葛根素眼部传递系统的研究
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摘要
眼部药物传递系统作为控制释放领域的重要分支一直备受人们的关注。眼睛的有效保护机制与高度敏感性促使人们探索开发安全合理的给药系统，同时又限制了许多药剂学手段的应用，为此类剂型的设计带来了极大的困难。本文选择葛根素(puerarin)作为模型药物，采用亲水性高分子材料制备了眼用凝胶和眼用原位凝胶传递系统。本论文包括以下内容：
1．在论文的处方前研究工作中，本文分别建立了两种紫外分光光度法，用于葛根素基本理化性质的测定，并对葛根素的溶解度和油水分配系数进行了测定。同时绘制了葛根素在不同浓度羟丙基-β-环糊精的相溶解度图。
2．采用离体兔角膜透过实验，考察了多种渗透促进剂对PUE角膜透过性的影响，其中乙二胺四乙酸(EDTA)可显著增加PUE的角膜透过量，并且未见角膜毒性，同时，由于其又能与金属离子络合，可保护PUE。所以选择乙二胺四乙酸(EDTA)为PUE的渗透促进剂。
3．用溶出度桨法，考察了以多种高分子材料为基质制备的PUE凝胶剂的体外释放行为，其中以3％的HPMC K4M和F4M为基质制备的PUE凝胶剂的体外释放呈现缓释行为；采用离体兔角膜透过实验，考察以K4M和F4M为基质制备的凝胶剂中PUE的角膜透过行为，其中以K4M为基质制备的凝胶剂中PUE的表观渗透系数(P_(app))是对照组的1.35倍，说明作为眼用凝胶基质K4M优于F4M。
4．用搅拌子法，考察不同浓度的泊洛沙姆溶液的相转变温度。同时，测定了不同温度下，不同浓度泊洛沙姆溶液的粘度；采用桨法测定了以泊洛沙姆为基质的PUE眼用原位凝胶的体外释放行为，与PUE滴眼液相比体外释放呈现明显的缓释行为；采用离体兔角膜透过实验，考察以泊洛沙姆为凝胶基质的原位凝胶剂中PUE的角膜透过行为，各种渗透参数与PUE滴眼液对照组相比没有显著性差异(p＞0.05)。综合考虑，选择20％的泊洛沙姆407和10％的泊洛沙姆188作为PUE眼用原位凝胶的凝胶基质较为理想。
5．对葛根素眼用凝胶剂和眼用原位凝胶剂进行了局部刺激性和兔眼滞留时间实验。结果表明，葛根素眼用凝胶剂和眼用原位凝胶剂对兔眼均无刺激性。与葛根素滴眼液相比显著延长了药物在眼部的滞留时间(P＜0.01)。
As one of the important fields of controlled release, ophthalmic drug delivery has received extensive interests. The sensitivity and protection mechanisms of the eye promote more effective drug delivery systems with better compliancy to be developed, however, the applications of numerous pharmaceutical methods are limited simultaneously, which presents great challenge in designing ophthalmic dosage forms. In this thesis, Puerarin (PUE) was selected as a model drug. A new controlled-release PUE gel and a new thermosensitive gel PUE were developed using hydrophilic polymers. A series of relative studies were carried out. 1.In the preformation study, two kinds of UV spectrophotometer were developed for assay during the studies of physicochemical properties. And in this part the basic physicochemical properties of PUE were determined. Meanwhile, phase solubility diagrams of PUE/HP-β-CD system was obtained.
2. The permeability characteristics of PUE across the cornea of an isolated rabbit were studied in vitro using a variety of permeability enhancers. EDTA showed a significant improvement permeability without toxity to the cornea of an isolated rabbit and could protect PUE by reacting with metal ion. For the above two reasons EDTA was chosen as permeability enhancers of PUE.
3. Release behavior of PUE from gel based on various kinds of hydrogel were explored in a dissolution apparatus under stirring conditions. The results showed the PUE release behavior from gel based on 3%K4M and 3%F4M were delayed release. The permeability of PUE gel across isolated rabbit cornea was measured using in vitro method .The apparent permeability of PUE from gel contained 3%K4M showed a improvement as much as 1.35 times, compared with that of in the control(P<0.05) .It indicates that K4M was a better hydrogel base for PUE
1．在论文的处方前研究工作中，本文分别建立了两种紫外分光光度法，用于葛根素基本理化性质的测定，并对葛根素的溶解度和油水分配系数进行了测定。同时绘制了葛根素在不同浓度羟丙基-β-环糊精的相溶解度图。
2．采用离体兔角膜透过实验，考察了多种渗透促进剂对PUE角膜透过性的影响，其中乙二胺四乙酸(EDTA)可显著增加PUE的角膜透过量，并且未见角膜毒性，同时，由于其又能与金属离子络合，可保护PUE。所以选择乙二胺四乙酸(EDTA)为PUE的渗透促进剂。
3．用溶出度桨法，考察了以多种高分子材料为基质制备的PUE凝胶剂的体外释放行为，其中以3％的HPMC K4M和F4M为基质制备的PUE凝胶剂的体外释放呈现缓释行为；采用离体兔角膜透过实验，考察以K4M和F4M为基质制备的凝胶剂中PUE的角膜透过行为，其中以K4M为基质制备的凝胶剂中PUE的表观渗透系数(P_(app))是对照组的1.35倍，说明作为眼用凝胶基质K4M优于F4M。
4．用搅拌子法，考察不同浓度的泊洛沙姆溶液的相转变温度。同时，测定了不同温度下，不同浓度泊洛沙姆溶液的粘度；采用桨法测定了以泊洛沙姆为基质的PUE眼用原位凝胶的体外释放行为，与PUE滴眼液相比体外释放呈现明显的缓释行为；采用离体兔角膜透过实验，考察以泊洛沙姆为凝胶基质的原位凝胶剂中PUE的角膜透过行为，各种渗透参数与PUE滴眼液对照组相比没有显著性差异(p＞0.05)。综合考虑，选择20％的泊洛沙姆407和10％的泊洛沙姆188作为PUE眼用原位凝胶的凝胶基质较为理想。
5．对葛根素眼用凝胶剂和眼用原位凝胶剂进行了局部刺激性和兔眼滞留时间实验。结果表明，葛根素眼用凝胶剂和眼用原位凝胶剂对兔眼均无刺激性。与葛根素滴眼液相比显著延长了药物在眼部的滞留时间(P＜0.01)。
As one of the important fields of controlled release, ophthalmic drug delivery has received extensive interests. The sensitivity and protection mechanisms of the eye promote more effective drug delivery systems with better compliancy to be developed, however, the applications of numerous pharmaceutical methods are limited simultaneously, which presents great challenge in designing ophthalmic dosage forms. In this thesis, Puerarin (PUE) was selected as a model drug. A new controlled-release PUE gel and a new thermosensitive gel PUE were developed using hydrophilic polymers. A series of relative studies were carried out. 1.In the preformation study, two kinds of UV spectrophotometer were developed for assay during the studies of physicochemical properties. And in this part the basic physicochemical properties of PUE were determined. Meanwhile, phase solubility diagrams of PUE/HP-β-CD system was obtained.
2. The permeability characteristics of PUE across the cornea of an isolated rabbit were studied in vitro using a variety of permeability enhancers. EDTA showed a significant improvement permeability without toxity to the cornea of an isolated rabbit and could protect PUE by reacting with metal ion. For the above two reasons EDTA was chosen as permeability enhancers of PUE.
3. Release behavior of PUE from gel based on various kinds of hydrogel were explored in a dissolution apparatus under stirring conditions. The results showed the PUE release behavior from gel based on 3%K4M and 3%F4M were delayed release. The permeability of PUE gel across isolated rabbit cornea was measured using in vitro method .The apparent permeability of PUE from gel contained 3%K4M showed a improvement as much as 1.35 times, compared with that of in the control(P<0.05) .It indicates that K4M was a better hydrogel base for PUE
引文
[1] 梁文权．生物药剂学与药物动力学．第一版，北京：人民卫生出版社，2000，72～75．
[2] 陆彬．药物新剂型与新技术．第一版，北京：人民卫生出版社，1998，416～420．
[3] Lang JC. Ocular drug delivery conventional ocular formulations. Adv Drug Delivery Rev, 1995, 16:39-43.
[4] Zimmer A, Kreuter J. Microspheres and nanoparticles used in ocular delivery systems. Adv Drug Delivery Rev, 1995, 16: 61-73.
[5] Saettone MF, Salminen L. Ocular inserts for topical delivery. Adv Drug Delivery Rev, 1995, 16:95-106.
[6] Eun-Young Kim, Zhong-Gao Gao, Jeong-Sook Park .rhEGF/HP-β-CD complex in poloxamer gel for ophthalmic delivery. Inter J Pharm, 2002, 233:159-167.
[7] 周亮．青光眼的防治．中国乡村医药，1997，4，9：43～44．
[8] 王建萍．青光眼的预防及临床展望．中国初级卫生保健，2003，17，10：71～72．
[9] 张好琳，梁敬钰．葛根素的研究进展．海峡药学，2005，17，1：2～5．
[10] 吴正红，朱延勤，严汉英等．葛根素滴眼液的药效学研究．中国药理学通报，1998，14(6)，569～570．
[11] Ashim K.Mitra.Ophthalmic Drug Delivery Systems,New York,Marcel Dekker Inc,second edition,282～307
[12] Luc Van Santvliet, Annick Ludwig.The influence of penetration enhancers on the volume instilled of eye drops.Eur J Pharm Biopharm, 1998,45:189-198.
[13] M.Fabrizio Saettone,Patrizia Chetoni,Riccardo Cerbai etal.Evaluation of ocular permeation enhancers:in vitro effects on corneal transport of fourβ-blockers and invitro/in vivo toxic activity, Int J Pharma, 1996,142:103-113.
[1]．国家药典委员会，中华人民共和国药典一部，北京：化学工业出版社，2000，273。
[2] Helene HagerstrOm, Mattias Paulsson, Katarina Edsman. Evaluation of mucoadhesion for two polyelectrolyte gels in simulated physiological conditions using a rheological method. Eur J Pharma Sci.2000, 9: 301-309.
[3] 国家药典委员会，中华人民共和国药典二部，北京：化学工业出版社，2000，附录174。
[4]. Hong-Ru Lin,K.C Sung, Carbopol/pluronic phase change solutions for ophthalmic drug delivery. J.Con.Res. 2000, 69 : 379-388.
[5]．张朝佑主编．人体解剖学(下册，第二版)．北京：人民卫生出版社，1998，pp．1739～1767．
[6] Higuchi, T., Connors, K.A.,1965. Phase-solubility techniques. Adv. Anal Chem. Instr. 4,117-212.
[7] Higuchi, T., Kristiansen, H., 1970. Binding specificity between small organic in aqueous solution: Classification of small organic solutes into two groups according to binding tendencies. J. Pharm.Sci., 59, 1601-1608.
[1] Yoshida F, Topliss JG.Unified model for the corneal permeability of related and diverse compounds with respect to their physicochemical properties. J Pharm Sci, 1996, 85:819-823.
[2] Liaw J, Robinson JR. The effect of polyethylene glycol molecular weight on corneal transport and the related influence of penetration enhancers. Int J Pharm, 1992, 88: 125-140.
[3] Prausnitz MR, Noonan JS. Permeability of cornea, sclera, and conjunctiva: A literature analysis for drug delivery to the eye. J Pharm Sci, 1998, 87: 1479-1488.
[4] Mitra AK, Mikkelson TJ. Mechanism of transcorneal permeation of pilocarpine. J Pharm Sci, 1988, 77: 771-775.
[5] Sasaki H, Ichikawa M, Kawakami S, et al. In situ ocular absorption of tilisolol through ocular membranes in albino rabbits. J Pharm Sci, 1996, 85: 940-943.
[6] Suhunen P, Jarvinen T, Koivisto S, Urtti A. Different effects of pH on the permeation of pilocarpine and pilocarpine prodrugs across the isolated rabbit cornea. Eur J Pharm Sci, 1998, 6:169-176.
[7] Gupta M, Majumdar DK. Effect of concentration, pH, and preservative on in vitro transcorneal permeation of ibuprofen and flurbiprofen from non-buffered aqueous drops. Indian J Exp Biol, 1997, 35: 844-849.
[8] Conroy CW, Maren TH. Effect of pH on the ocular distribution of a topical carbonic anhydrase inhibitor. Exp Eye Res, 1995, 61: 213-222.
[9] Saettone MF, Chetoni P, Cerbai R, et al. Evaluation of ocular permeation enhancers: in vitro effects on corneal transport of four β-blockers, and in vitro/in vivo toxic activity. Int J Pharm, 1996, 142: 103-113.
[10] Sasaki H, Igarashi Y, Nagano T, et al. Different effects of absorption promoters on corneal and conjunctival penetration of ophthalmic bata-blockers. Pharm Res, 1995, 12: 1146-1150.
[11] Tang-Liu DD, Richman JB, Weinkam RJ, Takruri H. Effects of four penetration enhancers on corneal permeability of drngs in vitro. J Pharm Sci, 1994, 83: 85-90.
[12] Ismail IM, Chen C-C, Richman JB, et al. Comparison of azone and hexamethylene lauramide in toxicologic effects and penetration enhancement of cimetidine in rabbit eyes . Pharm Res, 1992, 9: 817-821.
[13] Rojanasakul Y, Liaw J, Robinson JR. Mechanisms of action of some penetration enhancers in the cornea: laser scanning confocal microscopic and eletrophysiology studies. Int J Pharm, 1990, 66:131-142.
[14] 魏刚，温度敏感眼用原位凝胶的研究．沈阳药科大学博士学位论文，pp．17．
[15] 李江英，张亚军．注射用葛根素的质量标准研究，中成药 2003，25，8：622-624．
[16] 崔福德．药剂学．第五版北京：人发卫生出版社，2005，pp245．
[17] Rojanasakul Y, Robinson JR. The cytoskeleton of the cornea and its role in tight junction permeability.Int J Pharm, 1991, 68: 135-149.
[1] 陆彬主编．药物新剂型与新技术．第一版，北京：人民卫生出版社，1998，pp．415．
[2] Helene HagerstrOm, Mattias Paulsson, Katarina Edsman. Evaluation of mucoadhesion for two polyelectrolyte gels in simulated physiological conditions using a rheological method. European Journal of Pharmaceutical Sciences. 2000, 9, 301-309.
[3] Theodore Moore. Scott Croy.et al, Experimental investigation and mathematical modeling of Pluronic F127 gel dissolution: drug release in stirred systems, J Cont Rel. 2000(67): 191-202
[4] 吴正红，朱延勤，严汉英．葛根素的溶解性及高分子聚合物助溶作用的研究，江苏药学与临床研究，1999，7(1)：9-11．
[5] 郑俊民主编．药用高分子材料学．北京：中国医药科技出版社，2000，pp．131．
[1].choi HG,Jung JH,Ryu JM.et al. Development of in situ-gelling and mucoadhesive acetaminophen liquid suppository.Int J Pharm, 1998,165(1):33-44.
[2]. Helene HagerstrOm, Mattias Paulsson, Katarina Edsman. Evaluation of mucoadhesion for two polyelectrolyte gels in simulated physiological conditions using a rheological method. Eur J Pharma Sci.2000,9,301-309.
[3]．陆彬主编．药物新剂型与新技术．第一版，北京：人民卫生出版社，1998，416．
[1] 国家食品药品监督管理局编，化学药物刺激性、过敏性和溶血性研究技术指导原则．2005年3月，14～16．
[2] Th.F. Vandamme, L. Brobeck. Poly(amidoamine) dendrimers as ophthalmic vehicles for ocular delivery of pilocarpine nitrate and tropicamide. J Cont Rel. 2005, 102, 23-38.
[2] 陆彬．药物新剂型与新技术．第一版，北京：人民卫生出版社，1998，416～420．
[3] Lang JC. Ocular drug delivery conventional ocular formulations. Adv Drug Delivery Rev, 1995, 16:39-43.
[4] Zimmer A, Kreuter J. Microspheres and nanoparticles used in ocular delivery systems. Adv Drug Delivery Rev, 1995, 16: 61-73.
[5] Saettone MF, Salminen L. Ocular inserts for topical delivery. Adv Drug Delivery Rev, 1995, 16:95-106.
[6] Eun-Young Kim, Zhong-Gao Gao, Jeong-Sook Park .rhEGF/HP-β-CD complex in poloxamer gel for ophthalmic delivery. Inter J Pharm, 2002, 233:159-167.
[7] 周亮．青光眼的防治．中国乡村医药，1997，4，9：43～44．
[8] 王建萍．青光眼的预防及临床展望．中国初级卫生保健，2003，17，10：71～72．
[9] 张好琳，梁敬钰．葛根素的研究进展．海峡药学，2005，17，1：2～5．
[10] 吴正红，朱延勤，严汉英等．葛根素滴眼液的药效学研究．中国药理学通报，1998，14(6)，569～570．
[11] Ashim K.Mitra.Ophthalmic Drug Delivery Systems,New York,Marcel Dekker Inc,second edition,282～307
[12] Luc Van Santvliet, Annick Ludwig.The influence of penetration enhancers on the volume instilled of eye drops.Eur J Pharm Biopharm, 1998,45:189-198.
[13] M.Fabrizio Saettone,Patrizia Chetoni,Riccardo Cerbai etal.Evaluation of ocular permeation enhancers:in vitro effects on corneal transport of fourβ-blockers and invitro/in vivo toxic activity, Int J Pharma, 1996,142:103-113.
[1]．国家药典委员会，中华人民共和国药典一部，北京：化学工业出版社，2000，273。
[2] Helene HagerstrOm, Mattias Paulsson, Katarina Edsman. Evaluation of mucoadhesion for two polyelectrolyte gels in simulated physiological conditions using a rheological method. Eur J Pharma Sci.2000, 9: 301-309.
[3] 国家药典委员会，中华人民共和国药典二部，北京：化学工业出版社，2000，附录174。
[4]. Hong-Ru Lin,K.C Sung, Carbopol/pluronic phase change solutions for ophthalmic drug delivery. J.Con.Res. 2000, 69 : 379-388.
[5]．张朝佑主编．人体解剖学(下册，第二版)．北京：人民卫生出版社，1998，pp．1739～1767．
[6] Higuchi, T., Connors, K.A.,1965. Phase-solubility techniques. Adv. Anal Chem. Instr. 4,117-212.
[7] Higuchi, T., Kristiansen, H., 1970. Binding specificity between small organic in aqueous solution: Classification of small organic solutes into two groups according to binding tendencies. J. Pharm.Sci., 59, 1601-1608.
[1] Yoshida F, Topliss JG.Unified model for the corneal permeability of related and diverse compounds with respect to their physicochemical properties. J Pharm Sci, 1996, 85:819-823.
[2] Liaw J, Robinson JR. The effect of polyethylene glycol molecular weight on corneal transport and the related influence of penetration enhancers. Int J Pharm, 1992, 88: 125-140.
[3] Prausnitz MR, Noonan JS. Permeability of cornea, sclera, and conjunctiva: A literature analysis for drug delivery to the eye. J Pharm Sci, 1998, 87: 1479-1488.
[4] Mitra AK, Mikkelson TJ. Mechanism of transcorneal permeation of pilocarpine. J Pharm Sci, 1988, 77: 771-775.
[5] Sasaki H, Ichikawa M, Kawakami S, et al. In situ ocular absorption of tilisolol through ocular membranes in albino rabbits. J Pharm Sci, 1996, 85: 940-943.
[6] Suhunen P, Jarvinen T, Koivisto S, Urtti A. Different effects of pH on the permeation of pilocarpine and pilocarpine prodrugs across the isolated rabbit cornea. Eur J Pharm Sci, 1998, 6:169-176.
[7] Gupta M, Majumdar DK. Effect of concentration, pH, and preservative on in vitro transcorneal permeation of ibuprofen and flurbiprofen from non-buffered aqueous drops. Indian J Exp Biol, 1997, 35: 844-849.
[8] Conroy CW, Maren TH. Effect of pH on the ocular distribution of a topical carbonic anhydrase inhibitor. Exp Eye Res, 1995, 61: 213-222.
[9] Saettone MF, Chetoni P, Cerbai R, et al. Evaluation of ocular permeation enhancers: in vitro effects on corneal transport of four β-blockers, and in vitro/in vivo toxic activity. Int J Pharm, 1996, 142: 103-113.
[10] Sasaki H, Igarashi Y, Nagano T, et al. Different effects of absorption promoters on corneal and conjunctival penetration of ophthalmic bata-blockers. Pharm Res, 1995, 12: 1146-1150.
[11] Tang-Liu DD, Richman JB, Weinkam RJ, Takruri H. Effects of four penetration enhancers on corneal permeability of drngs in vitro. J Pharm Sci, 1994, 83: 85-90.
[12] Ismail IM, Chen C-C, Richman JB, et al. Comparison of azone and hexamethylene lauramide in toxicologic effects and penetration enhancement of cimetidine in rabbit eyes . Pharm Res, 1992, 9: 817-821.
[13] Rojanasakul Y, Liaw J, Robinson JR. Mechanisms of action of some penetration enhancers in the cornea: laser scanning confocal microscopic and eletrophysiology studies. Int J Pharm, 1990, 66:131-142.
[14] 魏刚，温度敏感眼用原位凝胶的研究．沈阳药科大学博士学位论文，pp．17．
[15] 李江英，张亚军．注射用葛根素的质量标准研究，中成药 2003，25，8：622-624．
[16] 崔福德．药剂学．第五版北京：人发卫生出版社，2005，pp245．
[17] Rojanasakul Y, Robinson JR. The cytoskeleton of the cornea and its role in tight junction permeability.Int J Pharm, 1991, 68: 135-149.
[1] 陆彬主编．药物新剂型与新技术．第一版，北京：人民卫生出版社，1998，pp．415．
[2] Helene HagerstrOm, Mattias Paulsson, Katarina Edsman. Evaluation of mucoadhesion for two polyelectrolyte gels in simulated physiological conditions using a rheological method. European Journal of Pharmaceutical Sciences. 2000, 9, 301-309.
[3] Theodore Moore. Scott Croy.et al, Experimental investigation and mathematical modeling of Pluronic F127 gel dissolution: drug release in stirred systems, J Cont Rel. 2000(67): 191-202
[4] 吴正红，朱延勤，严汉英．葛根素的溶解性及高分子聚合物助溶作用的研究，江苏药学与临床研究，1999，7(1)：9-11．
[5] 郑俊民主编．药用高分子材料学．北京：中国医药科技出版社，2000，pp．131．
[1].choi HG,Jung JH,Ryu JM.et al. Development of in situ-gelling and mucoadhesive acetaminophen liquid suppository.Int J Pharm, 1998,165(1):33-44.
[2]. Helene HagerstrOm, Mattias Paulsson, Katarina Edsman. Evaluation of mucoadhesion for two polyelectrolyte gels in simulated physiological conditions using a rheological method. Eur J Pharma Sci.2000,9,301-309.
[3]．陆彬主编．药物新剂型与新技术．第一版，北京：人民卫生出版社，1998，416．
[1] 国家食品药品监督管理局编，化学药物刺激性、过敏性和溶血性研究技术指导原则．2005年3月，14～16．
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