复方盐酸二甲双胍人体药代动力学及生物等效性研究
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摘要
建立人血浆中二甲双胍和格列本脲浓度的测定方法。用所建立的方法研究复方盐酸二甲双胍人体药代动力学，并评价制剂间的生物等效性。
一 血浆中二甲双胍浓度测定方法
采用反相离子对色谱-紫外法，测定人血浆中二甲双胍的浓度。以3mmol.L~(-1)十二烷基磺酸钠(含5％的三乙胺，用磷酸调pH至4.0)：乙腈(65:35，v／v)为流动相，检测波长为232nm；血浆经乙腈沉淀蛋白后上样。血浆中二甲双胍的线性范围：0.03～1.50μg.mL~(-1)，相关系数0.9998。方法回收率在80～120％之间，变异系数为2.59～14.14％。该方法灵敏、准确、简便，适合盐酸二甲双胍药代动力学的研究。
二 血浆中格列本脲浓度测定的HPLC-MS法
血浆经固相萃取后，以乙腈：0.2％甲酸(60:40，v／v)为流动相，采用Waters Xterra MS C_(18)柱(150mm×2.1mm，3.5μm)进行分离。质谱检测模式为选择离子监测(SIM)，以碎片离子m／z 369强度定量，外标法定量，血浆中格列本脲在2.5～160.0ng.mL~(-1)浓度范围内线性关系良好(γ=0.9997)，最低检测限为0.5ng.mL~(-1)，本方法灵敏、专属、重现性好，可满足格列本脲药物代谢动力学的研究。
三 复方盐酸二甲双胍药代动力学研究
18名男性健康志愿者，交叉单剂量口服复方盐酸二甲双胍片、复方盐酸二甲双胍胶囊，或盐酸二甲双胍片联用格列本脲片。用上述建立的方法测定受试者血浆中的二甲双胍和格列本脲的浓度。结果表明，三种制剂在人体内具有相似的药动学过程。试验片剂、试验胶囊和参比制剂中格列本脲主要药动学参数t_(max)(2.50±0.69)h，(2.89±0.83)h，(2.67±0.75)h，C_(max)分别为(75.05±19.67)ng.mL~(-1)，(69.85±17.89)ng.mL~(-1)，(68.96±18.36)ng.mL~(-1)，t_(1/2)分别为(2.73±0.83)h，(3.21±1.20)h，(3.36+1.33)h；AUC_(o→tn)分别为(300.54±86.47)h.ng.mL~(-1)，(280.93±84.26)h.ng.mL~(-1)，(297.50±93.50)
h.ng.毗一’;Aue。一分别为(305.02士90.10)h.ng.mL一‘，(290.15士57.41)
h.ng .mL一’，(3 n .40土1 01 .65)h，ng .rnL一，。相应地，二甲双肌的主要药
代动力学参数分别为tm。;(1.53士0.93)h，(l，94士0.76)h，(1.77
士0.85)h，Cma二分别为(0.89士0.22)p g.mL一‘，(0.82士0.14)。g.mL一‘，
(0名9士0.22)协g.mL一’，tl/2分别为(6.25士2.19)h，(6.05士2.63)
h，(5.55土2.65)h;^ue。一t。分别为(5.66士1.31)h.p 9.11止一，，(5.40
士1.20)h.o g.mL一，，(5.30士1.10)h.p 9.毗一‘;^ue。一分别为(6.17
士1 .77)h.0 9.毗一l，(5.59士1.73)h.p g.rnL一l，(5.75土1.63)h.o g.mL一l。
三种制剂中两种药物成分的tma二，Cma:，AuCO一tn，AUC。一均无显著性
差异(p>0.05)，但两种药物均表现出较大的个体差异(p<0.01)。
四生物停效性研究
相对生物利用度以AUCO_t。计算，试验片剂和试验胶囊中格列本
脉的相对生物利用度分别为(102.36士13.48)%，(95.31土12.09)%;
相应地，二甲双肌的相对生物利用度分别为(107 .55士16.18)%，
(102.90土16.69)%。经等效性检验，结果表明:两种试验制剂(复方
盐酸二甲双肌片，复方盐酸二甲双肌胶囊)与这两种药物单方的联合
使用时，在健康人体内生物等效。
This study introduced analytical methods to measure plasma concentration of metformin and glibenclamide, and put the methods into application for the pharmacokinetic research of compound metformin in healthy volunteers, meanwhile, the bioequivalence between formulations were investigated.
1. HPLC-UV method for measurement metformin concentration in plasma
A reversed phase ion-pair HPLC-UV method was used to analyze metformin concentration. The mobile phase was composed of 3mmol.L-1 SDS ( containing 5% triethylamine, adjusted pH to 4.0 with phosphate acid) :acetonitrile (65: 35, v/v), the detection wavelength was set 232nm. The plasma sample was precipitated protein before injection into chromatography. The plasma drug concentration kept a good linear relationship with response in the range of 0.03 - 1.50@g.mL-1.The recovery was 80 - 120%, and the variance coefficient was 2.59~ 14.14%. The method was proved rapid, sensitive and accurate enough for metformin pharmacokinetic research.
2. HPLC-MS for determination glibenclamide concentration in plasma
A highly sensitive, specific and reproducible high performance liquid chromatography combined with mass spectrometry has been developed for the quantitative analysis of glibenclamide hi plasma. Plasma was processed with liquid-solid extraction. The mobile phase consists of acetonitrile: 0.2% formic acid (60:40,v/v) with a flow rate of 0.2mL.min-1. The separation procedure was performed on chromatographic column of Waters Xterra MS C18 ( 150mmX 2.1mm, 3.5@m) . A Waters high performance liquid chromatography joint with mass spectrometer equipped with electrospray ionization source was used for glibenclamide concentration determination. Selected ion monitoring (SIM) using fragment ion m/z 369 was applied to quantify glibenclamide. The linear for glibenclamide was 2.5-160ng,mL-1, (γ =0.9997), and the LOD was 0.5ng.mL-1. It was suitable
for glibenclamide pharmacokinetic research.
3. Pharmacokinetic study of compound metformin
18 male healthy volunteers were given an orally single dose of test formulation or reference formulation. And the described methods were employed to measure plasma concentration of metformin and glibenclamide, respectively. Three formulations had similar pharmacokinetic characteristics. As to glibenclamide (tablet, capsule, reference): tmax (2.50±0.69)h, (2.89±0.83)h, and (2.67 ± 0.75)h; Cmax (75.05 ±19.67)ng.mL-1, (69.85± 17.89)ng.mL-1,and (68.96 ±18.36)ng.mL-1; t1/2 (2.73±0.83)h, (3.21 ± 1.20)h, and (3.36±1.33)h; AUC0-tn (300.54± 86.47)h.ng.mL-1, (280.93 ± 84.26)h.ng.mL-1, and (297.50±93.50)h.ng.mL-1; AUC0-~ (308.02 ± 90. 10)h.ng.mL-1, (290.18 ±
87.41)h.ng.mL-1, and(311.40±101.65)h.ng.mL-1, respectively. As to metformin (tablet, capsule, reference): tmax (1.83±0.93)h, (1.94±0.76)h, and (1.77 ± 0.85)h; Cmax (0.89 ± 0.22) μg.mL-1 , (0.82 ± 0.14) μ
g.mL-1, and (0.89±0.22)μ g.mL-1; t1/2 (6.25±2.19)h, (6.05±2.63)h, and (5.88±2.68)h; AUC0-tn (5.66± 1.31)h.μg.mL-1, (5.40±1.20)h.μg-mL-1, and (5.30±1.10)h.μg-mL-1; AUC0-∞ (6.17± 1.77)h.P g.mL-1, (5.89± 1.73)h. μ g-mL-1 and (5.75±1.63)h.μg.mL-1; respectively. The main pharmacokinetic parameters such as tmax, Cmaxm, AUCo-tn, AUC0-∞ had no significant differences in three formulations(p>0.05), and the same condition occurred on metformin. However, there had significantly statistic differences between individuals on both glibenclamide and metformin (p<0.01). 4. Bioequivalent analyses
Calculated by AUCo-tn the relative bioavailability of tablet and capsule were
( 102.36± 13.48) %, (95.31±12.09)% for glibenclamide, and (107.55
±16.18) %, (102.90±16.69) % for metformin, respectively. The results
ultimately showed the two test preparations were both bioequivalent with reference
preparation based on two one-sided t tests and 90% confidence interval.
一 血浆中二甲双胍浓度测定方法
采用反相离子对色谱-紫外法，测定人血浆中二甲双胍的浓度。以3mmol.L~(-1)十二烷基磺酸钠(含5％的三乙胺，用磷酸调pH至4.0)：乙腈(65:35，v／v)为流动相，检测波长为232nm；血浆经乙腈沉淀蛋白后上样。血浆中二甲双胍的线性范围：0.03～1.50μg.mL~(-1)，相关系数0.9998。方法回收率在80～120％之间，变异系数为2.59～14.14％。该方法灵敏、准确、简便，适合盐酸二甲双胍药代动力学的研究。
二 血浆中格列本脲浓度测定的HPLC-MS法
血浆经固相萃取后，以乙腈：0.2％甲酸(60:40，v／v)为流动相，采用Waters Xterra MS C_(18)柱(150mm×2.1mm，3.5μm)进行分离。质谱检测模式为选择离子监测(SIM)，以碎片离子m／z 369强度定量，外标法定量，血浆中格列本脲在2.5～160.0ng.mL~(-1)浓度范围内线性关系良好(γ=0.9997)，最低检测限为0.5ng.mL~(-1)，本方法灵敏、专属、重现性好，可满足格列本脲药物代谢动力学的研究。
三 复方盐酸二甲双胍药代动力学研究
18名男性健康志愿者，交叉单剂量口服复方盐酸二甲双胍片、复方盐酸二甲双胍胶囊，或盐酸二甲双胍片联用格列本脲片。用上述建立的方法测定受试者血浆中的二甲双胍和格列本脲的浓度。结果表明，三种制剂在人体内具有相似的药动学过程。试验片剂、试验胶囊和参比制剂中格列本脲主要药动学参数t_(max)(2.50±0.69)h，(2.89±0.83)h，(2.67±0.75)h，C_(max)分别为(75.05±19.67)ng.mL~(-1)，(69.85±17.89)ng.mL~(-1)，(68.96±18.36)ng.mL~(-1)，t_(1/2)分别为(2.73±0.83)h，(3.21±1.20)h，(3.36+1.33)h；AUC_(o→tn)分别为(300.54±86.47)h.ng.mL~(-1)，(280.93±84.26)h.ng.mL~(-1)，(297.50±93.50)
h.ng.毗一’;Aue。一分别为(305.02士90.10)h.ng.mL一‘，(290.15士57.41)
h.ng .mL一’，(3 n .40土1 01 .65)h，ng .rnL一，。相应地，二甲双肌的主要药
代动力学参数分别为tm。;(1.53士0.93)h，(l，94士0.76)h，(1.77
士0.85)h，Cma二分别为(0.89士0.22)p g.mL一‘，(0.82士0.14)。g.mL一‘，
(0名9士0.22)协g.mL一’，tl/2分别为(6.25士2.19)h，(6.05士2.63)
h，(5.55土2.65)h;^ue。一t。分别为(5.66士1.31)h.p 9.11止一，，(5.40
士1.20)h.o g.mL一，，(5.30士1.10)h.p 9.毗一‘;^ue。一分别为(6.17
士1 .77)h.0 9.毗一l，(5.59士1.73)h.p g.rnL一l，(5.75土1.63)h.o g.mL一l。
三种制剂中两种药物成分的tma二，Cma:，AuCO一tn，AUC。一均无显著性
差异(p>0.05)，但两种药物均表现出较大的个体差异(p<0.01)。
四生物停效性研究
相对生物利用度以AUCO_t。计算，试验片剂和试验胶囊中格列本
脉的相对生物利用度分别为(102.36士13.48)%，(95.31土12.09)%;
相应地，二甲双肌的相对生物利用度分别为(107 .55士16.18)%，
(102.90土16.69)%。经等效性检验，结果表明:两种试验制剂(复方
盐酸二甲双肌片，复方盐酸二甲双肌胶囊)与这两种药物单方的联合
使用时，在健康人体内生物等效。
This study introduced analytical methods to measure plasma concentration of metformin and glibenclamide, and put the methods into application for the pharmacokinetic research of compound metformin in healthy volunteers, meanwhile, the bioequivalence between formulations were investigated.
1. HPLC-UV method for measurement metformin concentration in plasma
A reversed phase ion-pair HPLC-UV method was used to analyze metformin concentration. The mobile phase was composed of 3mmol.L-1 SDS ( containing 5% triethylamine, adjusted pH to 4.0 with phosphate acid) :acetonitrile (65: 35, v/v), the detection wavelength was set 232nm. The plasma sample was precipitated protein before injection into chromatography. The plasma drug concentration kept a good linear relationship with response in the range of 0.03 - 1.50@g.mL-1.The recovery was 80 - 120%, and the variance coefficient was 2.59~ 14.14%. The method was proved rapid, sensitive and accurate enough for metformin pharmacokinetic research.
2. HPLC-MS for determination glibenclamide concentration in plasma
A highly sensitive, specific and reproducible high performance liquid chromatography combined with mass spectrometry has been developed for the quantitative analysis of glibenclamide hi plasma. Plasma was processed with liquid-solid extraction. The mobile phase consists of acetonitrile: 0.2% formic acid (60:40,v/v) with a flow rate of 0.2mL.min-1. The separation procedure was performed on chromatographic column of Waters Xterra MS C18 ( 150mmX 2.1mm, 3.5@m) . A Waters high performance liquid chromatography joint with mass spectrometer equipped with electrospray ionization source was used for glibenclamide concentration determination. Selected ion monitoring (SIM) using fragment ion m/z 369 was applied to quantify glibenclamide. The linear for glibenclamide was 2.5-160ng,mL-1, (γ =0.9997), and the LOD was 0.5ng.mL-1. It was suitable
for glibenclamide pharmacokinetic research.
3. Pharmacokinetic study of compound metformin
18 male healthy volunteers were given an orally single dose of test formulation or reference formulation. And the described methods were employed to measure plasma concentration of metformin and glibenclamide, respectively. Three formulations had similar pharmacokinetic characteristics. As to glibenclamide (tablet, capsule, reference): tmax (2.50±0.69)h, (2.89±0.83)h, and (2.67 ± 0.75)h; Cmax (75.05 ±19.67)ng.mL-1, (69.85± 17.89)ng.mL-1,and (68.96 ±18.36)ng.mL-1; t1/2 (2.73±0.83)h, (3.21 ± 1.20)h, and (3.36±1.33)h; AUC0-tn (300.54± 86.47)h.ng.mL-1, (280.93 ± 84.26)h.ng.mL-1, and (297.50±93.50)h.ng.mL-1; AUC0-~ (308.02 ± 90. 10)h.ng.mL-1, (290.18 ±
87.41)h.ng.mL-1, and(311.40±101.65)h.ng.mL-1, respectively. As to metformin (tablet, capsule, reference): tmax (1.83±0.93)h, (1.94±0.76)h, and (1.77 ± 0.85)h; Cmax (0.89 ± 0.22) μg.mL-1 , (0.82 ± 0.14) μ
g.mL-1, and (0.89±0.22)μ g.mL-1; t1/2 (6.25±2.19)h, (6.05±2.63)h, and (5.88±2.68)h; AUC0-tn (5.66± 1.31)h.μg.mL-1, (5.40±1.20)h.μg-mL-1, and (5.30±1.10)h.μg-mL-1; AUC0-∞ (6.17± 1.77)h.P g.mL-1, (5.89± 1.73)h. μ g-mL-1 and (5.75±1.63)h.μg.mL-1; respectively. The main pharmacokinetic parameters such as tmax, Cmaxm, AUCo-tn, AUC0-∞ had no significant differences in three formulations(p>0.05), and the same condition occurred on metformin. However, there had significantly statistic differences between individuals on both glibenclamide and metformin (p<0.01). 4. Bioequivalent analyses
Calculated by AUCo-tn the relative bioavailability of tablet and capsule were
( 102.36± 13.48) %, (95.31±12.09)% for glibenclamide, and (107.55
±16.18) %, (102.90±16.69) % for metformin, respectively. The results
ultimately showed the two test preparations were both bioequivalent with reference
preparation based on two one-sided t tests and 90% confidence interval.
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[52] 李焱，钟光恕，程桦等．重新评估格列本脲(优降糖)最大剂量的初步研究．中华内分泌代谢杂志，1997(13)：30-33．
[53] Naji Najib, Nasir Idkaidek, M. Beshtawi, et al. Bioequivalence Evaluation of Two Brands of Metformin 500mg Tablets (Dialon & Glucophage)-in Healthy Human Volunteers. Biopharmaeeutics & Drug Disposition, 2002(23):301-306.
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[55] Enose Appavoo Arno, Prithiviraj Anand, Kesavan Bhaskar, et al. Eudragit NE30D Based Metformin/Glielazide Extended Release Tablets: Formulation, Characterisation and in Vitro Release Studies. Chemical Pharmaceutical Bulletin, 2002(50): 1495-1498.
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