硫柳汞和苯扎氯铵联合用药的抗真菌协同作用和机制研究及对真菌性角膜炎模型治疗作用和对兔的毒性作用研究

Our former study confirmed that thimerosal exhibited potent in vitro activity against broad-spectrum ocular pathogenic fungi. Recently, our primary work based on animal experiment and clinical trials addressed the suitability of thimerosal for the treatment of keratomycosis with its concentration as a preservative（0.01％）. Since the antifungal eye drops are typically administered with a dosing frequency of one drop every 1 or 2 hours and the average duration of administration of the antifungal eye drops is 6 weeks during treatment keratomycosis, the need for high dosing frequency and long treatment duration will increase a possibility of toxicity of thimerosal. It is tempting to speculate if there is synergism when thimerosal is combined with some antifungals, concentration of thimerosal in topical formulation may be decreased to much lower than its concentration as a preservative, therefore, the safety of topical thimerosal will be improved. Based on the hypothesis, the in vitro activity of thimerosal in combination with different antifungals was assessed against some isolates of ocular pathogenic fungi. The results showed that there was significant synergism when thimerosal was combined with benzalkonium chloride. The present study will be performed to determine the antifungal activity of a combination of thimerosal and benzalkonium chloride against broad-spectrum ocular pathogenic fungi in vitro, the efficacy of the combined application in keratomycosis in guinea pigs, and the toxic action of the combined application in rabbits. The present study will also be performed to study the antifungal mechanism of the combined application. The aim of this study is to evaluate the safety and efficacy of the combined application. Results obtained in this study may contribute to the development of new antifungal eye drops.

我们已经证明了防腐剂硫柳汞对眼部致病真菌有非常强的体外抗菌活性。初步的实验和临床研究显示以硫柳汞用作眼药防腐剂的常规浓度（0.01％）治疗真菌性角膜炎疗效显著。由于治疗真菌性角膜炎的用药频次较高，用药时间较长，为防止长期用药可能引起的毒副作用，我们设想如果硫柳汞与其它药物联合应用有明显的抗真菌协同作用，制备联合用药制剂则可使制剂中硫柳汞的浓度明显低于其用作防腐剂的浓度，使其用药的安全性进一步提高。按此思路，我们将硫柳汞与多种药物组合进行体外抗真菌活性的小样本筛选，结果显示硫柳汞和苯扎氯铵联合用药有明显的抗真菌协同作用。本项目拟从体外药效、体内药效及体内长期毒性三个方面对硫柳汞和苯扎氯铵联合用药进行系统的疗效及毒性评估，并探讨其抗真菌的作用机制，旨在判断硫柳汞和苯扎氯铵联合用药是否有作为新的抗真菌眼药进行开发的可能性。本项目将为开发高效、广谱、低毒的抗真菌眼药提供新思路及充分的科学依据。

本项目原申请面上项目,拟从体内外药效及长毒三方面对硫柳汞联合苯扎氯铵进行系统的疗效及毒性评估,并探讨其抗真菌机制,旨在判断硫柳汞联合苯扎氯铵是否有作为新抗真菌眼药开发的可能性。因本项目由面上项目改为主任基金项目,我们按1年资助期将项目的研究内容和目标修订为体外药效研究：1) 评估硫柳汞等10种药物单独用药的抗真菌活性；2) 评估硫柳汞联合苯扎氯铵的抗真菌协同作用。. 我们完成了硫柳汞等10种药物单独用药和硫柳汞联合苯扎氯铵对300株眼部致病真菌的体外抗菌活性研究。结果显示: . 1) 对于我国真菌性角膜炎的主要致病菌镰刀菌属和曲霉菌属,单独用药时硫柳汞抑制90%菌株的最低抑菌浓度(MIC90)分别为0.0313和0.0625μg/ml,硝酸苯汞的MIC90均为0.0313μg/ml,纳米银的MIC90均为1μg/ml,特比奈芬的MIC90分别为4和0.0625μg/ml,益康唑的MIC90分别为4和0.25μg/ml,那他霉素的MIC90分别为8和32μg/ml,酮康唑的MIC90分别为32和2μg/ml,二性霉素B的MIC90均为2μg/ml,苯扎氯铵的MIC90均为32μg/ml,氟康唑的MIC90分别为512和256μg/ml。. 2) 硫柳汞联合苯扎氯铵时对镰刀菌属的分级抑制浓度指数(fractional inhibitory concentration index,FICI)范围为0.375～0.5,对曲霉菌属的FICI范围为0.25～0.375。硫柳汞联合苯扎氯铵对镰刀菌属和曲霉菌属的协同作用均为100%。. 联合用药研究结果提示硫柳汞联合苯扎氯铵有作为新抗真菌眼药开发的可能性。. 单独用药的研究结果提示: 1) 对于镰刀菌属和曲霉菌属,硫柳汞和硝酸苯汞都有非常强的抗菌活性,纳米银有较强的抗菌活性。2) 特比奈芬和益康唑对曲霉菌属的抗菌活性非常强,对镰刀菌属的抗菌活性明显减弱。3) 那他霉素对镰刀菌属有抗菌活性,对绝大部分曲霉菌属无抗菌活性;酮康唑对曲霉菌属有抗菌活性,对绝大部分镰刀菌属无抗菌活性。4) 镰刀菌属和曲霉菌属对二性霉素B的耐药菌株明显增多;苯扎氯铵对镰刀菌属和曲霉菌属的抗菌活性非常弱,氟康唑对镰刀菌属和曲霉菌属基本无抗菌活性。. 本研究结果为开发高效的抗真菌眼药提供了新思路及实验依据。