氨氯地平贝那普利胶囊在中国健康受试者体内的药动学研究
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摘要
1目的
建立一种可靠、高效、简便的方法,测定氨氯地平、贝那普利及其代谢产物贝那普利拉在人血浆中的浓度,进行单次及多次口服氨氯地平贝那普利胶囊在中国健康受试者体内的药物动力学研究,考察氨氯地平、贝那普利及其代谢产物贝那普利拉药动学的剂量间差异、线性特征、性别差异、蓄积情况,并观察健康志愿者服药后的不良反应,为临床合理用药提供参考依据。2方法2.1 UPLC-ESI-MS/MS法测定人血浆中氨氯地平、贝那普利及贝那普利拉2.1.1氨氯地平血样测定：
血浆样品经固相萃取后真空挥干浓缩十倍处理,以卡马西平作为内标(IS),用色谱柱(ACQUITY UPLC(?) BEH C18,2.1 mm×50 mm,1.7μm)进行分离,柱温：40℃,进样室温度：10℃,流动相组成为乙腈：水溶液(含0.1%甲酸)=(35：65,V/V),流速：0.3 mL·min-1。UPLC-ESI-MS/MS法选择性监测被测物质的准分子离子峰。2.1.2贝那普利及贝那普利拉测定：
血浆样品经蛋白沉淀法处理,以依那普利作为内标(IS),用色谱柱(ACQUITY UPLC(?) BEH C18,2.1 mm×50 mm,1.7μm)进行分离,柱温：40℃,进样室温度：10℃,流动相组成为甲醇：水溶液(含0.1%甲酸)=(50：50,V/V),流速：0.25 mL·min-1。UPLC-ESI-MS/MS法选择性监测被测物质的准分子离子峰。2.2药物动力学研究2.2.1单次给药
24名健康志愿者,男女各半,采用随机区组设计,男女受试者各分为3组,分别单次口服氨氯地平贝那普利胶囊1、2、3粒(5mg/10mg,10mg/20mg,15mg/30mg；氨氯地平贝那普利胶囊每粒含苯磺酸氨氯地平5mg/盐酸贝那普利10mg),给药物前和给药后15.0、30.0、45.0 min,1.0、1.5、2.0、3.0、4.0、6.0、8.0、10.0、12.0、24.0、36.0、48.0、72.0、96.0、120.0、144.0 h(共20点)由静脉取血4 mL,置肝素化抗凝试管中,分离血浆存于-70℃冰箱中,以备血药浓度测定。2.2.2多次给药
采用中剂量(10 mg/20 mg),8人(男女各半)。服药方法为每日1次,一次2粒(规格：每粒含苯磺酸氨氯地平5 mg／盐酸贝那普利10 mg),连续给药14天。给药时间为8：00 AM。于第1天多次给药前、第11、12、13、14日给药前,第14日在服药后按照单次给药设计采集血样。各取血点经静脉留置针取前臂静脉血4.0 mL,置肝素化抗凝试管中,分离血浆存于-70℃冰箱中,以备血药浓度测定。3结果3.1方法学结果3.1.1UPLC-ESI-MS/MS法测定人血浆中氨氯地平
氨氯地平及内标在C18柱上保留较好,分离完全,氨氯地平的线性范围为0.09～30.0 ng·mL-1,r2=0.999302,最低定量限为0.09 ng·mL-1。氨氯地平的平均萃取回收率都大于85%,方法回收率都在107.7%～111.9%之间,日内日间RSD均小于9%。3.1.2UPLC-ESI-MS/MS法同时测定人血浆中贝那普利及贝那普利拉
贝那普利、贝那普利拉及内标依那普利在C18柱上保留较好,分离完全,贝那普利及贝那普利拉的线性范围为1.0～1000.0 ng·mL-r2=0.997250和0.999730,最低定量限为1.0 ng·mL-1。贝那普利和贝那普利拉的平均萃取回收率都大于93.8%,方法回收率都在100.4%～103.3%之间,日内日间RSD均小于9.3%。贝那普利拉的平均萃取回收率都大于87.8%,方法回收率都在98.1%～108.8%之间,日内日间RSD均小于9.5%。3.2氨氯地平贝那普利的药物动力学3.2.1单次给药药动学参数及其在剂量间的差异3.2.1.1氨氯地平单次给药药动学参数及其在剂量间的差异
单剂量口服氨氯地平贝那普利胶囊1、2、3粒(5 mg/10 mg,10mg/20 mg,15 mg/30 mg；氨氯地平贝那普利胶囊每粒含苯磺酸氨氯地平5mg/盐酸贝那普利10 mg)后氨氯地平(5 mg、10 mg、15 mg)的Tmax分别为6.0±1.1 h、7.0±3.4 h和5.6±3.0 h,三种剂量口服的达峰时间相近(P>0.05)；Cmax分别为4.37±1.37 ng·mL-1、7.23±1.81ng·mL-1和14.71±3.14 ng·mL-1,AUC0-144分别为167.7±35.9 ng·h·mL-1、283.8±47.6 ng·h·mL-1和574.9±159.0 ng·h·mL-1,AUC0-∞分别为176.8±40.2 ng·h·mL-1、304.1±54.8 ng·h·mL-1和628.2±197.5 ng·h·mL-1。说明在5-15mg剂量范围内,氨氯地平Cmax、AUC0-144、AUC0-∞与剂量相关,但其与剂量不成正比关系。t1/2分别为33.5±6.2 h、36.1±7.6 h和39.8±12.5 h,低、中、高剂量的消除半衰期相近(P>0.05),但有逐渐增加的趋势；V/F分别为1389.6±231.5 L、1724.4±269.2 L和1414.2±342.6 L；CL/F分别为29.5±6.5 L/h、34.0±7.2 L/h和25.9±7.6L/h；MRT分别为47.8±4.0 h、51.1±9.9 h和52.9±13.9 h,低、中、高三种剂量的V/F、CL/F、MRT相近,差异无统计学意义(P>0.05)。3.2.1.2贝那普利单次给药药动学参数及其在剂量间的差异
单次口服1粒、2粒、3粒氨氯地平贝那普利胶囊后贝那普利(10mg、20 mg、30 mg)的Tmax分别为0.50±0.19 h、0.81±0.51 h和0.63±0.40h,三种剂量口服的达峰时间相近(P>0.05)；Cmax分别为264.10±84.95ng·mL-1、376.85±173.59 ng·mL-1和642.42±331.14 ng·mL-1,AUC0～8分别为245.9±87.7 ng·h·mL-1、450.5±122.8 ng·h·mL-1和557.6±73.5ng·h·mL-1,AUC0-∞分别为248.7±87.9 ng·h·mL-1、477.2±130.8 ng·h·mL-1和580.2±71.1 ng·h·mL-1。说明在给10-30 mg剂量范围内,贝那普利Cmax、AUC0-8、AUC0-∞与剂量呈线性相关关系,但不成正比关系,这与文献报道一致。t1/2分别为0.76±0.19 h、2.85±1.70 h和2.91±1.65 h,V/F分别为44.4±11.5 L、186.6±128.8 L和217.9±122.6 L,MRT分别为1.0±0.3 h、2.3±0.7 h和1.8±0.6 h,低剂量与中、高剂量的消除半衰期、表观分布容积、体内平均滞留时间的差异有统计学意义(P<0.05)。CL/F分别为44.5±14.7 L/h、44.6±11.3 L/h和52.5±7.7 L/h,低、中、高剂量的清除率相近,各剂量组间无统计学差异(P>0.05)；3.2.1.3贝那普利拉单次给药药动学参数及其在剂量间的差异
单次口服1粒、2粒、3粒氨氯地平贝那普利胶囊后贝那普利的活性代谢产物贝那普利拉的Tmax分别为1.4±0.5 h、2.1±1.3 h和1.4±0.4h,Tmax各剂量组之间无统计学差异(P>0.05)；Cmax分别为232.60±100.46 ng·mL-1、323.48±61.58 ng·mL-1和615.91±188.23ng·mL-1,AUC0～24分别为1025.9±172.2 ng·h·mL-1、1924.8±314.4ng·h·mL-1和3095.0±713.4 ng·h·mL-1,AUC0-∞分别为1053.6±177.1ng·h·mL-1、1989.8±320.9 ng·h·mL-1和3150.0±715.6 ng·h·mL-1。贝那普利拉的Cmax、AUC与贝那普利的剂量呈线性相关,但其Cmax与贝那普利的剂量不成正比关系。t1/2分别为4.9±0.3 h、5.0±0.7 h和4.3±0.4 h；V/F分别为69.3±11.5 L、75.0±16.5 L和62.0±13.8 L；CL/F分别为9.7±1.6 L/h、10.3±1.4 L/h和9.9±2.0 L/h；MRT分别为7.8±1.5 h、8.7±1.4 h和7.3±1.1 h,低、中、高三种剂量的V/F、CL/F、MRT相近,差异无统计学意义(P>0.05)。3.2.2多次给药药动学参数及其在剂量间的差异3.2.2.1氨氯地平单次给药药动学参数及其在剂量间的差异
连续多次口服氨氯地平贝那普利胶囊(每日早8点一次、每次2粒)14天后达到稳态,氨氯地平的Tmax为6.3±3.3 h；Cmax为24.00±5.58ng·mL-1,AUC0～144为1264.1±376.7 ng·h·mL-1,AUC0-∞为1343.8±405.6ng·h·mL-1；t1/2为33.6±10.9h；CL/F为23.0±6.5 L/h；V/F为1137.4±527.3L；Cmaxss为24.03±5.56 ng·mL-1；Cminss为15.64±3.93 ng·mL-1；Cav为19.32±5.05 ng·mL-1；DF为0.4±0.1。
多次给药组和单次给药的中剂量组的Tmax、t1/2比较无统计学差异(P>0.05),CL/F、V/F、Cmax比较有统计学差异(P<0.05)。多次给药组AUC0～24ss(463.7±121.1 ng·h·mL-1)和单次中剂量组的AUC0～∞(304.2±54.9 ng·h·mL-1)比较有统计学差异(P<0.05)。多次服用氨氯地平贝那普利胶囊后氨氯地平存在蓄积现象。3.2.2.2贝那普利单次给药药动学参数及其在剂量间的差异
多次连续口服2粒氨氯地平贝那普利胶囊后贝那普利的Tmax为0.47±0.16 h；Cmax为434.28±90.70 ng·mL-1,AUC0～8为504.2±118.2ng·h·mL-1,AUC0-∞为519.1±112.9 ng·h·mL-1；t1/2为2.30±1.02 h；CL/F为42.0±11.7 L/h；V/F为151.6±111.6L；Cmaxss为434.28±90.70 ng·mL-1；Cminss为3.95±1.40 ng·mL-1。Cav为21.0±4.9 ng·mL-1;DF为21.6±7.9。
多次给药组和单次给药的中剂量组的Tmax、t1/2、CL/F、V/F、Cmax比较无统计学差异(P>0.05)。多次给药组AUC0～8ss (504.2±118.2 ng·h·mL-1)和单次中剂量组的AUC0-∞(477.2±130.8 ng·h·mL-1)比较无统计学差异(P>0.05)。多次服用氨氯地平贝那普利胶囊后贝那普利无蓄积现象。3.2.2.3贝那普利拉单次给药药动学参数及其在剂量间的差异
多次连续口服2粒氨氯地平贝那普利胶囊后贝那普利代谢物贝那普利拉血药浓度达峰时间Tmax为1.6±0.4 h；Cmax为401.58±76.09ng·mL-1,AUC0～24为2338.9±326.3ng·h·mL-1,AUC0-∞为2412.3±329.3 ng·h·mL-1;t1/2为5.1±0.5 h; CL/F为8.7±1.3L/h; V/F为64.4±13.8L;Cmaxss为401.58±76.09 ng·mL-1；Cminss为9.35±1.90 ng·mL-1；Cav为97.45±13.59 ng·mL-1：DF为4.0±0.5。
多次给药组和单次给药中剂量组的Tmax、t1/2、V/F比较无统计学差异(P>0.05),CL/F、Cmax比较有统计学差异(P<0.05)。多次给药组AUC0～24ss(2338.9±326.3 ng·h·mL-1)和单次中剂量组的AUC0～∞(1989.8±320.9 ng·h·mL-1)比较有统计学差异(P<0.05)。多次服用氨氯地平贝那普利胶囊后贝那普利拉有轻微蓄积现象。3.3安全性评价
单次给药试验中无受试者出现不良反应现象。试验完成后受试者进行第二次体检,体检结果均无异常。
多次给药试验中个别受试者在试验过程中出现不良反应现象。试验完成后,受试者进行第二次体检,体检结果均无异常。4结论4.1测定方法
本实验方法可准确、快速测定氨氯地平、贝那普利及其活性代谢物贝那普利拉的血药浓度,用以研究单次给药药动学及多次给药的稳态药动学的考察。4.2药物动力学
口服氨氯地平贝那普利胶囊后氨氯地平和贝那普利药物动力学在人体内的线性相关性不是很好,贝那普利拉在人体内呈现良好的线性药物动力学特征；多次给药后氨氯地平在人体内有明显的蓄积作用,贝那普利无蓄积作用,贝那普利拉有轻微蓄积作用。4.3安全性
受试者口服氨氯地平贝那普利胶囊后耐受性、安全性良好。
1 OBJECTIVES
To establish an reliable、high-efficiency、easy ultra high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method for determination of amlodipine,benazepril and it's metabolite benazeprilat in human plasma,and to study the pharmacokinetics of amlodipine benazepril capsule in Chinese healthy volunteers via single and multiple dose medicine oral administration,to observe the inter-dose differences,linear characteristics,gender differences,accumulations,and the ADRs in Chinese healthy volunteers, to supply reference for it's clinical rational use. 2 METHODS
2.1 Ultra high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method for determination of amlodipine,benazepril and it's metabolite benazeprilat in human plasma 2.1.1 Determination of amlodipine in human plasma
Plasma samples were conducted by solid phase extraction and then evaporated ten folds by vacuum sublimation.Carbamazepine was used as the internal standard (IS).The UPLC separation of the analytes were performed on an ACQUITY UPLC(?) BEH C18,2.1 mm×50mm,1.7μm) column,column temperature was 40℃,sample acquisition room temperature is 10℃,with a mobile phase of a mixture of acetonitrile:water (formic acid:0.1%) (35:65,v/v) at a flow rate of 0.3 mL·min-1.Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used for determination,with positive ion SIM detection of amllodipine. 2.1.2 Determination of benazepril and it's metabolite benazeprilat in human plasma
Plasma samples were conducted by protein precipitation.Enalapril was used as the internal standard (IS).The UPLC separation of the analytes were performed on an ACQUITY UPLC(?) BEH C18,2.1 mm×50 mm,1.7μm) column,column temperature was 40℃,sample acquisition room temperature is 10℃,with a mobile phase of a mixture of methanol:water (formic acid:0.1%) (50:50,v/v) at a flow rate of 0.25 mL·min-1. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used for determination,with positive ion SIM detection of benazepril and it's metabolite benazeprilat. 2.2 Pharmacokinetics of amlodipine benazepril capsules 2.2.1 Single doses medicine
In this study,twenty four healthy volunteers(male and female,half by half) were divided into three groups (each group consisting of 4 males and 4 females) by randomized block design. Single doses of 1、2、3 capsules(5 mg/10 mg,10 mg/20 mg,15 mg/30 mg;each amlodipine benazepril capsule contain 5 mg of amlodipine and 10 mg of benazepril) were administered to three groups respectively.Venous blood samples (4.0 mL) were collected immediately before dose and at15.0,30.0,45.0 min,1.0、1.5、2.0、3.0、4.0、6.0、8.0、10.0、12.0、24.0、36.0、48.0、72.0、96.0、120.0、144.0 h after administration.The blood samples were collected in heparinized collection tubes and then were gently mixed.The plasma was separated by centrifugation at 3000 rpm for 5 min.Aliquots of the plasma were transferred to eppendorf tubes,and store at-70℃until analysis. 2.2.2 Multiple dose medicine
Employing middle dose group(10 mg/20 mg) and eight healthy volunteers (4 males and 4 females each) to carry out multiple doses trail.Eight healthy volunteers (4 females and 4 males) were given amlodipine benazepril capsules for 14 days (once per day,2 capsules each time,oral administration,every 8:00 AM). The attainment of steady states were evaluated from the plasma trough concentrations (Cmin) measured immediately prior to dosing on days 11,12 and 13. On the 14th day, the last 10 mg of amlodipine and 20 mg of benazepri doses was given at 8:00 AM.The blood samples were collected at the same time as in the single-dose study,and all other experimental conditions were also the same as in the single doses study. 3 RESULTS 3.1 Methodology results 3.1.1 UPLC-ESI-MS/MS determination of amlodipine in human plasma
Amlodipine and IS were well retained in the C18 column,and fully seperated.The calibration curve was linear in the range of 0.09-30.0 ng-mL-1 for amlodipine,coefficient was 0.999302,the lower limit of quantification (LLOQ) was 0.09 ng-mL-1 for amlodipine.The average extraction recoveries for amlodipine was above 85%.The methodology recoveries were between 107.7%～111.9%.The intra-day and inter-day relative standard deviations were less than 9%. 3.1.2 UPLC-ESI-MS/MS simultaneously determination of benazepril and it's metabolite benazeprilat in human plasma
Benazepril and it's metabolite benazeprilat and IS were well retained in the C18 column,and fully seperated.The calibration curve was linear in the range of 1.0～1000.0 ng·mL-1 for benazepril and it's metabolite benazeprilat,coefficient were 0.997250 and 0.999730 respectively,the lower limit of quantification (LLOQ) was 1.0 ng·mL-1.The average extraction recoveries for benazepril was above 93.8%.The methodology recoveries for benazepril were between 100.4%～103.3%.The intra-day and inter-day relative standard deviations for benazepril were less than 9.3%.The average extraction recoveries for benazeprilat was above 87.8%.The methodology recoveries for benazeprilat were between 98.1%～108.8%.The intra-day and inter-day relative standard deviations for benazeprilat were less than 9.5%. 3.2 Pharmacokinetics of amlodipine benazepril capsule 3.2.1 Pharmacokinetic parameters of single doses medicine and there's differences in three groups 3.2.1.1 Pharmacokinetic parameters of single doses medicine of amlodipine and there's differences in three groups
After single doses oral administration of amlodipine benazepril capsule of 1、2、3 capsules(5 mg/10 mg,10 mg/20 mg,15 mg/30 mg;each amlodipine benazepril capsule contain 5 mg of amlodipine and 10mg of benazepril),the main pharmacokinetic parameters found for amlodipine at doses of 5,10,15 mg were as follows:tmax were 6.0±1.1h> 7.0±3.4 h and 5.6±3.0 h, respectively, tmax were close in three groups;Cmax were 4.37±1.37ng·mL-1、7.23±1.81 ng·mL-1 and 14.71±3.14 ng·mL-1, respectively.AUC0-144 were 167.7±35.9 ng·h·mL-1、283.8±47.6 ng·h·mL-1 and 574.9±159.0 ng·h·mL-1,respectively.AUC0-∞were 176.8±40.2 ng·h·mL-1、304.1±54.8 ng·h·mL-1 and 628.2±197.5 ng·h·mL-1, respectively,which means that between the doses of 5 mg to 15 mg,pharmacokinetic parameters of Cmax、AUC0-144、AUC0-∞of amlodipine depended on doses,but it did not show a direct ratio relationship.t1/2 were 33.5±6.2h、36.1±7.6h and 39.8±12.5 h,respectively.t1/2 were close in three groups(P>0.05),while it showed an increasing trend;V/F were 1389.61231.5L、1724.4±269.2L and 1414.2±342.6 L,respectively; CL/F were 29.5±6.5 L/k 34.0±7.2 L/h and 25.9±7.6 L/h, respectively; MRT were 47.8±4.0h、51.1+9.9h and 52.9113.9h,respectively; pharmacokinetic parameters of V/F、CL/F、MRT of low,middle and high dose groups were close, the differences had no statistics significance(P>0.05). 3.2.1.2 Pharmacokinetic parameters of single doses medicine of benazepril and there's differences in three groups
After single doses oral administration of amlodipine benazepril capsules of 1、2、3 capsules(5mg/10 mg,10 mg/20mg,15 mg/30 mg; each amlodipine benazepril capsule contain 5 mg of amlodipine and 10mg of benazepril), the main pharmacokinetic parameters found for benazepril at doses of 10,20,30 mg were as follows:tmax were 0.50±0.19h,0.81±0.51h and 0.63±0.40 h, respectively, tmax were close in three groups(P>0.05); Cmax were 264.10±84.95 ng·mL-1、376.85±1173.59 ng·mL-1 and 642.42±331.14 ng·mL-1, respectively.AUC0-8 were 245.9+87.7 ng·h·mL-1、450.5±122.8 ng·h·mL-1 and 557.6±73.5 ng·h·mL-respectively.AUC0-∞were 248.7±87.9 ng·h·mL-1、477.2±130.8 ng·h·mL-1 and 580.2±71.1 ng·h·mL-1, respectively, which meaned that between the doses of 10 mg to 30 mg,pharmacokinetic parameters of Cmax、AUC0-8、AUC0-∞of benazepril were linear with doses,but it did not show a direct ratio relationship,which fitted the literatures reported.t1/2 were 0.76±0.19 h、2.85±1.70 h and 2.91±1.65 Irrespectively.t1/2 were close in three groups(P>0.05),while it showed an increasing trend;V/F were 44.4±11.5 L、186.6±128.8Land217.9±122.6L,respectively; MRT were 1.0±0.3h 2.3±0.7 h and 1.8±0.6 h,respectively;the pharmacokinetic parameters of t1/2,V/F,MRT of benazepril of low-dose group differ with middle and high-dose groups, the differences had statistics significance(P<0.05). CL/F were 44.5±14.7 L/h、44.6±11.3 L/h and 52.5±7.7 L/h,respectively; the pharmacokinetic parameters of CL/F of benazepril of low,middle and high dose groups were close, the differences had no statistics significance(P>0.05). 3.2.1.3 Pharmacokinetic parameters of single doses medicine of benazeprilat and there's differences in three groups
After single doses oral administration of amlodipine benazepril capsule of 1、2、3 capsules(5 mg/10 mg,10 mg/20 mg,15 mg/30 mg;each amlodipine benazepril capsule contain 5 mg of amlodipine and 10mg of benazepril), the main pharmacokinetic parameters found for benazeprilat at doses of 10,20,30 mg were as follows:tmax were 1.4±0.5h、2.1±1.3h and 1.4±0.4 h,respectively,tmax were close in three groups(P>0.05); Cmax were 232.60±100.46 ng·mL-1、323.48±61.58 ng·mL-1 and 615.91±188.23 ng·mL-1, respectively.AUC0-24 were 1025.9±172.2 ng·h·mL-1924.8±314.4 ng·h·mL、and 3095.0±713.4 ng·h·mL-1, respectively. AUC0-∞were 1053.6±177.1 ng·h·mL-1、1989.8±320.9 ng·h·mL-1 and 3150.0±715.6 ng·h·mL-1，respectively.Pharmacokinetic parameters of AUC of benazeprilat was linear with the doses of benazepril,while Cmax did not show a direct ratio relationship with the doses of benazepril.t1/2 were 4.9±0.3h、5.0±0.7h and 4.3±0.4h h,respectively.t1/2 were close in three groups(P>0.05). V/F were 69.3±11.5 L、75.0±16.5 L and 62.0±13.8 L,respectively;MRT were 7.8±1.5h、8.7±1.4h and 7.3±1.1 h,respectively;CL/F were 9.7±1.6 L/h、10.3±1.4 L/h and 9.9±2.0 L/h,respectively;the pharmacokinetic parameters of t1/2,V/F,MRT, CL/F of benazeprilat of low,middle and high-dose groups were close,the differences had no statistics significance(P>0.05). 3.2.2 Pharmacokinetic parameters of multiple doses medicine and there's differences in three groups 3.2.2.1 Pharmacokinetic parameters of multiple doses medicine of amlodipine and there's differences between multiple dose group and single middle-dose group
The main pharmacokinetic parameters found for amlodipine at dose of 10 mg (2 capsules each time per day for 14 days of oral administration) were as follows:tmax was 6.3±3.3 h,Cmax was 24.00±5.58 ng·mL-1,AUCo-144 was 1264.1±376.7 ng-h-mL-1,AUC0-∞was 1343.8±405.6 ng-h-mL-1;t1/2 was 33.6±10.9 h;CL/F was 23.0±6.5 L/h;V/F was1137.4±527.3 L;Cmaxss was 24.03±5.56 ng-mL-1;Cminss was 15.64±3.93 ng·mL-1;Cav was 19.32±5.05 ng·mL-1;DF was 0.4±0.1.
The pharmacokinetic parameters of Tmax、t1/2 of amlodipine of single middle-dose group and multiple dose were close, the differences had no statistics significance(P>0.05).The pharmacokinetic parameters of CL/F、V/F、Cmax of amlodipine multiple dose differ with single middle-dose group, the differences had statistics significance(P<.05).
The pharmacokinetic parameters of AUC0～24SS (463.7±121.1 ng-h-mL-1) of amlodipine multiple dose differ with single middle-dose group of AUC0-∞(304.2±54.9 ng·h·mL-1), the differences had statistics significance(P<0.05),which meaned that after multiple-dose medicine of amlodipine benazepril capsules,amlodipine had an accumulation. 3.2.2.2 Pharmacokinetic parameters of multiple doses medicine of benazepril and there's differences between multiple dose group and single middle-dose group
The main pharmacokinetic parameters found for benazepril at dose of 20 mg(2 capsules each time per day for 14 days of oral administration) were as follows:tmax was 0.47±0.16 h,Cmax was 434.28±90.70 ng·mL-1，AUC0～8 was 504.2±118.2 ng·h·mL-1,AUC0-∞was 519.1±112.9 ng·h·mL-1;t1/2 was 2.30±1.02 h;CL/F was 42.0±11.7 L/h;V/F was 151.6±111.6 L;Cmaxss was 434.28±90.70 ng·mL-1;Cminss was 3.95±1.40 ng·mL-1;Cav was 21.0±4.9 ng·mL-1;DF was 21.6±7.9.The pharmacokinetic parameters of Tmax、t1/2、CL/F、V/F、Cmax of benazepril of single middle-dose group and multiple dose were close, the differences had no statistics significance(P>0.05).
The pharmacokinetic parameters of AUC0-8 (477.2±130.8 ng·h·mL-1) of benazepril of single middle-dose group and multiple dose of AUC0～8SS(504.2±118.2 ng·h·mL-1)were close,the differences had no statistics significance(P>0.05),which meaned that after multiple-dose medicine of amlodipine benazepril capsules,no accumulation happened. 3.2.2.3 Pharmacokinetic parameters of multiple doses medicine of benazeprilat and there's differences between multiple dose group and single middle-dose group
The main pharmacokinetic parameters found for benazeprilat at dose of 20 mg (2 capsules each time per day for 14 days of oral administration) were as follows:tmax was 1.6±0.4 h,Cmax was 401.58±76.09 ng·mL-1,AUC0～24 was 2338.9±326.3 ng·h·mL-1,AUC0-∞was 2412.3±329.3 ng·h·mL-1;t1/2 was 5.1±0.5 h;CL/F was 8.7±1.3 L/h;V/F was 64.4±13.8 L;Cmaxsswas 401.58±76.09 ng·mL-1;Cminss was 9.35±1.90 ng·mL-1;Cav was 97.45±13.59 ng·mL-1;DF was 4.0±0.5.
The pharmacokinetic parameters of Tmax、t1/2、V/F of benazeprilat of single middle-dose group and multiple doses were close, the differences had no statistics significance(P>0.05).The pharmacokinetic parameters of CL/F、max of benazeprilat of multiple dose differ with single middle-dose group, the differences had statistics significance(P<0.05).
The pharmacokinetic parameters of AUC0～24ss(2338.9±326.3 ng·h·mL-1) of benazeprilat of multiple doses differ with the single middle-dose group of AUC0～∞(1989.8±320.9 ng·h·mL-1).The differences had statistics significance(P<0.05),which meaned that after multiple-dose medicine of amlodipine benazepril capsules,a slight accumulation of benazeprilat happened. 3.3 Assessment of the safety profiles of amlodipine benazepril capsule
During the single doses phase,no ADRs happened in Chinese healthy volunteers.Physical and laboratory examination results after single doses clinical trail finished were all normal.
During the multiple dose phase,ADRs happened in some volunteers. Physical and laboratory examination results after multiple dose medicine clinical trail finished were all normal. 4 CONCLUSIONS 4.1 Methods
The method was accurate and fast for determination of amlodipine,benazepril and it's metabolites benazeprilat in human plasma. It can be used for studying the pharmacokinetics of amlodipine,benazepril and it's metabolite benazeprilat of single doses medicine and steady state pharmacokinetics of multiple dose medicine. 4.2 Pharmacokinetics
After oral administration of single-dose of amlodipine benazepril capsules,amlodipine and benazepril showed partly linear pharmacokinetic characteristics, benazeprilat showed good linear pharmacokinetic characteristics; after oral administration of multiple-dose of amlodipine benazepril capsules, amlodipine had an obvious accumulation,benazepril had no accumulation, benazeprilat had a slight accumulation. 4.3 Safety profiles
After oral administration of amlodipine benazepril capsules in Chinese healthy volunteers,tolerance and safety profiles were well proved.
建立一种可靠、高效、简便的方法,测定氨氯地平、贝那普利及其代谢产物贝那普利拉在人血浆中的浓度,进行单次及多次口服氨氯地平贝那普利胶囊在中国健康受试者体内的药物动力学研究,考察氨氯地平、贝那普利及其代谢产物贝那普利拉药动学的剂量间差异、线性特征、性别差异、蓄积情况,并观察健康志愿者服药后的不良反应,为临床合理用药提供参考依据。2方法2.1 UPLC-ESI-MS/MS法测定人血浆中氨氯地平、贝那普利及贝那普利拉2.1.1氨氯地平血样测定：
血浆样品经固相萃取后真空挥干浓缩十倍处理,以卡马西平作为内标(IS),用色谱柱(ACQUITY UPLC(?) BEH C18,2.1 mm×50 mm,1.7μm)进行分离,柱温：40℃,进样室温度：10℃,流动相组成为乙腈：水溶液(含0.1%甲酸)=(35：65,V/V),流速：0.3 mL·min-1。UPLC-ESI-MS/MS法选择性监测被测物质的准分子离子峰。2.1.2贝那普利及贝那普利拉测定：
血浆样品经蛋白沉淀法处理,以依那普利作为内标(IS),用色谱柱(ACQUITY UPLC(?) BEH C18,2.1 mm×50 mm,1.7μm)进行分离,柱温：40℃,进样室温度：10℃,流动相组成为甲醇：水溶液(含0.1%甲酸)=(50：50,V/V),流速：0.25 mL·min-1。UPLC-ESI-MS/MS法选择性监测被测物质的准分子离子峰。2.2药物动力学研究2.2.1单次给药
24名健康志愿者,男女各半,采用随机区组设计,男女受试者各分为3组,分别单次口服氨氯地平贝那普利胶囊1、2、3粒(5mg/10mg,10mg/20mg,15mg/30mg；氨氯地平贝那普利胶囊每粒含苯磺酸氨氯地平5mg/盐酸贝那普利10mg),给药物前和给药后15.0、30.0、45.0 min,1.0、1.5、2.0、3.0、4.0、6.0、8.0、10.0、12.0、24.0、36.0、48.0、72.0、96.0、120.0、144.0 h(共20点)由静脉取血4 mL,置肝素化抗凝试管中,分离血浆存于-70℃冰箱中,以备血药浓度测定。2.2.2多次给药
采用中剂量(10 mg/20 mg),8人(男女各半)。服药方法为每日1次,一次2粒(规格：每粒含苯磺酸氨氯地平5 mg／盐酸贝那普利10 mg),连续给药14天。给药时间为8：00 AM。于第1天多次给药前、第11、12、13、14日给药前,第14日在服药后按照单次给药设计采集血样。各取血点经静脉留置针取前臂静脉血4.0 mL,置肝素化抗凝试管中,分离血浆存于-70℃冰箱中,以备血药浓度测定。3结果3.1方法学结果3.1.1UPLC-ESI-MS/MS法测定人血浆中氨氯地平
氨氯地平及内标在C18柱上保留较好,分离完全,氨氯地平的线性范围为0.09～30.0 ng·mL-1,r2=0.999302,最低定量限为0.09 ng·mL-1。氨氯地平的平均萃取回收率都大于85%,方法回收率都在107.7%～111.9%之间,日内日间RSD均小于9%。3.1.2UPLC-ESI-MS/MS法同时测定人血浆中贝那普利及贝那普利拉
贝那普利、贝那普利拉及内标依那普利在C18柱上保留较好,分离完全,贝那普利及贝那普利拉的线性范围为1.0～1000.0 ng·mL-r2=0.997250和0.999730,最低定量限为1.0 ng·mL-1。贝那普利和贝那普利拉的平均萃取回收率都大于93.8%,方法回收率都在100.4%～103.3%之间,日内日间RSD均小于9.3%。贝那普利拉的平均萃取回收率都大于87.8%,方法回收率都在98.1%～108.8%之间,日内日间RSD均小于9.5%。3.2氨氯地平贝那普利的药物动力学3.2.1单次给药药动学参数及其在剂量间的差异3.2.1.1氨氯地平单次给药药动学参数及其在剂量间的差异
单剂量口服氨氯地平贝那普利胶囊1、2、3粒(5 mg/10 mg,10mg/20 mg,15 mg/30 mg；氨氯地平贝那普利胶囊每粒含苯磺酸氨氯地平5mg/盐酸贝那普利10 mg)后氨氯地平(5 mg、10 mg、15 mg)的Tmax分别为6.0±1.1 h、7.0±3.4 h和5.6±3.0 h,三种剂量口服的达峰时间相近(P>0.05)；Cmax分别为4.37±1.37 ng·mL-1、7.23±1.81ng·mL-1和14.71±3.14 ng·mL-1,AUC0-144分别为167.7±35.9 ng·h·mL-1、283.8±47.6 ng·h·mL-1和574.9±159.0 ng·h·mL-1,AUC0-∞分别为176.8±40.2 ng·h·mL-1、304.1±54.8 ng·h·mL-1和628.2±197.5 ng·h·mL-1。说明在5-15mg剂量范围内,氨氯地平Cmax、AUC0-144、AUC0-∞与剂量相关,但其与剂量不成正比关系。t1/2分别为33.5±6.2 h、36.1±7.6 h和39.8±12.5 h,低、中、高剂量的消除半衰期相近(P>0.05),但有逐渐增加的趋势；V/F分别为1389.6±231.5 L、1724.4±269.2 L和1414.2±342.6 L；CL/F分别为29.5±6.5 L/h、34.0±7.2 L/h和25.9±7.6L/h；MRT分别为47.8±4.0 h、51.1±9.9 h和52.9±13.9 h,低、中、高三种剂量的V/F、CL/F、MRT相近,差异无统计学意义(P>0.05)。3.2.1.2贝那普利单次给药药动学参数及其在剂量间的差异
单次口服1粒、2粒、3粒氨氯地平贝那普利胶囊后贝那普利(10mg、20 mg、30 mg)的Tmax分别为0.50±0.19 h、0.81±0.51 h和0.63±0.40h,三种剂量口服的达峰时间相近(P>0.05)；Cmax分别为264.10±84.95ng·mL-1、376.85±173.59 ng·mL-1和642.42±331.14 ng·mL-1,AUC0～8分别为245.9±87.7 ng·h·mL-1、450.5±122.8 ng·h·mL-1和557.6±73.5ng·h·mL-1,AUC0-∞分别为248.7±87.9 ng·h·mL-1、477.2±130.8 ng·h·mL-1和580.2±71.1 ng·h·mL-1。说明在给10-30 mg剂量范围内,贝那普利Cmax、AUC0-8、AUC0-∞与剂量呈线性相关关系,但不成正比关系,这与文献报道一致。t1/2分别为0.76±0.19 h、2.85±1.70 h和2.91±1.65 h,V/F分别为44.4±11.5 L、186.6±128.8 L和217.9±122.6 L,MRT分别为1.0±0.3 h、2.3±0.7 h和1.8±0.6 h,低剂量与中、高剂量的消除半衰期、表观分布容积、体内平均滞留时间的差异有统计学意义(P<0.05)。CL/F分别为44.5±14.7 L/h、44.6±11.3 L/h和52.5±7.7 L/h,低、中、高剂量的清除率相近,各剂量组间无统计学差异(P>0.05)；3.2.1.3贝那普利拉单次给药药动学参数及其在剂量间的差异
单次口服1粒、2粒、3粒氨氯地平贝那普利胶囊后贝那普利的活性代谢产物贝那普利拉的Tmax分别为1.4±0.5 h、2.1±1.3 h和1.4±0.4h,Tmax各剂量组之间无统计学差异(P>0.05)；Cmax分别为232.60±100.46 ng·mL-1、323.48±61.58 ng·mL-1和615.91±188.23ng·mL-1,AUC0～24分别为1025.9±172.2 ng·h·mL-1、1924.8±314.4ng·h·mL-1和3095.0±713.4 ng·h·mL-1,AUC0-∞分别为1053.6±177.1ng·h·mL-1、1989.8±320.9 ng·h·mL-1和3150.0±715.6 ng·h·mL-1。贝那普利拉的Cmax、AUC与贝那普利的剂量呈线性相关,但其Cmax与贝那普利的剂量不成正比关系。t1/2分别为4.9±0.3 h、5.0±0.7 h和4.3±0.4 h；V/F分别为69.3±11.5 L、75.0±16.5 L和62.0±13.8 L；CL/F分别为9.7±1.6 L/h、10.3±1.4 L/h和9.9±2.0 L/h；MRT分别为7.8±1.5 h、8.7±1.4 h和7.3±1.1 h,低、中、高三种剂量的V/F、CL/F、MRT相近,差异无统计学意义(P>0.05)。3.2.2多次给药药动学参数及其在剂量间的差异3.2.2.1氨氯地平单次给药药动学参数及其在剂量间的差异
连续多次口服氨氯地平贝那普利胶囊(每日早8点一次、每次2粒)14天后达到稳态,氨氯地平的Tmax为6.3±3.3 h；Cmax为24.00±5.58ng·mL-1,AUC0～144为1264.1±376.7 ng·h·mL-1,AUC0-∞为1343.8±405.6ng·h·mL-1；t1/2为33.6±10.9h；CL/F为23.0±6.5 L/h；V/F为1137.4±527.3L；Cmaxss为24.03±5.56 ng·mL-1；Cminss为15.64±3.93 ng·mL-1；Cav为19.32±5.05 ng·mL-1；DF为0.4±0.1。
多次给药组和单次给药的中剂量组的Tmax、t1/2比较无统计学差异(P>0.05),CL/F、V/F、Cmax比较有统计学差异(P<0.05)。多次给药组AUC0～24ss(463.7±121.1 ng·h·mL-1)和单次中剂量组的AUC0～∞(304.2±54.9 ng·h·mL-1)比较有统计学差异(P<0.05)。多次服用氨氯地平贝那普利胶囊后氨氯地平存在蓄积现象。3.2.2.2贝那普利单次给药药动学参数及其在剂量间的差异
多次连续口服2粒氨氯地平贝那普利胶囊后贝那普利的Tmax为0.47±0.16 h；Cmax为434.28±90.70 ng·mL-1,AUC0～8为504.2±118.2ng·h·mL-1,AUC0-∞为519.1±112.9 ng·h·mL-1；t1/2为2.30±1.02 h；CL/F为42.0±11.7 L/h；V/F为151.6±111.6L；Cmaxss为434.28±90.70 ng·mL-1；Cminss为3.95±1.40 ng·mL-1。Cav为21.0±4.9 ng·mL-1;DF为21.6±7.9。
多次给药组和单次给药的中剂量组的Tmax、t1/2、CL/F、V/F、Cmax比较无统计学差异(P>0.05)。多次给药组AUC0～8ss (504.2±118.2 ng·h·mL-1)和单次中剂量组的AUC0-∞(477.2±130.8 ng·h·mL-1)比较无统计学差异(P>0.05)。多次服用氨氯地平贝那普利胶囊后贝那普利无蓄积现象。3.2.2.3贝那普利拉单次给药药动学参数及其在剂量间的差异
多次连续口服2粒氨氯地平贝那普利胶囊后贝那普利代谢物贝那普利拉血药浓度达峰时间Tmax为1.6±0.4 h；Cmax为401.58±76.09ng·mL-1,AUC0～24为2338.9±326.3ng·h·mL-1,AUC0-∞为2412.3±329.3 ng·h·mL-1;t1/2为5.1±0.5 h; CL/F为8.7±1.3L/h; V/F为64.4±13.8L;Cmaxss为401.58±76.09 ng·mL-1；Cminss为9.35±1.90 ng·mL-1；Cav为97.45±13.59 ng·mL-1：DF为4.0±0.5。
多次给药组和单次给药中剂量组的Tmax、t1/2、V/F比较无统计学差异(P>0.05),CL/F、Cmax比较有统计学差异(P<0.05)。多次给药组AUC0～24ss(2338.9±326.3 ng·h·mL-1)和单次中剂量组的AUC0～∞(1989.8±320.9 ng·h·mL-1)比较有统计学差异(P<0.05)。多次服用氨氯地平贝那普利胶囊后贝那普利拉有轻微蓄积现象。3.3安全性评价
单次给药试验中无受试者出现不良反应现象。试验完成后受试者进行第二次体检,体检结果均无异常。
多次给药试验中个别受试者在试验过程中出现不良反应现象。试验完成后,受试者进行第二次体检,体检结果均无异常。4结论4.1测定方法
本实验方法可准确、快速测定氨氯地平、贝那普利及其活性代谢物贝那普利拉的血药浓度,用以研究单次给药药动学及多次给药的稳态药动学的考察。4.2药物动力学
口服氨氯地平贝那普利胶囊后氨氯地平和贝那普利药物动力学在人体内的线性相关性不是很好,贝那普利拉在人体内呈现良好的线性药物动力学特征；多次给药后氨氯地平在人体内有明显的蓄积作用,贝那普利无蓄积作用,贝那普利拉有轻微蓄积作用。4.3安全性
受试者口服氨氯地平贝那普利胶囊后耐受性、安全性良好。
1 OBJECTIVES
To establish an reliable、high-efficiency、easy ultra high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method for determination of amlodipine,benazepril and it's metabolite benazeprilat in human plasma,and to study the pharmacokinetics of amlodipine benazepril capsule in Chinese healthy volunteers via single and multiple dose medicine oral administration,to observe the inter-dose differences,linear characteristics,gender differences,accumulations,and the ADRs in Chinese healthy volunteers, to supply reference for it's clinical rational use. 2 METHODS
2.1 Ultra high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method for determination of amlodipine,benazepril and it's metabolite benazeprilat in human plasma 2.1.1 Determination of amlodipine in human plasma
Plasma samples were conducted by solid phase extraction and then evaporated ten folds by vacuum sublimation.Carbamazepine was used as the internal standard (IS).The UPLC separation of the analytes were performed on an ACQUITY UPLC(?) BEH C18,2.1 mm×50mm,1.7μm) column,column temperature was 40℃,sample acquisition room temperature is 10℃,with a mobile phase of a mixture of acetonitrile:water (formic acid:0.1%) (35:65,v/v) at a flow rate of 0.3 mL·min-1.Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used for determination,with positive ion SIM detection of amllodipine. 2.1.2 Determination of benazepril and it's metabolite benazeprilat in human plasma
Plasma samples were conducted by protein precipitation.Enalapril was used as the internal standard (IS).The UPLC separation of the analytes were performed on an ACQUITY UPLC(?) BEH C18,2.1 mm×50 mm,1.7μm) column,column temperature was 40℃,sample acquisition room temperature is 10℃,with a mobile phase of a mixture of methanol:water (formic acid:0.1%) (50:50,v/v) at a flow rate of 0.25 mL·min-1. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used for determination,with positive ion SIM detection of benazepril and it's metabolite benazeprilat. 2.2 Pharmacokinetics of amlodipine benazepril capsules 2.2.1 Single doses medicine
In this study,twenty four healthy volunteers(male and female,half by half) were divided into three groups (each group consisting of 4 males and 4 females) by randomized block design. Single doses of 1、2、3 capsules(5 mg/10 mg,10 mg/20 mg,15 mg/30 mg;each amlodipine benazepril capsule contain 5 mg of amlodipine and 10 mg of benazepril) were administered to three groups respectively.Venous blood samples (4.0 mL) were collected immediately before dose and at15.0,30.0,45.0 min,1.0、1.5、2.0、3.0、4.0、6.0、8.0、10.0、12.0、24.0、36.0、48.0、72.0、96.0、120.0、144.0 h after administration.The blood samples were collected in heparinized collection tubes and then were gently mixed.The plasma was separated by centrifugation at 3000 rpm for 5 min.Aliquots of the plasma were transferred to eppendorf tubes,and store at-70℃until analysis. 2.2.2 Multiple dose medicine
Employing middle dose group(10 mg/20 mg) and eight healthy volunteers (4 males and 4 females each) to carry out multiple doses trail.Eight healthy volunteers (4 females and 4 males) were given amlodipine benazepril capsules for 14 days (once per day,2 capsules each time,oral administration,every 8:00 AM). The attainment of steady states were evaluated from the plasma trough concentrations (Cmin) measured immediately prior to dosing on days 11,12 and 13. On the 14th day, the last 10 mg of amlodipine and 20 mg of benazepri doses was given at 8:00 AM.The blood samples were collected at the same time as in the single-dose study,and all other experimental conditions were also the same as in the single doses study. 3 RESULTS 3.1 Methodology results 3.1.1 UPLC-ESI-MS/MS determination of amlodipine in human plasma
Amlodipine and IS were well retained in the C18 column,and fully seperated.The calibration curve was linear in the range of 0.09-30.0 ng-mL-1 for amlodipine,coefficient was 0.999302,the lower limit of quantification (LLOQ) was 0.09 ng-mL-1 for amlodipine.The average extraction recoveries for amlodipine was above 85%.The methodology recoveries were between 107.7%～111.9%.The intra-day and inter-day relative standard deviations were less than 9%. 3.1.2 UPLC-ESI-MS/MS simultaneously determination of benazepril and it's metabolite benazeprilat in human plasma
Benazepril and it's metabolite benazeprilat and IS were well retained in the C18 column,and fully seperated.The calibration curve was linear in the range of 1.0～1000.0 ng·mL-1 for benazepril and it's metabolite benazeprilat,coefficient were 0.997250 and 0.999730 respectively,the lower limit of quantification (LLOQ) was 1.0 ng·mL-1.The average extraction recoveries for benazepril was above 93.8%.The methodology recoveries for benazepril were between 100.4%～103.3%.The intra-day and inter-day relative standard deviations for benazepril were less than 9.3%.The average extraction recoveries for benazeprilat was above 87.8%.The methodology recoveries for benazeprilat were between 98.1%～108.8%.The intra-day and inter-day relative standard deviations for benazeprilat were less than 9.5%. 3.2 Pharmacokinetics of amlodipine benazepril capsule 3.2.1 Pharmacokinetic parameters of single doses medicine and there's differences in three groups 3.2.1.1 Pharmacokinetic parameters of single doses medicine of amlodipine and there's differences in three groups
After single doses oral administration of amlodipine benazepril capsule of 1、2、3 capsules(5 mg/10 mg,10 mg/20 mg,15 mg/30 mg;each amlodipine benazepril capsule contain 5 mg of amlodipine and 10mg of benazepril),the main pharmacokinetic parameters found for amlodipine at doses of 5,10,15 mg were as follows:tmax were 6.0±1.1h> 7.0±3.4 h and 5.6±3.0 h, respectively, tmax were close in three groups;Cmax were 4.37±1.37ng·mL-1、7.23±1.81 ng·mL-1 and 14.71±3.14 ng·mL-1, respectively.AUC0-144 were 167.7±35.9 ng·h·mL-1、283.8±47.6 ng·h·mL-1 and 574.9±159.0 ng·h·mL-1,respectively.AUC0-∞were 176.8±40.2 ng·h·mL-1、304.1±54.8 ng·h·mL-1 and 628.2±197.5 ng·h·mL-1, respectively,which means that between the doses of 5 mg to 15 mg,pharmacokinetic parameters of Cmax、AUC0-144、AUC0-∞of amlodipine depended on doses,but it did not show a direct ratio relationship.t1/2 were 33.5±6.2h、36.1±7.6h and 39.8±12.5 h,respectively.t1/2 were close in three groups(P>0.05),while it showed an increasing trend;V/F were 1389.61231.5L、1724.4±269.2L and 1414.2±342.6 L,respectively; CL/F were 29.5±6.5 L/k 34.0±7.2 L/h and 25.9±7.6 L/h, respectively; MRT were 47.8±4.0h、51.1+9.9h and 52.9113.9h,respectively; pharmacokinetic parameters of V/F、CL/F、MRT of low,middle and high dose groups were close, the differences had no statistics significance(P>0.05). 3.2.1.2 Pharmacokinetic parameters of single doses medicine of benazepril and there's differences in three groups
After single doses oral administration of amlodipine benazepril capsules of 1、2、3 capsules(5mg/10 mg,10 mg/20mg,15 mg/30 mg; each amlodipine benazepril capsule contain 5 mg of amlodipine and 10mg of benazepril), the main pharmacokinetic parameters found for benazepril at doses of 10,20,30 mg were as follows:tmax were 0.50±0.19h,0.81±0.51h and 0.63±0.40 h, respectively, tmax were close in three groups(P>0.05); Cmax were 264.10±84.95 ng·mL-1、376.85±1173.59 ng·mL-1 and 642.42±331.14 ng·mL-1, respectively.AUC0-8 were 245.9+87.7 ng·h·mL-1、450.5±122.8 ng·h·mL-1 and 557.6±73.5 ng·h·mL-respectively.AUC0-∞were 248.7±87.9 ng·h·mL-1、477.2±130.8 ng·h·mL-1 and 580.2±71.1 ng·h·mL-1, respectively, which meaned that between the doses of 10 mg to 30 mg,pharmacokinetic parameters of Cmax、AUC0-8、AUC0-∞of benazepril were linear with doses,but it did not show a direct ratio relationship,which fitted the literatures reported.t1/2 were 0.76±0.19 h、2.85±1.70 h and 2.91±1.65 Irrespectively.t1/2 were close in three groups(P>0.05),while it showed an increasing trend;V/F were 44.4±11.5 L、186.6±128.8Land217.9±122.6L,respectively; MRT were 1.0±0.3h 2.3±0.7 h and 1.8±0.6 h,respectively;the pharmacokinetic parameters of t1/2,V/F,MRT of benazepril of low-dose group differ with middle and high-dose groups, the differences had statistics significance(P<0.05). CL/F were 44.5±14.7 L/h、44.6±11.3 L/h and 52.5±7.7 L/h,respectively; the pharmacokinetic parameters of CL/F of benazepril of low,middle and high dose groups were close, the differences had no statistics significance(P>0.05). 3.2.1.3 Pharmacokinetic parameters of single doses medicine of benazeprilat and there's differences in three groups
After single doses oral administration of amlodipine benazepril capsule of 1、2、3 capsules(5 mg/10 mg,10 mg/20 mg,15 mg/30 mg;each amlodipine benazepril capsule contain 5 mg of amlodipine and 10mg of benazepril), the main pharmacokinetic parameters found for benazeprilat at doses of 10,20,30 mg were as follows:tmax were 1.4±0.5h、2.1±1.3h and 1.4±0.4 h,respectively,tmax were close in three groups(P>0.05); Cmax were 232.60±100.46 ng·mL-1、323.48±61.58 ng·mL-1 and 615.91±188.23 ng·mL-1, respectively.AUC0-24 were 1025.9±172.2 ng·h·mL-1924.8±314.4 ng·h·mL、and 3095.0±713.4 ng·h·mL-1, respectively. AUC0-∞were 1053.6±177.1 ng·h·mL-1、1989.8±320.9 ng·h·mL-1 and 3150.0±715.6 ng·h·mL-1，respectively.Pharmacokinetic parameters of AUC of benazeprilat was linear with the doses of benazepril,while Cmax did not show a direct ratio relationship with the doses of benazepril.t1/2 were 4.9±0.3h、5.0±0.7h and 4.3±0.4h h,respectively.t1/2 were close in three groups(P>0.05). V/F were 69.3±11.5 L、75.0±16.5 L and 62.0±13.8 L,respectively;MRT were 7.8±1.5h、8.7±1.4h and 7.3±1.1 h,respectively;CL/F were 9.7±1.6 L/h、10.3±1.4 L/h and 9.9±2.0 L/h,respectively;the pharmacokinetic parameters of t1/2,V/F,MRT, CL/F of benazeprilat of low,middle and high-dose groups were close,the differences had no statistics significance(P>0.05). 3.2.2 Pharmacokinetic parameters of multiple doses medicine and there's differences in three groups 3.2.2.1 Pharmacokinetic parameters of multiple doses medicine of amlodipine and there's differences between multiple dose group and single middle-dose group
The main pharmacokinetic parameters found for amlodipine at dose of 10 mg (2 capsules each time per day for 14 days of oral administration) were as follows:tmax was 6.3±3.3 h,Cmax was 24.00±5.58 ng·mL-1,AUCo-144 was 1264.1±376.7 ng-h-mL-1,AUC0-∞was 1343.8±405.6 ng-h-mL-1;t1/2 was 33.6±10.9 h;CL/F was 23.0±6.5 L/h;V/F was1137.4±527.3 L;Cmaxss was 24.03±5.56 ng-mL-1;Cminss was 15.64±3.93 ng·mL-1;Cav was 19.32±5.05 ng·mL-1;DF was 0.4±0.1.
The pharmacokinetic parameters of Tmax、t1/2 of amlodipine of single middle-dose group and multiple dose were close, the differences had no statistics significance(P>0.05).The pharmacokinetic parameters of CL/F、V/F、Cmax of amlodipine multiple dose differ with single middle-dose group, the differences had statistics significance(P<.05).
The pharmacokinetic parameters of AUC0～24SS (463.7±121.1 ng-h-mL-1) of amlodipine multiple dose differ with single middle-dose group of AUC0-∞(304.2±54.9 ng·h·mL-1), the differences had statistics significance(P<0.05),which meaned that after multiple-dose medicine of amlodipine benazepril capsules,amlodipine had an accumulation. 3.2.2.2 Pharmacokinetic parameters of multiple doses medicine of benazepril and there's differences between multiple dose group and single middle-dose group
The main pharmacokinetic parameters found for benazepril at dose of 20 mg(2 capsules each time per day for 14 days of oral administration) were as follows:tmax was 0.47±0.16 h,Cmax was 434.28±90.70 ng·mL-1，AUC0～8 was 504.2±118.2 ng·h·mL-1,AUC0-∞was 519.1±112.9 ng·h·mL-1;t1/2 was 2.30±1.02 h;CL/F was 42.0±11.7 L/h;V/F was 151.6±111.6 L;Cmaxss was 434.28±90.70 ng·mL-1;Cminss was 3.95±1.40 ng·mL-1;Cav was 21.0±4.9 ng·mL-1;DF was 21.6±7.9.The pharmacokinetic parameters of Tmax、t1/2、CL/F、V/F、Cmax of benazepril of single middle-dose group and multiple dose were close, the differences had no statistics significance(P>0.05).
The pharmacokinetic parameters of AUC0-8 (477.2±130.8 ng·h·mL-1) of benazepril of single middle-dose group and multiple dose of AUC0～8SS(504.2±118.2 ng·h·mL-1)were close,the differences had no statistics significance(P>0.05),which meaned that after multiple-dose medicine of amlodipine benazepril capsules,no accumulation happened. 3.2.2.3 Pharmacokinetic parameters of multiple doses medicine of benazeprilat and there's differences between multiple dose group and single middle-dose group
The main pharmacokinetic parameters found for benazeprilat at dose of 20 mg (2 capsules each time per day for 14 days of oral administration) were as follows:tmax was 1.6±0.4 h,Cmax was 401.58±76.09 ng·mL-1,AUC0～24 was 2338.9±326.3 ng·h·mL-1,AUC0-∞was 2412.3±329.3 ng·h·mL-1;t1/2 was 5.1±0.5 h;CL/F was 8.7±1.3 L/h;V/F was 64.4±13.8 L;Cmaxsswas 401.58±76.09 ng·mL-1;Cminss was 9.35±1.90 ng·mL-1;Cav was 97.45±13.59 ng·mL-1;DF was 4.0±0.5.
The pharmacokinetic parameters of Tmax、t1/2、V/F of benazeprilat of single middle-dose group and multiple doses were close, the differences had no statistics significance(P>0.05).The pharmacokinetic parameters of CL/F、max of benazeprilat of multiple dose differ with single middle-dose group, the differences had statistics significance(P<0.05).
The pharmacokinetic parameters of AUC0～24ss(2338.9±326.3 ng·h·mL-1) of benazeprilat of multiple doses differ with the single middle-dose group of AUC0～∞(1989.8±320.9 ng·h·mL-1).The differences had statistics significance(P<0.05),which meaned that after multiple-dose medicine of amlodipine benazepril capsules,a slight accumulation of benazeprilat happened. 3.3 Assessment of the safety profiles of amlodipine benazepril capsule
During the single doses phase,no ADRs happened in Chinese healthy volunteers.Physical and laboratory examination results after single doses clinical trail finished were all normal.
During the multiple dose phase,ADRs happened in some volunteers. Physical and laboratory examination results after multiple dose medicine clinical trail finished were all normal. 4 CONCLUSIONS 4.1 Methods
The method was accurate and fast for determination of amlodipine,benazepril and it's metabolites benazeprilat in human plasma. It can be used for studying the pharmacokinetics of amlodipine,benazepril and it's metabolite benazeprilat of single doses medicine and steady state pharmacokinetics of multiple dose medicine. 4.2 Pharmacokinetics
After oral administration of single-dose of amlodipine benazepril capsules,amlodipine and benazepril showed partly linear pharmacokinetic characteristics, benazeprilat showed good linear pharmacokinetic characteristics; after oral administration of multiple-dose of amlodipine benazepril capsules, amlodipine had an obvious accumulation,benazepril had no accumulation, benazeprilat had a slight accumulation. 4.3 Safety profiles
After oral administration of amlodipine benazepril capsules in Chinese healthy volunteers,tolerance and safety profiles were well proved.
引文
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2. Faulkner JK, McGibney D, Chasseaud LF, et al.The pharmacokinetics of amlodipine in healthy volunteers after single intravenous and oral doses and after 14 repeated oral doses given once daily. Br J Clin Pharmacol.1986;22(1):21-5.
3. Kaiser G, Ackermann R, Brechbuhler S, et al.Pharmacokinetics of the angiotensin converting enzyme inhibitor benazepril.HCl(CGS14824 A) in healthy volunteers after single and repeated administration. Biopharm Drug Dispos.1989; 10(4): 365-76.
4. Kaiser G, Ackermann R, Sioufi A, et al.Pharmacokinetics of a new ngiotensin-converting enzyme inhibitor, benazepril hydrochloride, in special populations. Am Heart J.1989; 117(3):746-51.
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2. Faulkner JK, McGibney D, Chasseaud LF, et al.The pharmacokinetics of amlodipine in healthy volunteers after single intravenous and oral doses and after 14 repeated oral doses given once daily. Br J Clin Pharmacol.1986;22(1):21-5.
3. Kaiser G, Ackermann R, Brechbuhler S, et al.Pharmacokinetics of the angiotensin converting enzyme inhibitor benazepril.HCl(CGS14824 A) in healthy volunteers after single and repeated administration. Biopharm Drug Dispos.1989; 10(4): 365-76.
4. Kaiser G, Ackermann R, Sioufi A, et al.Pharmacokinetics of a new ngiotensin-converting enzyme inhibitor, benazepril hydrochloride, in special populations. Am Heart J.1989; 117(3):746-51.
5. Sun JX, Cipriano A, Chan K, et al.Pharmacokinetic interaction study between benazepril and amlodipine in healthy subjects. Eur J Clin Pharmacol. 1994;47:285-289.
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