Study Of The Pharmacokinetics Of Amlodipine Besilate Plus Benazepril Hydrochloride Capsule In Chinese Healthy Volunteers

1 OBJECTIVESTo 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 METHODS2.1 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 plasmaPlasma 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 plasmaPlasma 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 medicineIn 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 medicineEmploying 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 plasmaAmlodipine 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 plasmaBenazepril 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 groupsAfter 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 groupsAfter 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 groupsAfter 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 groupThe 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 groupThe 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 capsuleDuring 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 PharmacokineticsAfter 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 profilesAfter oral administration of amlodipine benazepril capsules in Chinese healthy volunteers,tolerance and safety profiles were well proved.