门冬氨酸左氧氟沙星眼部给药系统的研究
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摘要
大多眼部疾病的治疗需局部用药，使药物进入眼部空腔及组织。而传统眼用制剂往往由于眼部特殊的生理屏障及保护机制使其生物利用度极低。本文以门冬氨酸左氧氟沙星为模型药物，从研究其亲脂性入手，首先尝试将其制备为壳聚糖纳米粒，然后将壳聚糖纳米粒混悬液与泊洛沙姆热敏型凝胶结合制备复合型给药体系，最后对此两种制剂与普通滴眼液进行了家兔泪液中的消除动力学及眼内组织分布的比较。
采用紫外分光光度法测定了药物的解离常数：pK_1=5.63±0.03，pK_2=8.52±0.05；在此基础上测定了药物的pH值依赖性的表观油水分配系数，并对药物离体角膜透过性进行考察。结果表明在等电点附近药物表观油水分配系数最大，角膜透过性最好。
尝试采用离子交联法制备载水溶性药物的壳聚糖纳米粒。通过单因素考察及正交试验优化处方工艺，制得纳米粒平均粒径357nm，zeta电位+35mv，包封率为14.01％。与左氧氟沙星包封率的比较表明通过多元阴离子对阳离子药物进行络合，降低药物荷正电的程度是提高此类药物包封率的一条可能的途径。
制备了泊洛沙姆热敏型凝胶，壳聚糖纳米粒的加入使溶液粘度及胶凝温度升高，壳聚糖分子与泊洛沙姆的PEO嵌段发生氢键络合阻碍胶束缠结可能是其原因所在。以无膜溶出模型考察体外释放表明单纯泊洛沙姆凝胶体系中药物释放符合零级动力学过程，复合给药体系的药物释放稍有延缓。
对制剂在家兔泪液中的消除动力学及眼内组织分布进行了研究。复合给药体系药-时曲线下面积(AUC)为普通制剂的1.8倍；壳聚糖纳米粒混悬液稍高于普通制剂，但两者无显著差异(P＞0.05)，与纳米粒包封率低有关。眼内组织分布表明药物在眼表滞留时间的长短可显著影响眼内组织的浓度。
Most ocular treatments call for the topical administration to introduce active drugs into the tissues around the ocular cavity. However the special physical barrier and protection mechanisms of the eye reduce the bioavailability drastically. In this thesis, aspartic levofloxacin(ASP-LEF) was selected as a model drug, and a series of relative researches were carried out, including the lipophilicity of the drug, trying to prepare chitosan nanoparticle, compounding the chitosan nanoparitcle with thermosensitive gel to prepare a kind of multiple drug delivery system and finally comparing the pharmcokinetics in the tears of white rabbits and the drug concentrations in the ocular tissues of the different preparations.The dissociation constants of pK_1 and pK_2 for aspartic levofloxacin were determined as 5.63±0.03 and 8.52±0.05 respectively by the UV spectrophotometry method using nonlinear regression analysis of origin 7.0 program. On the basis of that, the pH-dependent apparent distribution coefficient(Papp) of the drug in n-octanol/buffer system was determined, and the drug permeability across isolated cornea was studied. The results showed that the maximum value of Papp and the best permeability could be acquired at the isoelectric point of the drug.An attempt on preparing chitosan nanoparticle for a cationic hydrophilic drug by ionic gelation method was carried out. The research of single factors and the orthogonal design experiments were performed to optimize the conditions for the process and the formulation so that the chitosan nanoparticles with mean diameter of 357nm, zeta potential of +35mv, encapsulation efficiency of 14.01% were attained. The enhancement of encapsulation efficiency for a cationic hydrophilic drug could be realized by masking the positive charge of the model drug using multi-anion compound.
The thermosensitive gel of poloxamer was developed for ophthalmic use. As the chitosan nanoparticles were added, the viscosity and the gel temperature of the polymer solution both increased. It may be attributed to the chitosan as a kind of obstacle to the micellar structure of the poloxamer gel. A membraneless model was applied to study the gel dissolution and drug release. The drug release followed zero-order kinetics, and was completely controlled by gel dissolution as far as simple poloxamer gel. While the drug release in the multiple drug delivery system was delayed slightly.
The study on the drug elimination in the tears of white rabbits and the drug concentrations in the ocular tissues of the various preparations was performed. The area under the tears concentration vs time curve (AUC) for the multiple drug delivery system improved 0.8 folds in comparison with the conventional eyedrop; no significant difference statistically (P＞0.05) was observed in the AUC of the chitosan nanoparticle suspension. The residence time of drug before the cornea can significantly affect the drug concentration in the inter-ocular tissue.
采用紫外分光光度法测定了药物的解离常数：pK_1=5.63±0.03，pK_2=8.52±0.05；在此基础上测定了药物的pH值依赖性的表观油水分配系数，并对药物离体角膜透过性进行考察。结果表明在等电点附近药物表观油水分配系数最大，角膜透过性最好。
尝试采用离子交联法制备载水溶性药物的壳聚糖纳米粒。通过单因素考察及正交试验优化处方工艺，制得纳米粒平均粒径357nm，zeta电位+35mv，包封率为14.01％。与左氧氟沙星包封率的比较表明通过多元阴离子对阳离子药物进行络合，降低药物荷正电的程度是提高此类药物包封率的一条可能的途径。
制备了泊洛沙姆热敏型凝胶，壳聚糖纳米粒的加入使溶液粘度及胶凝温度升高，壳聚糖分子与泊洛沙姆的PEO嵌段发生氢键络合阻碍胶束缠结可能是其原因所在。以无膜溶出模型考察体外释放表明单纯泊洛沙姆凝胶体系中药物释放符合零级动力学过程，复合给药体系的药物释放稍有延缓。
对制剂在家兔泪液中的消除动力学及眼内组织分布进行了研究。复合给药体系药-时曲线下面积(AUC)为普通制剂的1.8倍；壳聚糖纳米粒混悬液稍高于普通制剂，但两者无显著差异(P＞0.05)，与纳米粒包封率低有关。眼内组织分布表明药物在眼表滞留时间的长短可显著影响眼内组织的浓度。
Most ocular treatments call for the topical administration to introduce active drugs into the tissues around the ocular cavity. However the special physical barrier and protection mechanisms of the eye reduce the bioavailability drastically. In this thesis, aspartic levofloxacin(ASP-LEF) was selected as a model drug, and a series of relative researches were carried out, including the lipophilicity of the drug, trying to prepare chitosan nanoparticle, compounding the chitosan nanoparitcle with thermosensitive gel to prepare a kind of multiple drug delivery system and finally comparing the pharmcokinetics in the tears of white rabbits and the drug concentrations in the ocular tissues of the different preparations.The dissociation constants of pK_1 and pK_2 for aspartic levofloxacin were determined as 5.63±0.03 and 8.52±0.05 respectively by the UV spectrophotometry method using nonlinear regression analysis of origin 7.0 program. On the basis of that, the pH-dependent apparent distribution coefficient(Papp) of the drug in n-octanol/buffer system was determined, and the drug permeability across isolated cornea was studied. The results showed that the maximum value of Papp and the best permeability could be acquired at the isoelectric point of the drug.An attempt on preparing chitosan nanoparticle for a cationic hydrophilic drug by ionic gelation method was carried out. The research of single factors and the orthogonal design experiments were performed to optimize the conditions for the process and the formulation so that the chitosan nanoparticles with mean diameter of 357nm, zeta potential of +35mv, encapsulation efficiency of 14.01% were attained. The enhancement of encapsulation efficiency for a cationic hydrophilic drug could be realized by masking the positive charge of the model drug using multi-anion compound.
The thermosensitive gel of poloxamer was developed for ophthalmic use. As the chitosan nanoparticles were added, the viscosity and the gel temperature of the polymer solution both increased. It may be attributed to the chitosan as a kind of obstacle to the micellar structure of the poloxamer gel. A membraneless model was applied to study the gel dissolution and drug release. The drug release followed zero-order kinetics, and was completely controlled by gel dissolution as far as simple poloxamer gel. While the drug release in the multiple drug delivery system was delayed slightly.
The study on the drug elimination in the tears of white rabbits and the drug concentrations in the ocular tissues of the various preparations was performed. The area under the tears concentration vs time curve (AUC) for the multiple drug delivery system improved 0.8 folds in comparison with the conventional eyedrop; no significant difference statistically (P＞0.05) was observed in the AUC of the chitosan nanoparticle suspension. The residence time of drug before the cornea can significantly affect the drug concentration in the inter-ocular tissue.
引文
[1] 张朝佑主编．人体解剖学(下册)．第二版[M]．北京，人民卫生出版社，1998，1739～1767．
[2] Lang JC. Ocular drug delivery conventional ocular formulations. Adv Drug Delivery Rev, 1995, 16: 39～43.
[3] Chien YW. Ocular Drug Delivery and Delivery Systems. In: Novel drug delivery Systems. (2nd) Marcel Dekker Inc., New York, 1992, pp. 269-300.
[4] Ludwig A, Van Ooteghem M. The evaluation of viscous ophthalmic vehicles by slit lamp fluorophotomctry in humans, Int J Pharm, 1989, 54: 95-102.
[5] Artursson P. Lindmark T, Davis SS, et al. Effect of chitosan on the permeability of intestinal epithelial cells (Caco2) [J]. Pharm Res, 1994, 11(9): 1358～1361
[6] Eun-Young Kim, Zhong-Gao Gao, Jeong-Sook Park. rhEGF/HP-β-CD complex in poloxamer gel for ophthalmic delivery[J]. Inter J Pharm, 2002, 233: 159-167.
[7] Gang Wei, Hui Xu, Ping Tian Ding, et al. Thermosetting gels with modulated gelation temperature for ophthalmic use: the rheological and gamma scintigraphic studies[J]. Journal of Controlled Release 2002, (83): 65～74.
[8] 徐连敏，陈改清．壳聚糖纳米粒的研究进展[J]．国外医学药学分册2002，29(6)：329～332
[9] Angela M. De Campos. Chitosan nanoparticles: a new vehicle for the improvement of the delivery of drugs to ocular surface. Application to cyclosporin A[J]. Int J Pharm 2001, 224: 159～168
[10] 郭炎荣．左氧氟沙星滴眼液及眼用凝胶的研究概述[J]．海峡药学，2003，15(4)：63～64
[11] 郑俊民，李启先，丁平田等．经皮给药新剂型[M]．北京：人民卫生出版社，1997．276～282
[12] 孙进．格帕沙星和HSR-903在肺上皮黏液层与肺泡巨噬细胞的分布[D]．博士学位论文
[13] 孙进，王淑君，陈丰等．在质子解离平衡中测定左氧氟沙星的亲脂性[J]．药学学报，2003，38(1)：57～61．
[14] 苏德森．王思玲．物理药剂学[M]．北京：化学工业出版社，2004．102～103
[15] Berthold A, Cremer K, Kreuter J. Preparation and characterization of chitosan microspheres as drug carrier for prednisolone sodium phosphate as model for anti-inflammatory drugs [J]. J. Control Release, 1996, 39: 17～25.
[16] Tian XX, Groves MJ. Formulation and biological activity of antineoplastic proteoglycans derived from Mycobacterium vaccae in chitosan nanoparticles [J]. J Phann Pharmacol, 1999, 51(2): 151～157.
[17] Cui Z, Mumper RJ. Chitosan based nanoparticles for topical genetic immunization [J]. J Control Release, 2001, 75(3): 409～410
[18] Ohya Y, Shiratani M, Kobayashi H, et al. Release beha-vior of 5-fluorouracil from chitosan-gei nanospheres immobilizing 5-fluorouracil coated with polysaccharides and their cell specific cytotoxicity [J]. Pure Appi Chem, 1994, A31: 629～642
[19] Kevin A. Janes, Fresneau MP, Marazuela A, et al. Chitosan nanoparticles as delivery systems for doxorubicin [J]. J Control Release, 2001, 73(2/3): 255～267.
[20] Calvo P, Re munan Lopez C, Vila-Jato JL, et al. Novel hydrophilic chitosan/ polyethylene oxide nanoparticles as proteins carders[J]. J Appl Polym Sci, 1997, 63: 125～132.
[21] Angela M., De Campos AM, Sanchez A, Alonso MJ. Chitosan nanoparticles: a new vehicle for the improvement of the delivery of drugs to the ocular surface. Application to cyclosporin A[J]. Int J Pharm, 2001, 224(1/2)159～168.
[22] 应晓英，胡富强，袁弘．胰岛素壳聚糖纳米粒的制备[J]．中国药学杂志2003，38(12)936～939
[23] Shikata F, Tokumitsu H, Ichikawa H, et al. In vitro cellular accumulation of gadolinium incorporated into chitosan nanoparticles designed for neutron-capture therapy of cancer[J]. Eur J Pharm Biopharm, 2002, 53(1): 57～63.
[24] 高晓黎，季兴梅．葡聚糖凝胶柱色谱法测定脂质体包封率的条件筛选[J]．中国药学杂志2003，38(7)：515～517
[25] 魏刚．体温敏感眼用凝胶的研究[D]．沈阳药科大学博士学位论文，2002．
[26] Pandit NK, Kisaka J. Loss of gelation ability of Pluronic(?) 127 in the presence of some salts. Int J Pharm, 1996, 145: 129～136.
[27] 刘志东，丁平田，李佳玮等．依诺沙星眼用缓释凝胶剂的体外研究[J]．中国药学杂志，2004，39(11)：834～836．
[28] 蔡鸿生，罗顺德，刘强等．乳酸左氧氟沙星滴眼液在兔眼中的药动学[J]．中国医院药学杂志，2001，21(7)：391～393
[29] 孙海燕，丁婉萍．唐星．利巴韦林原位胶化滴眼液在泪液中消除动力学的研究[J]．中国药剂学杂志．2005．3(1)：13～17
[30] 钟大放．以加权最小二乘法建立生物分析标准曲线的若干问题[J]．药物分析杂志，1996，16(5)：343～346．
[31] 陈悦，张俊杰，陈祖基．左氧氟沙星滴眼液的兔眼内药代动力学研究[J]．眼科研究2001，19(5)：429～431．
[2] Lang JC. Ocular drug delivery conventional ocular formulations. Adv Drug Delivery Rev, 1995, 16: 39～43.
[3] Chien YW. Ocular Drug Delivery and Delivery Systems. In: Novel drug delivery Systems. (2nd) Marcel Dekker Inc., New York, 1992, pp. 269-300.
[4] Ludwig A, Van Ooteghem M. The evaluation of viscous ophthalmic vehicles by slit lamp fluorophotomctry in humans, Int J Pharm, 1989, 54: 95-102.
[5] Artursson P. Lindmark T, Davis SS, et al. Effect of chitosan on the permeability of intestinal epithelial cells (Caco2) [J]. Pharm Res, 1994, 11(9): 1358～1361
[6] Eun-Young Kim, Zhong-Gao Gao, Jeong-Sook Park. rhEGF/HP-β-CD complex in poloxamer gel for ophthalmic delivery[J]. Inter J Pharm, 2002, 233: 159-167.
[7] Gang Wei, Hui Xu, Ping Tian Ding, et al. Thermosetting gels with modulated gelation temperature for ophthalmic use: the rheological and gamma scintigraphic studies[J]. Journal of Controlled Release 2002, (83): 65～74.
[8] 徐连敏，陈改清．壳聚糖纳米粒的研究进展[J]．国外医学药学分册2002，29(6)：329～332
[9] Angela M. De Campos. Chitosan nanoparticles: a new vehicle for the improvement of the delivery of drugs to ocular surface. Application to cyclosporin A[J]. Int J Pharm 2001, 224: 159～168
[10] 郭炎荣．左氧氟沙星滴眼液及眼用凝胶的研究概述[J]．海峡药学，2003，15(4)：63～64
[11] 郑俊民，李启先，丁平田等．经皮给药新剂型[M]．北京：人民卫生出版社，1997．276～282
[12] 孙进．格帕沙星和HSR-903在肺上皮黏液层与肺泡巨噬细胞的分布[D]．博士学位论文
[13] 孙进，王淑君，陈丰等．在质子解离平衡中测定左氧氟沙星的亲脂性[J]．药学学报，2003，38(1)：57～61．
[14] 苏德森．王思玲．物理药剂学[M]．北京：化学工业出版社，2004．102～103
[15] Berthold A, Cremer K, Kreuter J. Preparation and characterization of chitosan microspheres as drug carrier for prednisolone sodium phosphate as model for anti-inflammatory drugs [J]. J. Control Release, 1996, 39: 17～25.
[16] Tian XX, Groves MJ. Formulation and biological activity of antineoplastic proteoglycans derived from Mycobacterium vaccae in chitosan nanoparticles [J]. J Phann Pharmacol, 1999, 51(2): 151～157.
[17] Cui Z, Mumper RJ. Chitosan based nanoparticles for topical genetic immunization [J]. J Control Release, 2001, 75(3): 409～410
[18] Ohya Y, Shiratani M, Kobayashi H, et al. Release beha-vior of 5-fluorouracil from chitosan-gei nanospheres immobilizing 5-fluorouracil coated with polysaccharides and their cell specific cytotoxicity [J]. Pure Appi Chem, 1994, A31: 629～642
[19] Kevin A. Janes, Fresneau MP, Marazuela A, et al. Chitosan nanoparticles as delivery systems for doxorubicin [J]. J Control Release, 2001, 73(2/3): 255～267.
[20] Calvo P, Re munan Lopez C, Vila-Jato JL, et al. Novel hydrophilic chitosan/ polyethylene oxide nanoparticles as proteins carders[J]. J Appl Polym Sci, 1997, 63: 125～132.
[21] Angela M., De Campos AM, Sanchez A, Alonso MJ. Chitosan nanoparticles: a new vehicle for the improvement of the delivery of drugs to the ocular surface. Application to cyclosporin A[J]. Int J Pharm, 2001, 224(1/2)159～168.
[22] 应晓英，胡富强，袁弘．胰岛素壳聚糖纳米粒的制备[J]．中国药学杂志2003，38(12)936～939
[23] Shikata F, Tokumitsu H, Ichikawa H, et al. In vitro cellular accumulation of gadolinium incorporated into chitosan nanoparticles designed for neutron-capture therapy of cancer[J]. Eur J Pharm Biopharm, 2002, 53(1): 57～63.
[24] 高晓黎，季兴梅．葡聚糖凝胶柱色谱法测定脂质体包封率的条件筛选[J]．中国药学杂志2003，38(7)：515～517
[25] 魏刚．体温敏感眼用凝胶的研究[D]．沈阳药科大学博士学位论文，2002．
[26] Pandit NK, Kisaka J. Loss of gelation ability of Pluronic(?) 127 in the presence of some salts. Int J Pharm, 1996, 145: 129～136.
[27] 刘志东，丁平田，李佳玮等．依诺沙星眼用缓释凝胶剂的体外研究[J]．中国药学杂志，2004，39(11)：834～836．
[28] 蔡鸿生，罗顺德，刘强等．乳酸左氧氟沙星滴眼液在兔眼中的药动学[J]．中国医院药学杂志，2001，21(7)：391～393
[29] 孙海燕，丁婉萍．唐星．利巴韦林原位胶化滴眼液在泪液中消除动力学的研究[J]．中国药剂学杂志．2005．3(1)：13～17
[30] 钟大放．以加权最小二乘法建立生物分析标准曲线的若干问题[J]．药物分析杂志，1996，16(5)：343～346．
[31] 陈悦，张俊杰，陈祖基．左氧氟沙星滴眼液的兔眼内药代动力学研究[J]．眼科研究2001，19(5)：429～431．