相关趋势,但相关性无显著统计学意义。
4.中国汉族高血压患者中POR、CYP3A4、CYP3A5和MDR1基因多态性对氨氯地平药代动力学及降压疗效的影响
本研究共筛选了157例高血压患者,其中106进入洗脱期,67例入组进入氨氯地平治疗期,60例(男31人,女29人)完成4周治疗和随访及PK研究。治疗4周后坐位舒张压相对基线的变化绝对值为(7±6)mmHg,坐位收缩压相对基线的变化值为(18±10) mmHg,降压有效者32人,有效率53.3%。POR A503V、CYP3A4*1G、CYP3A5*3、 MDR1C3435T四种基因的多态性中除MDR1多态性对氨氯地平血药浓度具有一定影响外,其余三种多态性对氨氯地平血药浓度和疗效均无显著性影响。
5.氨氯地平干预中国汉族高血压患者的遗传及环境因素的PK/PD定量药理学研究
建立的群体药动学(PPK)模型和群体药效学(PPD)模型经验证稳定、准确。性别和MDR1基因多态性对清除率(CL/F)具有影响,总蛋白(TP)对清除率CL/F具有一定影响。氨氯地平的浓度-效应的最终模型的收缩压变化值与氨氯地平给药后达到稳态时的0-24小时药时曲线下面积(AUC0-24)呈正相关,AUCo-24越大,则收缩压降低值越大。
结论
1. POR A503V基因多态性可导致中国汉族男性健康志愿者肝脏CYP3A酶活性增加；
2. POR A503V多态性对中国汉族男性健康志愿者氨氯地平药代动力学影响不显著,POR A503V多态性对氨氯地平的吸收可能存在影响；
3.血浆中miR-34a的水平与中国汉族人群男性健康志愿者口服氨氯地平的药代动力学参数相关性不显著；
4.中国汉族原发性高血压患者应用氨氯地平降压治疗,女性患者口服氨氯地平后其血浆药物浓度高于男性,CL/F低于男性,但氨氯地平的降压疗效无显著性别差异；
5.高血压患者MDR1C3435T突变纯合子个体氨氯地平的血浆清除率升高,但该位点突变对氨氯地平的降压疗效影响不明显；
6. POR*28, CYP3A4*1G, CYP3A5*3基因多态性对氨氯地平药代动力学及降压疗效虽有一定的影响趋势,但无统计学意义的差异；
7.氨氯地平用药4周后其血药浓度与其降收缩压的效果相关,收缩压降低值与达到稳态后0-24小时药时曲线下面积(AUC0-24)呈正相关。
OBJECTIVES
1. To assess the effects of cytochrome P450oxidoreductase (POR) A503V polymorphism on CYP3A in vivo activity in healthy Chinese male volunteers.
2. To assess the effects of POR A503V polymorphism on the pharmacokinetics of amlodipine in healthy Chinese male volunteers.
3. To evaluate the correlationship between the miR-34a level in plasma and pharmacokinetics parameters of amlodipine in healthy Chinese male volunteers, and to investigate the potential mechanism how microRNA impact the pharmacokinetics of amlodipine.
4. To study the effect of POR, CYP3A4, CYP3A5and MDR1genetic polymorphisms on the pharmacokinetics and pharmacodynamics of amlodipine in Chinese essential hypertention (EH) patients.
5. To study the effect of genetic and environment factors on the pharmacokinetics and pharmacodynamics of amlodipine and to establish a mathematic PK/PD model for amlodipine in Chinese EH patients based on the method of pharmacometrics.
METHODS
1. Effects of POR A503V polymorphism on CYP3A activity in vivo
POR A503V polymorphism was genotyped in healthy Chinese Han subjects by PCR-based direct sequencing. Twenty-two healthy Chinese male subjects were randomly selected based on their POR A503V genotype, and were divided into two groups in a three-phase study. Oral (7.5mg) or intravenous (5mg) midazolam (MID) administration were performed in the first two-phase crossover study, while in the third phase, single oral dose of5mg amlodipine besylate administration was performed before collecting blood samples. Plasma concentrations of MID and1-hydroxy-midazolam (1-OH-MID) were determined by liquid chromatography-tandem mass spectrometry (LC-MS). The pharmacokinetic parameters were obtained by DAS Ver2.0, and statistical analysis was performed with SPSS13.0using. Differences among or between groups were compared with one-way ANOVA or paired sample t-test. P<0.05was regarded as significantly different.
2. Effects of POR A503V polymorphism on the pharmacokinetics of amlodipine in healthy Chinese male volunteers
Clinical trails were performed as previous study. Series venous blood samples were drawn after amlodipine administration. Plasma amlodipine concentrations were measured by method of HPLC-MS/MS. The pharmacokinetic parameters were obtained by DAS Ver2.0. The statistical analysis were performed with SPSS13.0. Differences in pharmacokinetic parameters of amlodipine among or between POR A503V genotypes were analyzed by one-way ANOVA or paired sample t-test.
3. Correlation between the plasma miR-34a level and pharmacokinetics of amlodipine in healthy Chinese male volunteers
Blood samples were collected before amlodipine administration in part two. Plasma RNA samples were extracted. The expression level of plasma miR-34a was determined by semi-quantitative real-time PCR. Amlodipine plasma concentration and the pharmacokinetics parameters detemined in part two were used. Statistical analysis was analyzed by the software SPSS13.0. Correlation between the expression level of miR-34a in plasma and the pharmacokinetics parameters of amlodipine was evaluated by non-parametric correlation analysis.
4. Influences of POR, CYP3A4, CYP3A5and MDR1genetic polymorphisms on the pharmacokinetics, pharmacodynamics and antihypertensive effects of amlodipine in Chinese EH patients.
Chinese Han Patients diagnosed as mild to moderate essential hypertension (EH) were recruited. After one week of placebo washout period, the subjects who met the clinical requirements and were willing to continue the study were given oral amlodipine besylate5mg once daily for four weeks. Serial blood samples were collected on day28just prior to the last amlodipine dosage and at2h,6h, and24h, respectively, after drug administration. Blood pressure (BP) was measured on day0(baseline BP) before initiation of amlodipine therapy and on day28after the final amlodipine dosage. The blood and urine samples were collected for the laboratory tests. Occurrence of adverse reactions were inquired and recorded. Concentrations of amlodipine in plasma was determined by HPLC-MS/MS. Genotyping for POR A503V、CYP3A4*1G、CYP3A5*3、 and MDR1C3435T polymorphisms were performed by direct sequencing of PCR-products. Statistical analysis was carried out with the software SPSS13.0. Differences in pharmacokinetics and pharmacodynamics parameters among or between genotypes were analyzed by one-way ANOVA test or paired sample t-test.
5. Evaluation of genetic and environmental factors on the pharmacokinetics and pharmacodynamics of amlodipine in Chinese Han patients with essential hypertension by pharmacometrics.
The database was established by using the clinical data from part four. PPK and PPD models were constructed by using the method of FOCEI with the software NONMEM7.2.
RESULTS
1. Effects of POR A503V polymorphism on CYP3A activity in vivo
Twenty-one CC homozygotes,21CT heterozygotes, and11TT homozygotes were observed for the73volunteers screened. The frequency of the POR*28T (503V) allele was43.2%. Seven A503V common homozygotes (CC genotype), eight heritozygotes (CT genotype), and seven rare homozygotess (TT genotype) were randomly selected. No significant difference in demographic characteristics, including age, height, body weight, was observed among the three genotype groups. As compared with CC homozygotes, TT homozygotes showed significantly increased AUC0-8of1-OH-MID after intravenous (P=0.026) but not oral MID administration. TT homozygotes also showed higher1-OH-MID Cmax than CC homozygotes (P=0.001) and CT heterozygotes (P=0.002) after intravenous MID injection. After intravenous MID injection, the MID metabolic ratio was significantly greater in the TT homozygotes compared with carriers of the C allele (P=0.031). No significantly difference in the overall CYP3A in vivo activity (hepatic plus intestinal) was observed among the POR*28genotypes.
2. Effects of POR A503V polymorphism on the pharmacokinetics of amlodipine in healthy Chinese Ha male volunteers
Tmax of amlodipine in individuals with POR A503V CC, CT, and TT genotypes was (5.43±3.21) h,(4.88±1.55) h and (4.57±1.51) h, respectively. AUC0→96of amlodipine in POR A503V CC, CT, and TT genotypes was (154.76±15.58) ng/mL/h,(126.41±37.88) ng/mL/h and (141.49±31.46) ng/mL/h, respectively. Cmax in POR A503V CC, CT, and TT genotypes was (4.87±0.99) ng/mL,(3.93±1.08) ng/mL and (5.47±0.91) ng/mL, respectively. t1/2were (38.94±15.81) h,(41.24±17.35)h and (33.66±13.19)h, respectively. Carriers of the POR A503V TT genotype showed significantly higher Cmax than the CC homozygotes (P=0.025). No significant difference in other pharmacokinetic parameters of amlodipine was observed among the POR A503V genotypes.
3. Correlation between the plasma miR-34a level and pharmacokinetics of amlodipine in healthy Chinese male volunteers
Plasma miR-34a expression was determined in21healthy, and the determination failed for one because of inconformity. Subjects were divided into two groups according to their plasma miR-34a level:higher than the median (n=11) and lower than the median (n=10). The Cmax of amlodipine in the group with high plasma miR-34a level (higher than the median) and the group with low plasma miR-34a level (lower than the median) were (4.62±1.02) ng/mL and(4.65±1.32) ng/mL, respectively. The AUC0-96were (151.61±28.67) ng/mL/h and (129.75±32.48) ng/mL/h in the two groups, respectively. There was no significant difference in the amlordipine pharmacokinetics between the two groups. The Non-parametric correlation analysis showed negative but not significant correlation between plasma miR-34a level and Cmax or AUC0-96of amlodipine (r=-0.215and-0.385, respectively, p>0.05).
4. Influences of POR A503V, CYP3A4, CYP3A5, and MDR1genetic polymorphisms on the pharmacokinetics, pharmacodynamics and antihypertensive effects of amlodipine in Chinese Han patients with hypertension.
A total of157EH patients were screened in our study,106patients and67patients entered into the washout period and the amlodipine treatment period, respectively. A total of60patients (31males,29females) completed the four-week treatment phase and the demanded PK study finally. After four-week amlodipine monotherapy, the absolute change in seated diastolic blood pressure (SeDBP) and seated systolic blood pressure (SeSBP) from baseline were7±6mmHg and18±10mmHg, respectively. Thirty-two patients were responder to amlodipine therapy, and the effect rate was53.3%. MDR1genetic polymorphisms of had a certain impact on the plasma amlodipine concentration. No association between genetic polymorphisms of POR A503V, CYP3A4, CYP3A5and either plasma amlodipine concentration or amlodipine therapeutic efficacy was observed (P>0.05).
5. Evaluation of genetic and environmental factors on the pharmacokinetics and pharmacodynamics of amlodipine in Chinese Han patients with essential hypertension by pharmacometrics.
The established PPK and PPD models were proved to be stable and accurate. Both gender and MDR1genetic polymorphism showed impacts on the CL/F of amlodipine. Total protein also showed a certain impact on the CL/F of amlodipine. In the final exposure-response model, the change in SeSBP from baseline correlated positively with the steady-state AUC0-24.
CONCLUSIONS
1. The POR A503V genetic polymorphism is associated with increased CYP3A in vivo activity in the liver in healthy Chinese Han subjects.
2. POR A503V genetic polymorphism has no significant effects on the pharmacokinetics of amliodipine in healthy Chinese Han subjects, but may affect the absorption of amlodipine.
3. There is no significant correlation between the expression level of plasma miR-34a and Cmax and AUC0-tof amlodipine after oral amlodipine administration.
4. Chinese Han EH patients should be administrated with amlodipine. Though the efficacy of amlodipine was no significant difference, the plasma amlodipine concentration trended to be higher, while the CL/F was lower in female EH patients than male patients,
5. Carriers of the MDR1C3435T mutation homozygote genotype showed increased clearance of amlodipine in hypertension patients, but the influence on the anti-hypertensive effects is negligible.
6. The polymorphisms of POR*28, CYP3A4*1G and CYP3A5*3trended to influence the pharmacokinetics and efficacy of amlodipine without statistically differences.
7. The efficacy of BP lowering effects of amlodipine correlates positively with its plasma concentration after4-week amlodipine therapy.
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