【作者】 伍海涛；

【导师】 唐由之； 王宁生；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2009， 博士

【摘要】 冰片是一味常用中药,常在复方中与其他药物联用,用于促进吸收、增加药物的药效,穿透某些生物屏障。冰片作为眼科外用药历史悠久。冰片在中医眼科的应用与其通透的药性有关;同时冰片有轻度的镇痛抗炎作用,并能抑制杀灭一些有害细菌,这些药效在眼科得到充分的应用。近年来研究表明冰片的促渗透作用与药物有关,并对冰片的促渗作用进行机制的探讨,认为冰片的作用与其开放细胞间隙有关,并可改善了角膜上皮细胞膜的流动性。以往的冰片药效评价都是以所实验的药物为评价指标,缺乏横向比较的可能。为了对冰片的眼科促渗透效果有一系统了解,有必要建立一个通用的评价指标,系统的评价冰片的药效,并用于比较冰片剂型改良后的药效,也可以作为冰片衍生物药效的一个粗筛模型。本课题在前期研究的基础上,改进建立了适合角膜的体外灌流装置和实验方法;用离体实验比较天然冰片(右旋龙脑)、异龙脑、水杨酸冰片酯促进荧光素钠透过角膜的效果;用离体实验考察天然冰片对普萘洛尔透过角膜的影响,探讨天然冰片促药物透过角膜的机制;通过离体实验考察了天然冰片与哌仑西平合用对后者的促通透作用,并用在体实验证实。1.离体角膜灌流装置的改进改良了角膜的体外灌流装置和建立相应的实验方法,采用荧光素钠作为作为统一的探针药物,测量给予不同浓度受试药后其在灌流接收池中的浓度变化,从而建立一个观察评价对冰片和其他促渗透剂的方法。2.天然冰片开放角膜屏障的机制及药效取新鲜无损新西兰兔角膜,分别损毁上皮层或内皮层,或采用从内侧灌流的方法,测量不同条件下荧光素钠透过量;兔角膜随机分为龙脑、异龙脑的高、中、低,溶剂对照组,和阳性对照组(氮酮)。置于体外灌流装置后,在给药池中加入不同浓度的药物和相同浓度的荧光素钠,在不同时点取接收池灌流液,以紫外分光光度法测量透过角膜的荧光素钠含量,评价药物的促通透效果。结果表明,天然冰片开放角膜屏障作用部位可能在角膜的上皮细胞层,与内皮细胞层无关。龙脑、异龙脑不同浓度给药,其促荧光素钠渗透效果与浓度呈正相关,与溶剂组有显著差异,与异龙脑间无显著差异。3.天然冰片对普萘洛尔透过角膜屏障的影响采用盐酸普萘洛尔作为作为探针药物,以荧光分光光度法测量给予冰片后其在灌流接收池中的浓度变化,考察冰片是否对其能促渗透,并推测冰片的作用机制。取新鲜无损新西兰兔角膜,在给药池中加入不同浓度的盐酸普萘洛尔,在不同时点取接收池灌流液,测量透过角膜的盐酸普萘洛尔含量,考察建立适宜的探针浓度;设立龙脑高、中、低、溶剂、空白对照组,放入体外灌流装置后在给药池中加入相应的药物和统一浓度的盐酸普萘洛尔,评价药物的促通透效果。结果显示:不同浓度的冰片对盐酸普萘洛尔透过角膜有抑制作用,其抑制效果随浓度增加而加强,溶剂对照组对盐酸普萘洛尔透过也有抑制作用,高、中浓度组与溶剂对照组有显著差别。4.水杨酸冰片酯对角膜通透性的影响采用荧光素钠作为探针药物,评价冰片衍生物水杨酸冰片酯的促透过能力,并考察其作用物质,推测其机制。取新鲜无损新西兰兔角膜,放入体外灌流装置后在给药池中加入不同浓度的水杨酸冰片酯和统一浓度的荧光素钠,在不同时点取接收池灌流液,以紫外分光光度法测量透过角膜的荧光素钠含量,评价药物的促通透效果。水杨酸冰片酯促荧光素钠渗透效果中、低浓度与龙脑相似,高浓度略差,其药效与原型直接作用角膜有关。5.天然冰片对派仑西平进入眼内的影响采用冰片作为促渗透剂与哌仑西平合用,研制一种能用于临床的滴眼剂。在离体和在体两个层次与单方的哌仑西平滴眼液作了对比实验。建立了哌仑西平的高效液相测量方法;取新鲜无损新西兰兔角膜,在给药池中加入含/不含冰片的盐酸哌仑西平滴眼液,在不同时点取接收池灌流液,测量透过角膜的盐酸哌仑西平含量;在活体新西兰兔一侧眼滴加含冰片哌仑西平滴眼液50μl,对测滴加等量哌仑西平滴眼液,每5min第一次,连续6次后过量戊巴比妥麻醉处死动物,摘取眼球,吸出房水,按部位解剖眼球,分别匀浆各部位,取上清液测量哌仑西平的含量。结果证明冰片可以有效的提高哌仑西平在眼内各部位的浓度,以接收池和房水中药物累积浓度计,离体增加19倍,在体增加13.3倍,除视网膜和晶状体外,各部位的药物含量均有不同程度的增加。给予冰片后不仅透过角膜吸收的哌仑西平数量增加,其延续时间也延长。从整个课题的研究结果,我们可以得到以下结论:1.天然冰片(右旋龙脑)、异龙脑对于荧光素钠的角膜渗透有促进作用,强度与局部应用的浓度成正相关,其作用部位可能在角膜上皮细胞层。表明右旋龙脑、异龙脑能促进经细胞间隙转运的药物透过角膜。水杨酸冰片酯的促渗透效果与右旋龙脑相似。2.天然冰片(右旋龙脑)可以抑制盐酸普萘洛尔透过角膜,作用与浓度成正相关。表明右旋龙脑对跨细胞转运的药物透过角膜有抑制作用。3.天然冰片能促进哌仑西平进入眼内,并延长其滞留时间,增加其生物利用度。通过本课题的研究,我们明确了冰片对于经细胞间隙转运药物透过角膜有促进作用,而对跨细胞转运药物透过角膜有抑制作用;比较了冰片衍生物水杨酸冰片酯的促渗透效果;验证了冰片哌仑西平滴眼液能促进哌仑西平进入眼内,改善哌仑西平的生物利用度。实验结果提示冰片与经细胞间隙转运的药物合用时会产生较好的促进作用,而对跨细胞转运的药物透过生物屏障则有消极的影响。实验结果对于临床联合用药有指导意义,可以采用监测荧光素钠透过率方法作为筛选促渗透剂的指标。更多还原

【Abstract】 Borneol is a common traditional Chinese medicine and few alone used in clinical therapy.It is often combined with other Chinese medicines to treating many kinds of diseases in the compound prescription.It is used to promote other drugs’ absorption, enhance their effects and help them pentrate the barrier.It is a long history for borneol as a external used ocular drug in China.The permeating property of borneol benefits its using on ophthalmology of TCM.Borneol has a low-grade analgesia and antiinflammatory effect. Besides these,it can inhabit some malignant bacteria.All these effects are used sufficiently at ophthalmology.Some studies show the effect of borneol changed with its partner,and its mechanism has been studied this year.The effect was presumed with its opening the paracellular gap and improving the flowability of corneal epithelium membranes.The evaluation of drug action were studied with the combined drug,which lead a deficiency of comparing with other drug.It is necessary to set up a public index to compare borneol’s drug action with others or its improved dosage form.It can be used as a coarse screen model of derivate of borneol too.The study invested the promotive effect of borneol in vitro and in vivo based on the previous studies.Different qualitative drugs were used as probes to presume the penetrant mechanism of borneol.The difference of drug action were studied among d-borneol,d-isoborneol and borneol salicylate.The promotive effect of bomeol to pirenzepin was proved,which founded the next study for clinic use.1.The Improvement of Corneal Diffusion ChamberA better corneal diffusion chamber was developed and method was set up.Fluorescein sodium was used as a common probe.The concentrations of fluorescein sodium in the perfusion chamber were measured at different dosage of borneol,and the dose-effect relationship was described which was used to observe and evaluate among bomeol and other penetrants.2.Mechanism of Borneol on Opening Corneal Barrier and Its EffectFresh and harmless corneas of Newzealand rabbit were destructed in epithelium or endothelium,or perfused from the inside of cornea.The concentration of fluorescein sodium was measured.Cornea were divided to different groups randomly.The groups included high,middle,low concentration of borneol,isoborneol,azone and solvent control group. Different concentration drugs and fluorescein sodium at a constancy concentration were added in administer chamber,the perfusate were collected at designed time.The concentration of fluorescein sodium was measured with ultraviolet spectrophotometry,and the effects of penetrants were evaluated.The results showed the possible site of action of borneol is the corneal epithelium,it has no relationship with endothelium.The effects of borneol and isoborneol promoting fluorescein sodium permeation were paralleled with their concentrations.The results increased significantly compared with solvent control group and showed no significant deviation to isoborneol.3.Effect of Borneol on Propranolol Penetrateing Corneal BarrierPropranolole hydrochloride was used as a common probe.The concentration of propranolole hydrochloride in the perfusion chamber was measured with fluorospectrophotometry at different dosage of borneol.The effect of penetrant was inspected and the mechanism of which was presumed.Fresh and harmless corneas of Newzealand rabbit were tested with different concentration of propranolole hydrochloride.The perfusate were collected at designed time and the concentration of propranolole hydrochloride in perfusate were measured.The suitable concentration of propranolole hydrochloride was selected as the concentration of probe.The high,middle,low concentration of borneol,solvent control and blank group were set.Different concentration borneol and fluorescein sodium at a constancy concentration were added in concentrationadminister chamber,and the effects of penetrants were evaluated.The results showed a depressant effect of borneol on propranolole hydrochloride permeation.The results depressed significantly at high and middle concentration compared with solvent control group and showed a negative correlation of borneol concentration.The solvent control group showed a depressant effect too.4.Effect of borneol salicylate on Corneal BarrierFluorescein sodium was used as a probe to evaluated permeation effect of borneol salicylate -a Borneol derivate.The material included was inspected and the mechanism of which was presumed.Fresh and harmless corneas of Newzealand rabbit were tested.Different concentration borneol derivate and fluorescein sodium at a constancy concentration were added in administer chamber,the perfusate were collected at designed time.The concentration of fluorescein sodium was measured with ultraviolet spectrophotometry,and the effects of penetrants were evaluated.The results showed a similar effect with borneol at middle and low concentration group and low at high concentration group.The effect was related with prototype of borneol salicylate.4.Influence of Borneol on Pirenzepine Penetrateing Corneal BarrierBorneol was combined with pirenzepine hydrochloride as a penetrant to produce a gutta for clinic.The complex gutta was tested compare with single pirenzepine hydrochloride gutta in vitro and in vivo.A HPLC method to calculate pirenzepine hydrochloride concentration.Fresh and harmless corneas of Newzealand rabbit were tested.Pirenzepine gutta with/without borneol were added in administer chamber,the perfusate were collected at designed time.The concentration of pirenzepine hydrochloride was measured with HPLC system.The alive Newzealand rabbit were droped with complex gutta 50μl per 5 min at one side saccus conjunctivae and the other side with single pirenzepine hydrochloride gutta.The rabbits were anaesthesia and sacrifaced with a over load pentobarbite after 6 droped gutta.The eyeballs were selected and aqueous humor was collected.The eyeballs were dissected refer to position,and homogenated.The clear supernatant of homogenate or perfusate was used to measured pirenzepine hydrochloride concentration with HPLC system.The results showed borneol can elevate pirenzepine concentration effectively at most regional of eyeball.The accumulation concentration in receive chamber was rasied to 19 times and 13.3 times at aqueous humor.Content of pirenzepine at most position increased at different level except retina and crystalline lens.The duration of pirenzepine in eyeball was prolonged as the content was raised.So,the following conclusions could be draw from the findings of the whole issue researches:1.Borneol and isoborneol can promote the corneal permeation of fluorescein sodium, the effect correlated with the concentration of local application positively.The possible site of action of borneol is the corneal epithelium.Borneol and isoborneol can promote those drug’ s permeation,which cross cornea via passive paracellular permeation.Borneol salicylate has(?) similar effect with borneol.2.A depressant effect of borneol was observed on propranolole hydrochloride permeation,the effect correlated with the concentration of local application positively. Borneol inhibit those drug’ s permeation,which cross cornea via transcellular.3.Borneol can promote pirenzepine’s permeation of cornea and prolong the resistance time.The bioavailability of pirenzepine was increased.In conclusion,it is clear that borneol can promote those drug’ s permeation,which cross cornea via passive paracellular permeation,Borneol inhibit those drug’ s permeation, which cross cornea via transcellular.The penetrant effect of borneol derivate- borneol salicylate,was compaired with borneol and isoborneol.It is peoved that borneol can promote pirenzepine’s permeation of cornea and increased.its bioavailability.The results hint that borneol can promote the drugs crossed cornea via passive paracellular permeation and inhibit the drugs crossed cornea via transcellular method The experiment offered a method to screen penetrant,the results could be a guide to clinical compound prescription.更多还原