加替沙星眼用凝胶不同点眼频次兔眼组织药代动力学比较

Comparison of the pharmacokinetics of gatifloxacin ophthalmic gel in rabbit ocular tissues with different dosing frequencies

目的建立一种测定兔眼组织中加替沙星浓度的方法,并比较加替沙星眼用凝胶兔眼单次和多次点眼后在眼组织及血浆中的药代动力学特征。方法取94只健康新西兰兔。任意选取10只新西兰兔不给予任何药物用于空白组织获取,将剩余84只按照随机数字表法随机分成单次给药组36只和多次给药组48只,雌雄各半,均取左眼为实验眼。单次给药组左眼给予1滴加替沙星眼用凝胶,分别于点眼后0.5、1、3、5、7、10 h收集泪液后进行心脏取血并处死,分别取房水、结膜、角膜、巩膜、虹膜-睫状体、晶状体、玻璃体、视网膜和脉络膜;多次给药组左眼每次给予1滴加替沙星眼用凝胶,每天3次,分别于连续给药第4和第6天首次给药后0.5 h,第7天首次给药后0.5、1、3、5、7、10 h进行心脏取血和眼组织收集。采用甲醇沉淀蛋白法预处理各样本,采用高效液相色谱串联质谱法测定并计算实验兔血浆及眼组织中加替沙星的达峰浓度(C_(max))、达峰时间(T_(max))、曲线下面积(AUC)等药代动力学参数,流动相采用甲醇-0.1%乙酸水溶液(体积比=70∶30),采用正离子多反应检测模式,以环丙沙星为内标物,并参照《中国药典》(2020年版)9012生物样品定量分析方法验证指导原则对方法的选择性、标准曲线和定量下限、准确度和精密度、提取回收率和基质效应、稳定性进行验证。结合加替沙星对眼部常见感染菌的最小抑菌浓度(MIC_(90)),计算各组织和血浆C_(max)/MIC_(90)和AUC/MIC_(90)值。结果加替沙星在各眼组织和血浆中线性关系良好;角膜组织中的日间准确度为-1.5%~6.0%,日间精密度≤15%;角膜组织中的提取回收率为92.0%~94.8%,经内标归一化计算得到的低、中、高浓度的基质效应精密度均不大于3.3%,单次给药后加替沙星在眼前节和后节组织均有较高的药物浓度分布,AUC 0-t从高到低分别为泪液、角膜、结膜、虹膜-睫状体、巩膜、房水、脉络膜、视网膜、晶状体和玻璃体,C_(max)分别为94.90μg/g、7.34μg/g、3.65μg/g、1.81μg/g、1.75μg/g、1.31μg/ml、0.86μg/g、0.53μg/g、0.13μg/g、0.07μg/ml,除晶状体、脉络膜和玻璃体液中的T_(max)为0.5 h,其余各组织T_(max)均为1 h。多次给药第4、6、7天的0.5 h各眼部组组织中加替沙星浓度比较,差异无统计学意义(P>0.05),且角膜、结膜和巩膜中AUC_(0-t)约为单次给药的2.04、2.12和2.32倍。单、多次给药后进入体循环的加替沙星浓度均低于25.00 ng/ml。对于金黄色葡萄球菌和表皮葡萄球菌,加替沙星眼用凝胶连续用药后在眼前节结膜、角膜、巩膜、虹膜-睫状体、房水和脉络膜中的药代动力学/药效学均可满足C_(max)/MIC_(90)≥10且AUC/MIC_(90)≥30。结论成功构建快速灵敏的眼组织加替沙星浓度测量方法。加替沙星眼用凝胶以每天3次点眼连续用药3 d后眼组织可达到稳态浓度,且比单次给药在眼组织浓度升高。局部使用加替沙星眼用凝胶可实现眼部结膜、角膜、巩膜、虹膜-睫状体常见感染细菌的有效治疗。

Objective To establish a method for determining the concentration of gatifloxacin in rabbit ocular tissue and compare the ocular pharmacokinetics of 0.3%gatifloxacin eye gel after a single and multiple topical instillations in rabbits.Methods Ninety-four healthy New Zealand rabbits were selected.Ten rabbits were randomly selected without any treatment for blank tissue collection,and the remaining 84 rabbits were randomly divided into a single-dose group(36 rabbits)and a multiple-dose group(48 rabbits)equally between males and females using a random number table.The left eye was taken as the experimental eye.The single-dose group was given one drop of 0.3%gatifloxacin eye gel into the left eyes,and the rabbits were divided evenly into six subgroups.In each subgroup,tear specimens and blood specimens were collected at 0.5,1,3,5,7,10 hours after gel application,then cardiac blood samples were taken,after which animals were sacrificed immediately to collect ocular tissue including aqueous humor,conjunctiva,cornea,sclera,iris-ciliary body,lens,vitreous body,retina,and choroid.The multiple-dose group was given 1 drop of gatifloxacin ophthalmic gel in the left eye three times a day.At 0.5 hour after the first administration days 4 and 6,and 0.5,1,3,5,7,and 10 hours after the first administration on day 7,the cardiac blood sampling and ocular tissue collection were performed.The methanol precipitation protein method was used to pretreat samples,and the concentration of gatifloxacin in rabbit plasma and eye tissue was measured and calculated by high-performance liquid chromatography-tandem mass spectrometry method to obtain pharmacokinetic-related parameters such as peak concentration(C_(max)),peak time(T_(max)),and area under curve(AUC).The mobile phase was a methanol-0.1%acetic acid aqueous solution(volume ratio=70∶30),and a positive ion multiple reaction detection mode was used.Ciprofloxacin was used as the internal standard,the selectivity,standard curve and lower limit of quantification,accuracy and precision,extraction recovery rate,matrix effect,and stability of the method were validated in accordance with the 9012 Guidelines for Validation of Quantitative Analysis Methods for Biological Samples in Chinese Pharmacopoeia(2020 edition).Combined with the minimum inhibitory concentration(MIC_(90))of gatifloxacin on common ocular infectious bacteria,C_(max)/MIC_(90)and AUC/MIC_(90)were calculated.The study protocol was reviewed and approved by the Animal Ethics Committee of Shenyang Xingqi Pharmaceutical Co.,Ltd.(No.XQ-2016-011).Results Gatifloxacin has a good linear relationship in various eye tissues and plasma.The between-run precision in corneal tissue is within the range of-1.5%-6.0%,and the daytime precision was not greater than 15%.The extraction recovery rate in corneal tissue ranged from 92.0%to 94.8%,and the precision of the matrix effect at low,medium,and high concentrations calculated by internal standard normalization was not greater than 3.3%.After a single topical instillation,gatifloxacin reached a high concentration in anterior and posterior segment ocular tissues and its distribution ranked in order from the highest to the lowest by AUC_(0-t)as follows,tears,cornea,conjunctiva,iris-ciliary body,sclera,aqueous humor,choroid,retina,lens and vitreous body,with the C_(max)of 94.90μg/g,7.34μg/g,3.65μg/g,1.81μg/g,1.75μg/g,1.31μg/ml,0.86μg/g,0.53μg/g,0.13μg/g and 0.07μg/ml,respectively.T_(max)was 1 hour in all ocular tissues except in the lens,choroid,and vitreous body fluid,where T_(max)was 0.5 hour.There was no significant difference among the concentrations of gatifloxacin in ocular tissues at 0.5 hour on days 4,6 and 7 after multiple dosing(P>0.05),and the AUC 0-t in the cornea,conjunctiva,and sclera was approximately 2.04,2.12,and 2.32 times that of the single dosing.The concentration of gatifloxacin released into the systemic circulation after single and multiple dosing was less than 25.00 ng/ml.For both Staphylococcus aureus and Staphylococcus epidermidis,pharmacokinetic/pharmacodynamics in the conjunctiva,cornea,sclera,iris-ciliary body,aqueous humor,and choroid were satisfied with C_(max)/MIC_(90)≥10 and AUC/MIC_(90)≥30 after continuous administration of gatifloxacin ophthalmic gel.Conclusions A rapid and sensitive method for measuring gatifloxacin concentration in ocular tissues is successfully constructed.Gatifloxacin ophthalmic gel administered three times a day for three days can achieve stable concentrations in ocular tissues,and the concentration of gatifloxacin in ocular tissues is increased compared with a single dose.Effective treatment of common bacterial infections of the conjunctiva,cornea,sclera,and iris-ciliary body can be achieved with topical application of gatifloxacin ophthalmic gel.