毛果芸香碱缓释药膜兔房水药代动力学及局部安全性研究
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摘要
目的：设计和制作符合结膜囊解剖结构的“半月形”药膜模板,制备1%半月形硝酸毛果芸香碱缓释眼用药膜；观察其在兔眼应用的药代动力学特征及局部毒性反应,评价其相对生物利用度和局部安全性；开发、研制硝酸毛果芸香碱新剂型,为青光眼的治疗提供安全、可靠、有效的缓释药物。
方法：1、健康新西兰白兔54只,雌雄不限,体重2-3kg,裂隙灯显微镜下检查无眼疾。随机分成9组,每组6只兔(6眼),每组兔右眼为实验组,左眼为对照组。右眼嵌入1%硝酸毛果芸香碱缓释药膜,左眼用微量取样器量取1%硝酸毛果芸香碱滴眼液50μl,滴入结膜囊。给药后分别于0.5、1、2、3、5、7、10、15及24h抽取房水进行定量分析。二氯甲烷提取房水中所含的药物,采用反相高效液相色谱(RP-HPLC)法测定房水中的药物浓度,药代动力学参数采用3P87药代动力学计算软件计算求得。比较两种剂型的毛果芸香碱药代动力学特征。
2、健康新西兰白兔12只,雌雄不限,体重2-3kg,裂隙灯显微镜下检查无眼疾。随机分成A、B两组,每组6只兔(6眼),每组兔右眼给予1%硝酸毛果芸香碱缓释药膜或滴眼液,左眼为空白对照组。单次给药刺激性实验：所有兔右眼给药一次后分别于1、2、4、24、48和72小时利用裂隙灯显微镜观察兔眼情况；多次给药刺激性试验：药膜组每24小时(根据药代动力学实验结果制定)给药一次,滴眼液组每天滴眼4次(根据临床用药特点：8、12、16和20点用药),连续给药5天,每天给药前及最后一次给药后1、2、4、24、48和72小时用裂隙灯显微镜观察兔眼结膜、角膜及虹膜情况,并按"Draize"标准进行刺激性评分。
3、健康新西兰白兔9只,雌雄不限,体重2-3kg,裂隙灯显微镜下检查无眼疾。随机分为正常组3只(6眼)；余6只兔(12眼)右眼为实验组,左眼为对照组,右眼嵌入1%硝酸毛果芸香碱缓释药膜,左眼用微量取样器量取1%硝酸毛果芸香碱滴眼液50μl,滴入结膜囊。药膜组每24小时给药一次,滴眼液组每天滴眼4次,连续用药30天后,经耳缘静脉空气栓塞处死兔子,取结膜和角膜组织标本,分别行光镜和扫描电镜检查,观察局部毒性反应。结果：1、反相高效液相色谱法检测可将毛果芸香碱与其它杂质很好分离,最低检测浓度为0.05μg/ml,毛果芸香碱在房水中的平均回收率为99.1%。根据房水药代动力学标准曲线,采用RP-HPLC法测得的用药后部分时间点1%硝酸毛果芸香碱缓释药膜组的房水药物浓明显高于1%硝酸毛果芸香碱滴眼液组,结果有明显统计学差异(P<0.05)。实验组和对照组用药后房水达峰时间分别为3h和0.5h,达峰浓度分别为6.55±0.32μg/ml和4.33±0.29μg/ml。药物消除半衰期分别为11.13h和1.93h。实验组药物的房水浓度-时间曲线下面积(AUC0-180)是对照组的1.92倍,实验组房水达峰浓度是对照组的1.51倍(P<0.05)。
2、单次给药刺激性实验,所有兔眼裂隙灯下检查眼部情况未见任何刺激性症状。多次给药刺激性实验,实验组1兔眼结膜轻度水肿；对照组1兔眼结膜轻度水肿、流泪,1兔眼出现少量白色分泌物；按"Draize"标准评分实验组与对照组分别为0.17和0.33,均小于3,认为两药对眼局部均无刺激性,两组评分差异无统计学意义(P>0.05)。
3、局部毒性反应实验：结膜组织光镜下炎症细胞计数,正常组、实验组和对照组炎症细胞数分别为35.83±5.98、58.17±6.55和113.17±6.85,经多样本均数间两两比较的SNK检验,按α=0.05水准,对照组均数最大,实验组次之,正常组最小,两两之间均数差别有统计学意义。角膜扫描电镜见角膜上皮细胞边缘不规则、细胞皱缩、细胞间距增宽、微绒毛丢失、细胞空洞和暗细胞增加,但对照组角膜上皮损伤较实验组明显(P<0.05)
结论：1%毛果芸香碱缓释药膜与1%毛果芸香碱滴眼液相比,达峰时间延后,达峰浓度提高,消除半衰期延长,提示其相对生物利用度提高,作用时间延长,起到缓释的作用,可明显减少用药次数；单次刺激性和多次刺激性实验均无刺激性反应；连续用药一个月后见结膜炎症细胞增生、角膜上皮微细结构改变,但较对照组为轻,说明其为一种局部安全、可靠的新剂型。
Objective Design and production the "half-moon" sustained-release film of Pilocarpine nitrate to meet the anatomy of conjunctival sac; Observe application of it in rabbit eyes pharmacokinetic characteristics and local toxicity; Evaluate the relative bioavailability and the local security; Research and development the new dosage form of nitrate Pilocarpine, to provide a safe, reliable and effective sustained release drugs on the treatment of glaucoma.
Methods 1.54 healthy New Zealand rabbits with either sex, weight 2-3kg and no eye disease by Slit-lamp microscope examination, were randomly divided into 9 groups of 6 rabbits (6 eyes), all the right eye were experimental group, and the left eye as the control. Experimental group conjunctival sac implanted in 1% pilocarpine sustained-release film, the left eye trickle-down 1% pilocarpine nitrate eye drops 50μl. After administration, respectively 0.5,1,2,3,5,7,10,15 and 24h quantitative analysis of aqueous extract. Dichloromethane extract aqueous humor contained in the drug, using reversed-phase high performance liquid chromatography (RP-HPLC) determination of the drug concentration in aqueous, the pharmacokinetic parameters calculated with 3P87 pharmacokinetic software to calculate obtain. Comparing the two formulations of pilocarpine pharmacokinetics features.
2.12 healthy New Zealand rabbits with either sex, weight 2-3kg and no eye disease by Slit-lamp microscope examination, were randomly divided into A, B two groups of 6 rabbits (6 eyes), Each rabbit's right eye to give 1% sustained-release film of Pilocarpine nitrate or eye drops, and the left eye as the blank control group. Single-dose stimulation test:after Single-dose was given to all the rabbits' right eyes, then at 1,2,4,24,48 and 72 hours respectively check rabbits' eyes by slit-lamp microscopy. Repeated stimulation test administration:sustained-release film was given every 24 hours once(based on experimental results of pharmacokinetics), eye drops was given four times a day (based on clinical treatment characteristics:8,12,16, and 20 o'clock medication), For 5 days, each time before and last time administered after at the 1,2,4,24,48 and 72 hours, respectively check rabbits' conjunctiva, cornea and iris by slit-lamp microscopy, In accordance with "Draize" standard irritation score.
3.9 healthy New Zealand rabbits with either sex, weight 2-3kg and no eye disease by Slit-lamp microscope examination, were randomly divided into A, B two groups of 6 rabbits (6 eyes), Were randomly divided into normal group 3 (6); the remaining 6 rabbits'(12 eyes) right eye as the experimental group, the left eye as the control, Experimental group conjunctival sac implanted in 1% pilocarpine sustained-release film, the left eye trickle-down 1% pilocarpine nitrate eye drops 50μl. sustained-release film was given every 24 hours once, eye drops was given four times a day,30 days after, killed the rabbit by ear vein air embolism, to take tissue samples of Conjunctiva and cornea then observe the local toxicity by light microscopy and scanning electron microscopy examination.
Result 1.RP-HPLC can be separated with Pilocarpine nitrate and other impurities.The minimum detectable concentration was 0.05μg/ml. The average recovery rate of pilocarpine nitrate from aqueous humor was 99.1%. The peak concentrations and half-life of pilocarpine in aqueous humor were 6.55±0.32μg/ml at 3h and 11.13h, respectively, in the experimental group. Whereas, the peak concentrations and half-life of pilocarpine in aqueous humor were 4.33±0.29μg/ml at 0.4h and 1.93h, respectively, in the control group. The peak concentration of pilocarpine in aqueous humor in the experimental group was 1.51(P<0.05) time higher than the control group.The area under the curve of drug concentration-time (AUCo-iso) in the experimental group was 1.92 time higher than the control group.
2. Single-dose stimulation test:There is no any irritation symptoms in all rabbit eyes by slit lamp examination. Repeated stimulation test: One rabbit conjunctival edema in experimental group; Control group, one rabbit conjunctival edema, tears, and with a little white discharge occurs in rabbit eyes. Accordance to the "Draize" criteria, the experimental group and the control group were 0.17 and 0.33, all less than 3. Therefore considered that neither sustained-release film nor eye drops is irritation to rabbit eyes and no significant difference between the two groups (P> 0.05).
3. Local toxicity test:Conjunctiva inflammatory cell counts by light microscope, In the normal group, experimental group and control group the number of inflammatory cells were 35.83±5.98,58.17±6.55 and 113.17±6.85, After SNK pairwise comparison tests, according to a= 0.05 level, the control group average maximum, followed by the experimental group and control group is the lowest, difference between the pairwise mean are statistically significant. The scanning electron microscope found that the edge of corneal epithelial cells are irregular, cell shrinkage, cell spacing widened, loss of microvilli, cell empty and dark cells increased, but the control group corneal epithelial damage badly than the experimental group (P<0.05).
Conclusion compared with 1% pilocarpine eye drops, the peak time of 1% pilocarpine nitrate half-moon-release film is delayed, peak concentration of it was increased, the elimination half-life of it is extended, suggesting its bioavailability increased, the action time prolonged, then played slow-release role. Either single or repeated stimulation test are no irritant reaction. Continuous administration a month later, conjunctivitis cell proliferation,corneal epithelial micro-structure changes, but lighter than the control group, indicating it was a local safe and reliable new formulations.
方法：1、健康新西兰白兔54只,雌雄不限,体重2-3kg,裂隙灯显微镜下检查无眼疾。随机分成9组,每组6只兔(6眼),每组兔右眼为实验组,左眼为对照组。右眼嵌入1%硝酸毛果芸香碱缓释药膜,左眼用微量取样器量取1%硝酸毛果芸香碱滴眼液50μl,滴入结膜囊。给药后分别于0.5、1、2、3、5、7、10、15及24h抽取房水进行定量分析。二氯甲烷提取房水中所含的药物,采用反相高效液相色谱(RP-HPLC)法测定房水中的药物浓度,药代动力学参数采用3P87药代动力学计算软件计算求得。比较两种剂型的毛果芸香碱药代动力学特征。
2、健康新西兰白兔12只,雌雄不限,体重2-3kg,裂隙灯显微镜下检查无眼疾。随机分成A、B两组,每组6只兔(6眼),每组兔右眼给予1%硝酸毛果芸香碱缓释药膜或滴眼液,左眼为空白对照组。单次给药刺激性实验：所有兔右眼给药一次后分别于1、2、4、24、48和72小时利用裂隙灯显微镜观察兔眼情况；多次给药刺激性试验：药膜组每24小时(根据药代动力学实验结果制定)给药一次,滴眼液组每天滴眼4次(根据临床用药特点：8、12、16和20点用药),连续给药5天,每天给药前及最后一次给药后1、2、4、24、48和72小时用裂隙灯显微镜观察兔眼结膜、角膜及虹膜情况,并按"Draize"标准进行刺激性评分。
3、健康新西兰白兔9只,雌雄不限,体重2-3kg,裂隙灯显微镜下检查无眼疾。随机分为正常组3只(6眼)；余6只兔(12眼)右眼为实验组,左眼为对照组,右眼嵌入1%硝酸毛果芸香碱缓释药膜,左眼用微量取样器量取1%硝酸毛果芸香碱滴眼液50μl,滴入结膜囊。药膜组每24小时给药一次,滴眼液组每天滴眼4次,连续用药30天后,经耳缘静脉空气栓塞处死兔子,取结膜和角膜组织标本,分别行光镜和扫描电镜检查,观察局部毒性反应。结果：1、反相高效液相色谱法检测可将毛果芸香碱与其它杂质很好分离,最低检测浓度为0.05μg/ml,毛果芸香碱在房水中的平均回收率为99.1%。根据房水药代动力学标准曲线,采用RP-HPLC法测得的用药后部分时间点1%硝酸毛果芸香碱缓释药膜组的房水药物浓明显高于1%硝酸毛果芸香碱滴眼液组,结果有明显统计学差异(P<0.05)。实验组和对照组用药后房水达峰时间分别为3h和0.5h,达峰浓度分别为6.55±0.32μg/ml和4.33±0.29μg/ml。药物消除半衰期分别为11.13h和1.93h。实验组药物的房水浓度-时间曲线下面积(AUC0-180)是对照组的1.92倍,实验组房水达峰浓度是对照组的1.51倍(P<0.05)。
2、单次给药刺激性实验,所有兔眼裂隙灯下检查眼部情况未见任何刺激性症状。多次给药刺激性实验,实验组1兔眼结膜轻度水肿；对照组1兔眼结膜轻度水肿、流泪,1兔眼出现少量白色分泌物；按"Draize"标准评分实验组与对照组分别为0.17和0.33,均小于3,认为两药对眼局部均无刺激性,两组评分差异无统计学意义(P>0.05)。
3、局部毒性反应实验：结膜组织光镜下炎症细胞计数,正常组、实验组和对照组炎症细胞数分别为35.83±5.98、58.17±6.55和113.17±6.85,经多样本均数间两两比较的SNK检验,按α=0.05水准,对照组均数最大,实验组次之,正常组最小,两两之间均数差别有统计学意义。角膜扫描电镜见角膜上皮细胞边缘不规则、细胞皱缩、细胞间距增宽、微绒毛丢失、细胞空洞和暗细胞增加,但对照组角膜上皮损伤较实验组明显(P<0.05)
结论：1%毛果芸香碱缓释药膜与1%毛果芸香碱滴眼液相比,达峰时间延后,达峰浓度提高,消除半衰期延长,提示其相对生物利用度提高,作用时间延长,起到缓释的作用,可明显减少用药次数；单次刺激性和多次刺激性实验均无刺激性反应；连续用药一个月后见结膜炎症细胞增生、角膜上皮微细结构改变,但较对照组为轻,说明其为一种局部安全、可靠的新剂型。
Objective Design and production the "half-moon" sustained-release film of Pilocarpine nitrate to meet the anatomy of conjunctival sac; Observe application of it in rabbit eyes pharmacokinetic characteristics and local toxicity; Evaluate the relative bioavailability and the local security; Research and development the new dosage form of nitrate Pilocarpine, to provide a safe, reliable and effective sustained release drugs on the treatment of glaucoma.
Methods 1.54 healthy New Zealand rabbits with either sex, weight 2-3kg and no eye disease by Slit-lamp microscope examination, were randomly divided into 9 groups of 6 rabbits (6 eyes), all the right eye were experimental group, and the left eye as the control. Experimental group conjunctival sac implanted in 1% pilocarpine sustained-release film, the left eye trickle-down 1% pilocarpine nitrate eye drops 50μl. After administration, respectively 0.5,1,2,3,5,7,10,15 and 24h quantitative analysis of aqueous extract. Dichloromethane extract aqueous humor contained in the drug, using reversed-phase high performance liquid chromatography (RP-HPLC) determination of the drug concentration in aqueous, the pharmacokinetic parameters calculated with 3P87 pharmacokinetic software to calculate obtain. Comparing the two formulations of pilocarpine pharmacokinetics features.
2.12 healthy New Zealand rabbits with either sex, weight 2-3kg and no eye disease by Slit-lamp microscope examination, were randomly divided into A, B two groups of 6 rabbits (6 eyes), Each rabbit's right eye to give 1% sustained-release film of Pilocarpine nitrate or eye drops, and the left eye as the blank control group. Single-dose stimulation test:after Single-dose was given to all the rabbits' right eyes, then at 1,2,4,24,48 and 72 hours respectively check rabbits' eyes by slit-lamp microscopy. Repeated stimulation test administration:sustained-release film was given every 24 hours once(based on experimental results of pharmacokinetics), eye drops was given four times a day (based on clinical treatment characteristics:8,12,16, and 20 o'clock medication), For 5 days, each time before and last time administered after at the 1,2,4,24,48 and 72 hours, respectively check rabbits' conjunctiva, cornea and iris by slit-lamp microscopy, In accordance with "Draize" standard irritation score.
3.9 healthy New Zealand rabbits with either sex, weight 2-3kg and no eye disease by Slit-lamp microscope examination, were randomly divided into A, B two groups of 6 rabbits (6 eyes), Were randomly divided into normal group 3 (6); the remaining 6 rabbits'(12 eyes) right eye as the experimental group, the left eye as the control, Experimental group conjunctival sac implanted in 1% pilocarpine sustained-release film, the left eye trickle-down 1% pilocarpine nitrate eye drops 50μl. sustained-release film was given every 24 hours once, eye drops was given four times a day,30 days after, killed the rabbit by ear vein air embolism, to take tissue samples of Conjunctiva and cornea then observe the local toxicity by light microscopy and scanning electron microscopy examination.
Result 1.RP-HPLC can be separated with Pilocarpine nitrate and other impurities.The minimum detectable concentration was 0.05μg/ml. The average recovery rate of pilocarpine nitrate from aqueous humor was 99.1%. The peak concentrations and half-life of pilocarpine in aqueous humor were 6.55±0.32μg/ml at 3h and 11.13h, respectively, in the experimental group. Whereas, the peak concentrations and half-life of pilocarpine in aqueous humor were 4.33±0.29μg/ml at 0.4h and 1.93h, respectively, in the control group. The peak concentration of pilocarpine in aqueous humor in the experimental group was 1.51(P<0.05) time higher than the control group.The area under the curve of drug concentration-time (AUCo-iso) in the experimental group was 1.92 time higher than the control group.
2. Single-dose stimulation test:There is no any irritation symptoms in all rabbit eyes by slit lamp examination. Repeated stimulation test: One rabbit conjunctival edema in experimental group; Control group, one rabbit conjunctival edema, tears, and with a little white discharge occurs in rabbit eyes. Accordance to the "Draize" criteria, the experimental group and the control group were 0.17 and 0.33, all less than 3. Therefore considered that neither sustained-release film nor eye drops is irritation to rabbit eyes and no significant difference between the two groups (P> 0.05).
3. Local toxicity test:Conjunctiva inflammatory cell counts by light microscope, In the normal group, experimental group and control group the number of inflammatory cells were 35.83±5.98,58.17±6.55 and 113.17±6.85, After SNK pairwise comparison tests, according to a= 0.05 level, the control group average maximum, followed by the experimental group and control group is the lowest, difference between the pairwise mean are statistically significant. The scanning electron microscope found that the edge of corneal epithelial cells are irregular, cell shrinkage, cell spacing widened, loss of microvilli, cell empty and dark cells increased, but the control group corneal epithelial damage badly than the experimental group (P<0.05).
Conclusion compared with 1% pilocarpine eye drops, the peak time of 1% pilocarpine nitrate half-moon-release film is delayed, peak concentration of it was increased, the elimination half-life of it is extended, suggesting its bioavailability increased, the action time prolonged, then played slow-release role. Either single or repeated stimulation test are no irritant reaction. Continuous administration a month later, conjunctivitis cell proliferation,corneal epithelial micro-structure changes, but lighter than the control group, indicating it was a local safe and reliable new formulations.
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[21]于世林编著《高效液相色谱方法及应用》(第二版)化学工业出版社2005年
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[7]陈祖基,眼置入剂——一种新的眼用药物长效定量释放制剂.国外医学-眼科分册.1997,21：176-181.
[8]徐岩,陈祖基,宋洁贞等.毛果芸香碱微乳滴眼剂及滴眼液在兔眼房水中的药代动力学研究.中华眼科杂志.1999,35：446448.
[9]Chastaing C, Rozier A, Plazonnet B. et al. Gelrit enhances the ocular penetratrion of pilocarpine in the pigmented rabbit, Invest Ophthalmol Vis Sci(ARVO),1995,36:sl59(680)
[10]Bourlais CL, Acar L, Zia H, et al. Ophthalmic drug delivery systems—Recent advance, Progress Retin Eye Res.1998,17:33-58.
[11]Belin M, Bar-Ilan A, Amselem S, et al. Ocular retention time of submicron emulsion(SME) and the miotic response to pilocarpine delivered in SME. Invest Ophthalmol Vis Sci.1995,36:s166.
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[14]Balazs EA, Gibbs DA. The rheological properties and biological function of hyaluronic acid. In:Balazs EA,(ed). Chemistry and molecular biology of the intercellular matrix. New York: Academic Press,1970.1241-1254.
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[17]凌沛学 主编 眼科药物的临床应用与研究.北京.中国医药科技出版社,2002.1：38.
[18]Lym HS, Suh Y, Park CK. Effects of hyaluronic acid on the polymorphonuclear leukocyte (PMN) release of active oxygen and protection of bocine comeal endothelial cells from activated PMNs [J] Korean J Ophthalmol,2004,18(1):23-28.
[19]颜耀东主编缓释控释制剂的设计与开发.北京.中国医药科技出版社,2006.5：3.
[20]黄丽娜,翁宏,成洪波,等.毛果芸香碱透明质酸钠药膜对兔眼的缩瞳作用.南方医科大学学报.2007,27：21-22
[21]于世林编著《高效液相色谱方法及应用》(第二版)化学工业出版社2005年
[1]黄丽娜,翁宏,成洪波,等.毛果芸香碱透明质酸钠药膜对兔眼的缩瞳作用.南方医科大学学报.2007,27：21-22
[2]中华人民共和国卫生部药政局,中国医院制剂规范.第二版.天津：天津科技翻译出版公司。1995：180.
[3]徐岩,宋洁贞,庞广仁,等.毛果芸香碱透明质酸钠缓释滴眼液在兔眼房水中的药代动力学研究.中华眼科杂志,2004,40(2)：87-89.
[4]朱玉云,高允生,孝建华.吲哚美辛滴眼液的毒性研究[J].泰山医学院学报,1995,16(3)：181-184.
[5]卫生部新药审批中心.新药(西药)药理毒理学研究的指导原则[M].北京：卫生部药政局,1993,208-9.
[6]毛真,刘杏,钟毅敏,等.毛果芸香碱和布林佐胺滴眼液对兔眼表组织的影响.中国病理生理杂志,2008,24(11)：2251-56.
[7]Berdy GJ, Abelson MB, Smith LM, et al. Preservativefree artificial tear preparations. Assessment of cor-neal epithelial toxic effects[J]. Arch Ophthalmol.1992,110(4):528-532.
[8]Spaeth GL, Visual loss in glaucoma clinic. I. Sociological considerations. Invest Ophthalmol.1970,9:73.
[9]Schoenwald RD, Ocular Phamrmacokinetics[A]. In: Zinmerman U. Textbook of ocular phar-macology[M]. New York: Lippincott-Raven publishers.1997,119-138.
[10]Naveh N, Muchtar S, Benita S. Pilocarpine incorporated into a submicron emulsion vehicle cause an un-expectedly prolonged ocular hypotensive effect in rabbits. J Ocular Pharmacol.1994,10:509-520.
[11]郭晓霞,寇咏梅.应用玻璃酸钠改进硝酸毛果芸香碱滴眼液.天津药学,2006,18：31-32.
[12]陈祖基,眼用脂质体的研究进展.眼科研究,1992,10：68-71.
[13]陈祖基,眼置入剂——一种新的眼用药物长效定量释放制剂.国外医学-眼科分册.1997,21：176-181.
[14]徐岩,陈祖基,宋洁贞等.毛果芸香碱微乳滴眼剂及滴眼液在兔眼房水中的药代动力学研究.中华眼科杂志.1999,35：446-448.
[15]Gurny R, Ibrahim H and Buri P. The development and use of in situ formed gels triggered by pH. In:Ed-man P. Eds. Biopharmaceutics of ocular drug delivery. Florida: CRC Press. Boca Raton.1993,81-90.
[16]Chastaing C, Rozier A, Plazonnet B. et al. Gelrit enhances the ocular penetratrion of pilocarpine in the pigmented rabbit, Invest Ophthalmol Vis Sci(ARVO),1995,36:sl59(680)
[17]Bourlais CL, Acar L, Zia H, et al. Ophthalmic drug delivery systems—Recent advance, Progress Retin Eye Res.1998,17:33-58.
[18]Belin M, Bar-llan A, Amselem S, et al. Ocular retention time of submicron emulsion(SME) and the miotic response to pilocarpine delivered in SME. Invest Ophthalmol Vis Sci.1995,36:s166.
[19]Bill A, Phillips CI. Ueoscleral drainage of apueous humor in human eye. Exp Eye Res.1971,12 (3):275
[20]惠延年主编.眼科学.第6版.北京：人民卫生出版社,2004.7
[21]蒋幼芹.青光眼的药物治疗.中华眼科杂志。2006,42(2)：190-192.
[22]徐岩,宋洁贞,庞广仁,等.毛果芸香碱透明质钠缓释滴眼剂在兔眼房水中的药代动力学研究.中华眼科杂志,2004,40：87-89.
[23]Mayer K, Palmer JW. The polysaccharide of the vitreous humor. J Biol Chem,1934,107:629.
[24]Rapport MM, Weissmann B, Linker A. et al. Isolation of a crystalline disaccharide, hyalobiuro-nic acid, from hyaluronic acid. Nature.1951,168(4284):996-7.
[25]Balazs EA, Gibbs DA. The rheological properties and biological function of hyaluronic acid. In:Balazs EA,(ed). Chemistry and molecular biology of the intercellular matrix. New York: Academic Press,1970.1241-54.
[26]Snibson GR, Greaves JL, Soper NDW, et al. Precorneal residence times of sodium hyaluronate solutions studied by quantitative gamma scintigraph. Eye,1990,4:594.
[27]Gurny R, Ryser JE, Tabatabay C, et al. Precorneal residence time in humans of sodium hyaluronate as measured by gamma scintigraph. Albrecht von Graefes Arch Ilin Exp Ophthalmol,1990,228:510.
[28]Camber O, Edman P. Sodium hyaluronate as an ophthalmic vehicle: some factors governing the effect of pilocarpine in rabbits. Curr Eye Res,1989,8:563.
[29]Pattone TP, Robinson JR. Ocular evaluation of polyvinylalcohol vehicle in rabbits. J Pharm Sci,1975,65: 1295.
[30]Saettone MF, Chetoni P, Torracca MT, et al. Evaluation of muco-adhesive properties and in vivo activity of ophthalmic vehicles based on hyaluronic acid. Int J Pharm,1989,51:203.
[31]Park K, Robinson JR. Bioadhesive polymers as platforms for oral-controlled drug delivery:method to study bioadhesion. Int J Pharm.1984,19:107.
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