Pharmacogenomics Study Of Amlodipine PK/PD In Chinese Han Population

OBJECTIVES1. 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. METHODS1. Effects of POR A503V polymorphism on CYP3A activity in vivoPOR 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 volunteersClinical 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 volunteersBlood 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.RESULTS1. 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 volunteersTmax 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 volunteersPlasma 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.CONCLUSIONS1. 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.