【作者】 刘洋；

【导师】 王丽平；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2019， 硕士

【摘要】 药物溶解度及肠渗透性是决定药物吸收的关键因素,生物药剂学分类系统(Biopharmaceutics classification system,BCS)即是基于这两个性质的药物预测系统,目前被FDA、WHO、EMA等权威机构认可并应用于仿制药生物等效性研究的豁免。近年来兽药研究者尝试将BCS引入兽用药物领域,但人与动物间存在种间差异,为保证分类的准确性及良好的体内外相关性,兽用药物的溶解度及渗透性测定均应考虑靶动物的生理条件,不能将人药BCS原则直接外推于动物。本研究旨在通过测定鸡的生理参数,以此为基础建立药物溶解度测定方法,利用过表达鸡源肠道外排蛋白的MDCK细胞系对鸡常用药物进行渗透性测定,同时探讨pH条件对药物渗透性的影响。最后选取提高溶解度的氟苯尼考微粉及晶型的优化制剂,评估其体内外吸收情况。1.鸡常用药物生理溶解度测定方法的建立BCS认为药物单次给药最高剂量能完全溶解于250 mL(50 mL胃液体积+200 mL送服药物的水)水中即为高溶解度药物。本实验选取1日龄AA白羽肉鸡,饲至8周龄,测定不同日龄鸡生理参数,包括体温、胃肠道pH值及胃肠容积,在此基础上初步建立鸡BCS溶解度测定方法并测定16种鸡常用药物的溶解度及分类。结果显示,不同日龄鸡体温约为41℃,胃肠道pH值范围为2.03～8.23,胃肠容积与体重成正相关,1 Kg体重鸡在进食条件容积为20.40 mL,而禁食条件为6.73 mL。本研究将溶解度测定条件建立在鸡体温及胃肠道pH值范围上,并结合胃肠容积及各药单次给药最高剂量进行分类。结果表明,16种药物中有3种药物在进食及禁食条件下的BCS分类不一致,另与已知人药BCS分类的7种药物比较,2种药物与鸡BCS分类不一致。说明温度及pH值影响药物的溶解度,而人与鸡的生理差异是不同溶解度表现及BCS分类差异的主要原因,所以人药BCS不能直接应用于鸡,并且在建立鸡BCS溶解度分类时应考虑进食与禁食条件下的差异。综上所述,鸡常用药物平衡溶解度在41℃及pH 1、3、5、7和8的缓冲溶液中测定,鸡BCS高溶解度定义为药物单次给药最高剂量能溶解于20.40 mL(进食)或6.73 mL(禁食)。2.鸡常用药物体外渗透性测定方法的建立渗透性测定的方法较为多样化,为保证形成良好的体内外相关性,同时考虑伦理,本实验选取MDCK、MDCK-chAbcg2及MDCK-chAbcg2/Abcb1细胞系进行药物渗透性测定,利用高渗透性药物酒石酸美托洛尔及盐酸普萘洛尔比较MDCK细胞系与Caco-2细胞之间是否存在差异性,结果发现同一药物在这两类型细胞中AP-BL侧及BL-AP侧渗透性均有不同程度的差异,并且不同药物在两细胞中表观渗透系数(Papp)的变化趋势也不相同,说明两细胞不能相互替代。再以各药物1/10单次给药最高剂量作为转运浓度,在MDCK、MDCK-chAbcg2及MDCK-chAbcg2/Abcb1细胞系上分别以5.5及7.4的pH条件进行渗透性测定,并以FDA推荐高渗透性药物酒石酸美托洛尔为标准药物分类,探讨不同细胞系及pH对渗透性测量的影响。结果显示pH对药物的渗透性存在一定程度的影响,在16种药物中有8种药物在不同pH条件下Papp存在显著或极显著的差异(P<0.05),但pH对药物渗透性分类的影响仅体现在个别药物,另比较同一个药物在不同细胞系上的Papp,pH 5.5和7.4条件下分别有10和13种药物在不同细胞系上的Papp存在显著或极显著的差异(P<0.05),但在16种药物中14种药物在不同细胞系上的分类完全相同,表明细胞系间差异并不明显,另比较分类结果与鸡生物利用度时发现相关性良好,16种药物中仅2种出现不符合的情况。综上所述,为更好的模拟鸡小肠,建立良好的体内外相关性,鸡常用药物渗透性测定方法为以各药物1/10单次给药最高剂量作为转运浓度,选取MDCK-chAbcg2/Abcb1细胞在pH 7.4条件下测定药物渗透性,鸡BCS高渗透性定义为表观渗透系数大于相同实验条件下酒石酸美托洛尔的Papp。3.基于BCS概念的氟苯尼考优化剂型的体外渗透性及体内药动学研究选取由吸收表现较差的氟苯尼考(FFC)制备而成的微粉及晶型制剂,通过双向转运实验及鸡体内药代动力学研究比较两种制剂与原料药在体内外吸收情况的差异。结果显示,在10 μmol/L浓度下,原料药在MDCK-chAbcb1/Abcg2细胞上的Papp(AP·BL)为0.27×10-6 cm/s,外排率ER为5.6,而微粉及晶型的Papp(AP·BL)分别为0.46及0.42×10-6 cm/s,均显著或极显著高于原料药(P<0.05),而外排率分别为3.35及3.27,显著或极显著低于原料药的ER(P<0.05)。50 μmol/L浓度下两个制剂与原料药差异程度与10μmol/L相似,原料药、微粉和晶型的Papp(AP-BL)分别为0.33、0.44和0.51(×10-6 cm/s),而外排率ER分别为5.11、4.12和3.95。另外同剂量灌服给鸡后,原料药的达峰浓度(Cmax)为 5.48 μg/mL,半衰期(T1/2 β)为 1.95 h,清除率(CL)为 1.09 mL/h/kg,药时曲线下面积AUC为18.67 μ·h/mL。而微粉和晶型的达峰浓度(Cmax)及半衰期(T1/2β)均高于原料药,Cmax分别为原料药的1.5和1.2倍,T1/2 β分别为1.1和1.9倍,而清除率(CL)则显著低于原料药(P<0.05),另外两种制剂具有更大的药时曲线下面积AUC,分别为原料药的1.2和1.3倍,相对生物利用度分别为121.4%及129.8%。以上结果均表明经过溶解度改造的制剂能有效改善FFC在鸡体内的吸收情况,进而提高生物利用度。综上结果,我们初步建立鸡的BCS溶解度及渗透性的分类方法并对16种鸡常用药物进行了分类,分析了鸡进食及禁食条件对药物溶解度分类方法的影响,以及不同细胞系及pH值对渗透性分类方法的影响,并分别与部分人药BCS分类结果进行对比,探讨建立鸡BCS分类方法的准确性及必要性,另对基于BCS概念改造的氟苯尼考制剂进行体内外吸收情况分析,讨论BCS科学框架对老药改造的指导作用。更多还原

【Abstract】 Solubility and permeability are the key factors which determine drug absorption.Biopharmaceutics classification system(BCS)is a drug classification and prediction system based on these factors.Currently,FDA,WHO and EMA have approved it to be applied as a science-based approach for the waiver of in vivo bioequivalence of the drugs used in human.In recent years,some researchers have tried to introduce BCS into the field of veterinary medicine.However,due to the interspecies differences,the BCS of human medicine cannot be directly extrapolated to animals,so the methods used to detect the dug solubility and permeability should be based on the physiological conditions of the target animals to ensure the accuracy of classification and the correlation between in vitro and in vivo.This study aims at establishing a method for determining the solubility of chicken BCS by measuring the physiological parameters of the chicken,the MDCK cell line overexpressing chicken-derived intestinal efflux protein is used as a tool for permeability measurement.The permeabilities of commonly used drugs are measured,and the effects of pH conditions on permeability detection are discussed.Finally,the absorption performance of florfenicol micro-powder and crystal was compared with API from in vitro and in vivo.1.Establishment of a method for determining the physiological solubility of commonly used drugs in chickensThe high physiological solubility is defined by FDA as the highest dose can be completely dissolved in 250 mL(50 mL of gastric fluid volume with 200 mL of water for drug delivery)in human drugs.In this experiment,AA white feather broilers aged 1-day were selected and raised to 8 weeks old.During this period,the physiological parameters including body temperature,pH of the gastrointestinal tract and gastrointestinal volume,were measured at different ages.Chicken BCS solubility classification method was initially established based on these,then conducted the measurement and classification of 16 drugs.The results showed that the body temperature of the chicken at different ages was 41℃,the pH of the gastrointestinal tract ranged from 2.03 to 8.23,and the gastrointestinal volume was positively correlated with the body weight,the volume of the chicken weighting 1 Kg in feding condition was 20.40 mL,while the fasting condition was 6.73 mL.Therefore,the condition of determining the solubility were based on chicken body temperature and the pH of the gastrointestinal tract.The results of solubility in this study were higher than the solubility data recorded in the pharmacopoeia due to the differences in experimental temperature and pH.When classified under the conditions of feding and fasting,3 of the 16 drugs were found inconsistent classification,which indicates that the different gastrointestinal volumes has the effect on the classification.when compared the "human BCS" and "chicken BCS",2 of the 7 drugs was found to be inconsistent in solubility classification.Therefore,the human BCS cannot be directly extrapolated to chicken,and the effect of fasting should be considered when establishing the classification of chicken BCS solubility.In summary,the solubility of commonly used drug in chicken is measured in 41℃ and buffer solutions which pH were 1,3,5,7 and 8,while the high solubility of chicken BCS is defined as the highest dose of the drug can be completely dissolved in 20.40 mL(feding)or 6.73 mL(fasting).2.Establishment of an in vitro permeability determination method for commonly used drugs in chickensThe methods for permeability measurement are diverse,considering ethical issues and in vitro-in vivo correlation with chicken,MDCK cell and MDCK cells overexpressing chicken-derived BCRP or P-gp were initially selected as the assay for measuring of drug permeability,firstly comparing the Papp of metoprolol tartrate or propranolol hydrochloride between MDCK cell lines and Caco-2 cell,the results showed that the Papp of AP-BL and BL-AP permeability in same drug were different,and the variety trend of different drugs Papp in the two cells is also different,indicating that the two cells cannot replace each other.Then the recommended high-permeability drug metoprolol tartrate is classified as a standard drug,the 1/10 highest dose of each drug was used as the transport concentration on the MDCK cell,MDCK-chAbcg2 cell,and MDCK-chAbcg2/Abcb1 cell at pH conditions of 5.5 and 7.4,respectively,to investigate the differences between cell lines and the effect of pH on permeability measurements.The results showed that pH would certain influence the apparent permeability coefficient of the drugs.8 of the 16 drugs were significant differences in Papp under different pH conditions,but the effect of pH on drug classification was only reflected in individual drugs.Then,on different cell lines,the Papp of 10 and 13 drugs were significant differences under pH 5.5 and 7.4,respectively,but 14 of 16 drugs were classified identically on different cell lines.In addition,when the classification results were compared with the chicken bioavailability,the correlation was found to be good,and only 2 of the 16 drugs showed non-conformity.In summary,in order to simulate the conditions of chicken small intestine and establish a good correlation between in vitro and in vivo,we chose an appropriate permeability measurement method for chicken commonly used drugs,which is the 1/10 highest dose of each drug was used as the transport concentration on the MDCK-chAbcg2/Abcb1 cell lines at pH 7.4.And the high permeability of chicken drugs was defined as the apparent permeability coefficient greater than the Papp of metoprolol tartrate under the same experimental conditions.3.In vitro permeability and in vivo pharmacokinetic study of florfenicol-optimized formula based on BCS conceptFlorfenicol was a poor absorption drug based on BCS concept,we compared the in vitro and in vivo absorption between florfenicol API and two kinds of formula including florfenicol micropowder or crysta1.The results showed that at 10 μmol/L,the Papp(AP-BL)of the drug substance on MDCK-chAbcb1/Abcg2 cells was 0.27×10-6 cm/s,and the efflux rate was 5.6,while the Papp(AP-BL)of micropowder and crystal were significantly higher than that of the drug substance(P<0.05),0.46 and 0.42×10-6 cm/s,respectively,and the efflux rate was 3.35 and 3.27,respectively,which were significantly lower than the ER of the drug substance(P<0.05).The difference between the two preparations and the drug substance was similar to 10 μmol/L at 50 μmol/L,and the Papp(AP-BL)of the drug substance,micropowder and crystal were 0.33,0.44 and 0.51(×10-6 cm/s),respectively.And the efflux rates were 5.11,4.12 and 3.95,respectively.In addition,after the same dose was administered to the chickens,the Cmax of the drug substance was 5.48(μg/mL,the T1/2β was 1.95 h,the CL was 1.09 mL/h/kg,and the AUC of the drug was 18.67μg·h/mL.The Cmax and T1/2β of the micropowder and crystal are higher than these of the drug substance,Cmax is 1.5-and 1.2-folds of the drug substance,respectively,and T1/2β is 1.1-and 1.9-folds,respectively,and the clearance was significantly lower than that of the drug substance(P<0.05),and the two formulations had a larger AUC,was 1.2-and 1.3-folds of the drug substance,respectively,and the relative bioavailability was up to 121.4 and 129.8%,respectively.The above results indicate that the solubility-modified florfenicol preparations can effectively improve the absorption and bioavailability of FFC in chickens.In summary,we initially established a classification method for solubility and permeability of chickens drugs applied to BCS and successfully classified 16 drugs.The effects of chicken feding and fasting conditions on solubility classification methods as well as different cell lines and pH values on permeability classification methods were analyzed and finally compared the results with human drug BCS.In all words,it is necessary to establish a physiologically based BCS classification method for chickens drugs.In addition,to discuss the guiding role of BCS in the transformation of old drugs,we compared the in vitro and in vivo absorption between florfenicol API and the FFC micropowder or crystal.更多还原