布洛伪麻分散片生物等效性研究及布洛芬在原代培养大鼠肝细胞中代谢的研究
|
摘要
目的：研究布洛伪麻分散片在健康人体内的生物等效性和布洛芬在大鼠原代培养正常和损伤肝细胞中的代谢情况。
方法：1．将22名健康男性志愿受试者，随机分为2组，分别于早晨一次空腹口服供试品(布洛伪麻分散片)或对照品(布洛伪麻片)4片(每片含布洛芬200mg和盐酸伪麻黄碱30mg)。建立高效液相色谱法，分别以萘普生、对硝基苯胺为内标，测定布洛芬和盐酸伪麻黄碱的血药浓度。药代动力学参数用3P97程序计算。2．建立大鼠肝细胞的分离及原代培养方法，初步研究了正常、损害后肝细胞对布洛芬的代谢过程，采用两步灌流技术并运用胶原酶消化法，分离肝细胞以正常、受损肝细胞分别与2.5μg／ml、5μg／ml、20μg／ml布洛芬进行悬浮共培养，实验分为正常组、CCl_4损伤组、H_2O_2损伤组三组，在不同时间点取出样本1.0ml，应用高效液相色谱法测定布洛芬的浓度。
结果：1供试品和对照品的布洛芬、盐酸伪麻黄碱均符合口服一级吸收一房室开放模型。主要参数：供试品和对照品AUC_((0-inf))(梯型法)、AUC_((0-t))(梯型法)、Cp(实测)、t_(1／2)(实测)，无显著性差异(P＞0.05)。药物动力学参数[AUC、Cp(实测值)]经对数转换后进行方差分析，并采用双单侧t检验进行生物等效性评价。统计分析结果表明两种制剂体内作用生物作用等效。2．CCl_4、H_2O_2损伤大鼠肝细胞与正常的肝细胞对2.5μg／ml、5μg／ml、20μg／ml布洛芬代谢的主要药动学参数有明显的差异，药物浓度越低，消除就越快，浓度高消除慢。
结论：1．22名健康男性志愿受试者口服供试品或对照品，以高效液相法测定布洛芬和盐酸伪麻黄碱血药浓度，经统计分析结果表明两种制剂体内作用生物等效
2．CCL_4或H_2O_2损伤与正常组比较药动学参数都有明显的改变，t_(1／2)延长，AUC增大，消除减慢。
Objective: To study the Bioequivalence of Ibuprofen and pseudoephedrine
dispersible tablets and Ibuprofen Metabolism in Primarily Cultured Rat
Hepatocytes.
Methods: l.In morning ,four tablets of test product (Ibuprofen and pseudoephedrine dispersible tablets, each tablet contains Ibuprofen 200mg pseudoephedrine hydrochloride 3Omg) or controlled product (Ibuprofen and pseudoephedrine tablets ) were given to 22 fasted healthy volunteers in a randomized study. Ibuprofen and pseudoephedrine hydrochloride concentration in plasma were determined by HPLC method and naproxen and para-nitroaniline were used as internal standards respectively. The pharmacokinetic parameters of Ibuprofen and pseudoephedrine hydrochloride were calculated by 3P97. 2.The metabolism of Ibuprofen in isolated rat hepatocytes was preliminary investigated. Hepatocytes were prepared from rats using two-step perfusion and collagenase digestion procedure. 2.5 u g/mK 5 u g/mK 20 U g/ml concentrations of ibuprofen were incubated with normal and injured hepatocytes.The tests were divided into normal hepatocytes group, CCU hepatocytes injured group and H2O2 hepatocytes injured group. 1ml Aliquots was wi
thdrawn from the incubation mixture at different time points of incubation. The concentrations of Ibuprofen in hepatocyte suspension were determined by HPLC method.
Results: 1 .On the plasma concentration-time curves of test product and controlled product , Ibuprofen and pseudoephedrine hydrochloride were fitted to the one compartment opened model with first-order absorption.The main pharmacokinetic parameters such as AUC(O-inf), AUC(O-t), Cp, Ti/2 had no significence (P>0.05) . The data of AUC and CP were log-transformed before anlalysis of variance , two sides t-test
justified bioequivalence.The statistical results showed two products had bioequivalence in vivo. 2.Compared with that of normal hepatocytes group, the main pharmacokinetic parameters of CCU hepatocytes injured group and H2O2 hepatocytes injured group which metalolized 2.5 ug/ml > 5 u g/ml > 20 u g/ml concentrations of Ibuprofen had signifence.At the same cell concentration, the lower the concentration of Ibuprofen, the fester the elimination is, and vice versa
Conclusion: 1. Affer 22 healthy volunteers orally taken test product and controlled product, the concentrations of Ibuprofen and pseudoephedrine hydrochloride were determined by HPLC method, the statistical results shows the test product is bioequivalent to the controlled product in vivo. 2. Pharmacokinetic parameters of injured groups have obvious changes, ti/2 delayed, AUC amplified, elimination decreased.
方法：1．将22名健康男性志愿受试者，随机分为2组，分别于早晨一次空腹口服供试品(布洛伪麻分散片)或对照品(布洛伪麻片)4片(每片含布洛芬200mg和盐酸伪麻黄碱30mg)。建立高效液相色谱法，分别以萘普生、对硝基苯胺为内标，测定布洛芬和盐酸伪麻黄碱的血药浓度。药代动力学参数用3P97程序计算。2．建立大鼠肝细胞的分离及原代培养方法，初步研究了正常、损害后肝细胞对布洛芬的代谢过程，采用两步灌流技术并运用胶原酶消化法，分离肝细胞以正常、受损肝细胞分别与2.5μg／ml、5μg／ml、20μg／ml布洛芬进行悬浮共培养，实验分为正常组、CCl_4损伤组、H_2O_2损伤组三组，在不同时间点取出样本1.0ml，应用高效液相色谱法测定布洛芬的浓度。
结果：1供试品和对照品的布洛芬、盐酸伪麻黄碱均符合口服一级吸收一房室开放模型。主要参数：供试品和对照品AUC_((0-inf))(梯型法)、AUC_((0-t))(梯型法)、Cp(实测)、t_(1／2)(实测)，无显著性差异(P＞0.05)。药物动力学参数[AUC、Cp(实测值)]经对数转换后进行方差分析，并采用双单侧t检验进行生物等效性评价。统计分析结果表明两种制剂体内作用生物作用等效。2．CCl_4、H_2O_2损伤大鼠肝细胞与正常的肝细胞对2.5μg／ml、5μg／ml、20μg／ml布洛芬代谢的主要药动学参数有明显的差异，药物浓度越低，消除就越快，浓度高消除慢。
结论：1．22名健康男性志愿受试者口服供试品或对照品，以高效液相法测定布洛芬和盐酸伪麻黄碱血药浓度，经统计分析结果表明两种制剂体内作用生物等效
2．CCL_4或H_2O_2损伤与正常组比较药动学参数都有明显的改变，t_(1／2)延长，AUC增大，消除减慢。
Objective: To study the Bioequivalence of Ibuprofen and pseudoephedrine
dispersible tablets and Ibuprofen Metabolism in Primarily Cultured Rat
Hepatocytes.
Methods: l.In morning ,four tablets of test product (Ibuprofen and pseudoephedrine dispersible tablets, each tablet contains Ibuprofen 200mg pseudoephedrine hydrochloride 3Omg) or controlled product (Ibuprofen and pseudoephedrine tablets ) were given to 22 fasted healthy volunteers in a randomized study. Ibuprofen and pseudoephedrine hydrochloride concentration in plasma were determined by HPLC method and naproxen and para-nitroaniline were used as internal standards respectively. The pharmacokinetic parameters of Ibuprofen and pseudoephedrine hydrochloride were calculated by 3P97. 2.The metabolism of Ibuprofen in isolated rat hepatocytes was preliminary investigated. Hepatocytes were prepared from rats using two-step perfusion and collagenase digestion procedure. 2.5 u g/mK 5 u g/mK 20 U g/ml concentrations of ibuprofen were incubated with normal and injured hepatocytes.The tests were divided into normal hepatocytes group, CCU hepatocytes injured group and H2O2 hepatocytes injured group. 1ml Aliquots was wi
thdrawn from the incubation mixture at different time points of incubation. The concentrations of Ibuprofen in hepatocyte suspension were determined by HPLC method.
Results: 1 .On the plasma concentration-time curves of test product and controlled product , Ibuprofen and pseudoephedrine hydrochloride were fitted to the one compartment opened model with first-order absorption.The main pharmacokinetic parameters such as AUC(O-inf), AUC(O-t), Cp, Ti/2 had no significence (P>0.05) . The data of AUC and CP were log-transformed before anlalysis of variance , two sides t-test
justified bioequivalence.The statistical results showed two products had bioequivalence in vivo. 2.Compared with that of normal hepatocytes group, the main pharmacokinetic parameters of CCU hepatocytes injured group and H2O2 hepatocytes injured group which metalolized 2.5 ug/ml > 5 u g/ml > 20 u g/ml concentrations of Ibuprofen had signifence.At the same cell concentration, the lower the concentration of Ibuprofen, the fester the elimination is, and vice versa
Conclusion: 1. Affer 22 healthy volunteers orally taken test product and controlled product, the concentrations of Ibuprofen and pseudoephedrine hydrochloride were determined by HPLC method, the statistical results shows the test product is bioequivalent to the controlled product in vivo. 2. Pharmacokinetic parameters of injured groups have obvious changes, ti/2 delayed, AUC amplified, elimination decreased.
引文
1．蒋曙光、屠锡德等．复方布洛芬缓释片研究[J]．中国药科大学学报，1998；29，422-426
2．黄胜炎．分散片进展[J]．中国药学杂志．1998；27(4)：226-228
3．潘永成 张寿熙等．布洛芬对创伤大鼠淋巴细胞内环核苷酸水平白细胞介素-2生成及NK细胞活性的影响[J]．中华创伤杂志．1996；12(5)：347-349
4. Allan M. Pharmacodynamics and Pharmacokinetics of the Profens: Enantioselectivity, Clinical Implications, and Special Refer ence to S(+) -Ibuprofen[J]. J Clin Pharmacl 1996; 36: 7s-15s
5. Joachim M. Ibuprofen Enantionmers and Lipid Metabolism[j]. Clin Pharmacl 1996; 36: 27s-32s
6．刘海风、郑一美·HPLC法测定布洛芬伪麻黄碱的布洛芬和盐酸麻黄碱含量[J]·．中国现代应用药学，1997，8(4)：38-39
7. Shao G, Goto J, Nambara T. Separation and determination of propranolol enantiomers in plasma by high-performance liqid chromatography with fluorescence detection, J liquid chromatogr, 1991, 14: 453
8．顾瑾、屠锡德等．复方盐酸伪麻黄双层缓释片研究[J]．中国药科大学学报，1996；27(12)：711-717．
9. Donato G M, Baeyens W, Van Den Boassche W et al. J pharm BIOMED Anal, 1994, 12: 21-26
10．陈恒武，黄隶华等[J]．毛细管电泳法快速测定复方布洛芬片的有效成分．色谱．1998；16(4)：289-292
11. G Shao、F WU DS Wang et al. Quantitatie analysis of (1)-EPHEDRINE and (d)-PSEUDOEPHEDRINE in plasma by high-performance liqid chromatography with fluorescence detection[J]. Acta Pharmaceutica Sininca 1995: 30(5): 384-389
12．金晓、崔凯荣．用(+)-FLEC手性试剂自动柱前衍生高效液相色谱法拆分麻黄碱类药物对映体[J]．药学学报．1994：29(2)：122-127
13．刘雁鸣，廖建容等．柱前荧光试剂衍生化RP-HPLC测定布洛芬对映体[J]．中国药科大学学报．1999，30(2)：124-126
14．王润玲、董伟林等．布洛芬糖浆剂药动学及相对生物利用度[J]．中国药举杂志．1997：32(10)：601-603
15．钟明康、施孝金等．盐酸伪麻黄碱缓释片在健康志愿者中的药动学[J]．中国医院药学杂志．2001；21(7)：395-397
16．李娟 等．HPLC同时测定布洛芬伪麻颗粒剂中布洛芬利伪麻黄碱含量[J]．中国药学杂志[J]，2000，35(9)：623-624
17．张晔、毛磊．高效液相色谱法测定糖浆剂中扑尔敏与伪麻黄碱含量[J]．中国药科大学学报．1994；25(1)：29-30
18. KU Bubhring and A Garbe. Simple and sensitie high-performance liquid chromatographic determinaton of methamphentamine in human urine as derivatie of 4-(4,5-diphey1-1H-imidazol-2-yl) benzoyl chloride, a new fluorescence
derivatization reagent [J].Journal of chromatography 1998; 712(13): 105-112
19．美L．R斯奈德，J．J柯克兰著．现代液相色谱法理论．第二版．化学工业出版社，1988，P276-280
20．梁宏曦、于古瑕等．HPLC测定止咳痰丸中麻黄碱、伪麻黄碱的含量[J]．中成药．1991，26(12)：34-36
21．陈坚、方维军等，盐酸伪麻黄碱在人体中的药物动力学[J]．中国医院药学杂志．1997；17：295-296．
22. Debra Ga. ibuprofen: A Inonograph. Amerian Pharmacy. 1991: 31-48
23. American soctety of hospital pharmacists. Pseudoephedrine hydrochioride. AHFS Drug information. 1994: 799
24．曹纯洁、皱洁等．布洛芬分散片的制备及其在家兔体内的生物利用度研究．第二军医大学学报．2000，21(10)：969-970．
25．陈新梅、方晓玲等．国内布洛芬剂型研究进展[J]．中国医院药学杂志．1197，17(1)：37-38
26．吴琳华、赵红光等．多剂量口服复方布洛芬缓释片与普通片的药代动力学及相对生物利用度．中国临床药理杂志[J]．2001，17(20)：129-132．
27．裘雁秋、粱德荣等．布洛芬伪麻黄胶囊临床研究[J]．华西医学，1996，11：254-256．
28. Hwang SS. In vitro and in vivo evaluation of a once-daily controlled-released pseudoephedine product[J]. J Clin pharmacol. 1995, 35: 259.
29. Tribble DL, AW TY and Jones DP. The pathophysiological singnificance of lipidperoxidation in oxidative cell injury. Hepatology, 1987: 7(2): 377-387
30. Seglen PO. Preparation of isolated rat liver cells. Methods in Cell biology. 1976; 13(1): 29-83
31. lpini G, Phillips J O, Vorman B et al. Recent advance in the isolation of liver cells. Hepatology. 1994; 20: 494-498
32．姚云清，张定凤，黄爱龙等．低浓度胶原酶原位循环灌流法用于原代大鼠肝细胞分离．现代医药卫生[J]．2001：17(2)：84-85
33．姜金兰，吕文富，胡春光等．大鼠肝细胞的分离及原代长期培养[J]．．白求恩医科大学学报．2000；26(6)：562-564
34. Puviani AC, Ottolenghi C, Tassinari B, et al. An update on high yield hepatocyte isolation methods and on the potential clinical use of isolated liver cells[J].. Comp Biochem Physiol A Mol Integr Physiol. 1998; 121(2): 99-103
35．刘小丽，李兰娟，陈智，大鼠肝细胞的原代培养[J]．．浙江医学．2001：23(4)：209-213
36. RaKba N, Melhaoui A, Loyer P, et al. Bgugaine, a pyrrolidine alkaloid from Arisarum vulgare, is a strong hepatotoxin in rat and human liver cell cultures. Toxicol Lert, 1999; 104(3): 239
37．杨雁，陈敏珠．体外大鼠肝细胞坏死性性损伤模型的建立[J]．．安徽医科大学学报．1999；34(6)：467-471
38．朱文忠，刘树孝，于布为．阿屈库铵对离体原代培养大鼠肝细胞的毒性研究[J]．中华麻醉学杂志．1997：17(2)：90-92
39． 薛俊峰，李桦，阮金秀等．氮烯乙茶在成年大鼠肝细胞中的生物转化[J]．药学学报．1999； 34(10) ：739． 743
40． Mc Queen CA. Hepatocytes in monolayer culture:an in vitro model for toxicity studies. In: Mc Queen CA,ed. In Vitro Toxicology.Model Systems and Methods.Caldwell New Jersey: The Telford Press,1989:131-155
41． 赵冬梅，李燕．药物代谢研究在新药开发中的作用．药学学报．2000；35(2) ：156-160
42． Riordan S,Williams R. Bioartificial liver support:developments in hepatocyte culture and bioreactor design.Br Med Bul!.1997;53(4) :730-734
43． Liu ML,Mars WM,Zarnegar R,et al.Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-a in adult rat liver.Hepatology. 1994; 19(6) : 1521-1530
44． Tribble DL, AW TY and Jones DP. The pathophysiological singnificance of lipidpheroxideatinal in oxidative cell injury. Hepatology, 1987:7(2) :377-387
45． Aguilar-Delfin I, Lopez-Barrera F, Hernaandez-Munoz R. Selective enhancement of liplid peroxidation in plasma membrane in two experimental models of liver regeneration: partial hepatectomy and acute ccl< administration. Hepatology. 1996:24(3) :657-662
2．黄胜炎．分散片进展[J]．中国药学杂志．1998；27(4)：226-228
3．潘永成 张寿熙等．布洛芬对创伤大鼠淋巴细胞内环核苷酸水平白细胞介素-2生成及NK细胞活性的影响[J]．中华创伤杂志．1996；12(5)：347-349
4. Allan M. Pharmacodynamics and Pharmacokinetics of the Profens: Enantioselectivity, Clinical Implications, and Special Refer ence to S(+) -Ibuprofen[J]. J Clin Pharmacl 1996; 36: 7s-15s
5. Joachim M. Ibuprofen Enantionmers and Lipid Metabolism[j]. Clin Pharmacl 1996; 36: 27s-32s
6．刘海风、郑一美·HPLC法测定布洛芬伪麻黄碱的布洛芬和盐酸麻黄碱含量[J]·．中国现代应用药学，1997，8(4)：38-39
7. Shao G, Goto J, Nambara T. Separation and determination of propranolol enantiomers in plasma by high-performance liqid chromatography with fluorescence detection, J liquid chromatogr, 1991, 14: 453
8．顾瑾、屠锡德等．复方盐酸伪麻黄双层缓释片研究[J]．中国药科大学学报，1996；27(12)：711-717．
9. Donato G M, Baeyens W, Van Den Boassche W et al. J pharm BIOMED Anal, 1994, 12: 21-26
10．陈恒武，黄隶华等[J]．毛细管电泳法快速测定复方布洛芬片的有效成分．色谱．1998；16(4)：289-292
11. G Shao、F WU DS Wang et al. Quantitatie analysis of (1)-EPHEDRINE and (d)-PSEUDOEPHEDRINE in plasma by high-performance liqid chromatography with fluorescence detection[J]. Acta Pharmaceutica Sininca 1995: 30(5): 384-389
12．金晓、崔凯荣．用(+)-FLEC手性试剂自动柱前衍生高效液相色谱法拆分麻黄碱类药物对映体[J]．药学学报．1994：29(2)：122-127
13．刘雁鸣，廖建容等．柱前荧光试剂衍生化RP-HPLC测定布洛芬对映体[J]．中国药科大学学报．1999，30(2)：124-126
14．王润玲、董伟林等．布洛芬糖浆剂药动学及相对生物利用度[J]．中国药举杂志．1997：32(10)：601-603
15．钟明康、施孝金等．盐酸伪麻黄碱缓释片在健康志愿者中的药动学[J]．中国医院药学杂志．2001；21(7)：395-397
16．李娟 等．HPLC同时测定布洛芬伪麻颗粒剂中布洛芬利伪麻黄碱含量[J]．中国药学杂志[J]，2000，35(9)：623-624
17．张晔、毛磊．高效液相色谱法测定糖浆剂中扑尔敏与伪麻黄碱含量[J]．中国药科大学学报．1994；25(1)：29-30
18. KU Bubhring and A Garbe. Simple and sensitie high-performance liquid chromatographic determinaton of methamphentamine in human urine as derivatie of 4-(4,5-diphey1-1H-imidazol-2-yl) benzoyl chloride, a new fluorescence
derivatization reagent [J].Journal of chromatography 1998; 712(13): 105-112
19．美L．R斯奈德，J．J柯克兰著．现代液相色谱法理论．第二版．化学工业出版社，1988，P276-280
20．梁宏曦、于古瑕等．HPLC测定止咳痰丸中麻黄碱、伪麻黄碱的含量[J]．中成药．1991，26(12)：34-36
21．陈坚、方维军等，盐酸伪麻黄碱在人体中的药物动力学[J]．中国医院药学杂志．1997；17：295-296．
22. Debra Ga. ibuprofen: A Inonograph. Amerian Pharmacy. 1991: 31-48
23. American soctety of hospital pharmacists. Pseudoephedrine hydrochioride. AHFS Drug information. 1994: 799
24．曹纯洁、皱洁等．布洛芬分散片的制备及其在家兔体内的生物利用度研究．第二军医大学学报．2000，21(10)：969-970．
25．陈新梅、方晓玲等．国内布洛芬剂型研究进展[J]．中国医院药学杂志．1197，17(1)：37-38
26．吴琳华、赵红光等．多剂量口服复方布洛芬缓释片与普通片的药代动力学及相对生物利用度．中国临床药理杂志[J]．2001，17(20)：129-132．
27．裘雁秋、粱德荣等．布洛芬伪麻黄胶囊临床研究[J]．华西医学，1996，11：254-256．
28. Hwang SS. In vitro and in vivo evaluation of a once-daily controlled-released pseudoephedine product[J]. J Clin pharmacol. 1995, 35: 259.
29. Tribble DL, AW TY and Jones DP. The pathophysiological singnificance of lipidperoxidation in oxidative cell injury. Hepatology, 1987: 7(2): 377-387
30. Seglen PO. Preparation of isolated rat liver cells. Methods in Cell biology. 1976; 13(1): 29-83
31. lpini G, Phillips J O, Vorman B et al. Recent advance in the isolation of liver cells. Hepatology. 1994; 20: 494-498
32．姚云清，张定凤，黄爱龙等．低浓度胶原酶原位循环灌流法用于原代大鼠肝细胞分离．现代医药卫生[J]．2001：17(2)：84-85
33．姜金兰，吕文富，胡春光等．大鼠肝细胞的分离及原代长期培养[J]．．白求恩医科大学学报．2000；26(6)：562-564
34. Puviani AC, Ottolenghi C, Tassinari B, et al. An update on high yield hepatocyte isolation methods and on the potential clinical use of isolated liver cells[J].. Comp Biochem Physiol A Mol Integr Physiol. 1998; 121(2): 99-103
35．刘小丽，李兰娟，陈智，大鼠肝细胞的原代培养[J]．．浙江医学．2001：23(4)：209-213
36. RaKba N, Melhaoui A, Loyer P, et al. Bgugaine, a pyrrolidine alkaloid from Arisarum vulgare, is a strong hepatotoxin in rat and human liver cell cultures. Toxicol Lert, 1999; 104(3): 239
37．杨雁，陈敏珠．体外大鼠肝细胞坏死性性损伤模型的建立[J]．．安徽医科大学学报．1999；34(6)：467-471
38．朱文忠，刘树孝，于布为．阿屈库铵对离体原代培养大鼠肝细胞的毒性研究[J]．中华麻醉学杂志．1997：17(2)：90-92
39． 薛俊峰，李桦，阮金秀等．氮烯乙茶在成年大鼠肝细胞中的生物转化[J]．药学学报．1999； 34(10) ：739． 743
40． Mc Queen CA. Hepatocytes in monolayer culture:an in vitro model for toxicity studies. In: Mc Queen CA,ed. In Vitro Toxicology.Model Systems and Methods.Caldwell New Jersey: The Telford Press,1989:131-155
41． 赵冬梅，李燕．药物代谢研究在新药开发中的作用．药学学报．2000；35(2) ：156-160
42． Riordan S,Williams R. Bioartificial liver support:developments in hepatocyte culture and bioreactor design.Br Med Bul!.1997;53(4) :730-734
43． Liu ML,Mars WM,Zarnegar R,et al.Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-a in adult rat liver.Hepatology. 1994; 19(6) : 1521-1530
44． Tribble DL, AW TY and Jones DP. The pathophysiological singnificance of lipidpheroxideatinal in oxidative cell injury. Hepatology, 1987:7(2) :377-387
45． Aguilar-Delfin I, Lopez-Barrera F, Hernaandez-Munoz R. Selective enhancement of liplid peroxidation in plasma membrane in two experimental models of liver regeneration: partial hepatectomy and acute ccl< administration. Hepatology. 1996:24(3) :657-662