【作者】 关延彬；

【导师】 李高；

【作者基本信息】 华中科技大学 ， 药理学， 2010， 博士

【摘要】 影响口服给药吸收的三个主要因素是溶出率、溶解度及通透性。疏水性药物的溶出通常是其吸收的限速阶段,因此,提高疏水性药物的溶解度和溶出速度可解决此类药物低口服吸收的难题,但是即使这样促吸收作用也不一定十分明显。近年来大量研究已发现,肠上皮细胞膜上的P-糖蛋白（P-glycoprotein, P-gp）药泵作用和细胞色素P₄₅₀（CYP）药物代谢酶系统对口服药物的跨膜转运和药物的生物利用度可产生重大影响。P-gp可能量依赖性地将吸收的药物重新泵回肠腔中去, P-gp的这一作用又称为“药物溢出泵”（drug efflux pump）,是限制P-gp底物药物吸收的主要因素,也是药物口服吸收的重要屏障。近年来不断有研究报道一些在在制剂中常常使用的普通辅料同样具有P-gp的抑制作用,这些辅料的优势是安全、不会被胃肠道吸收,以往常常被当作增溶剂或稳定剂使用。这些发现对克服P-gp药泵作用,提高口服药物生物利用度提供了美好的前景。但是在研究过程中也发现,许多具有P-gp药泵抑制作用的药用辅料虽然在体外得到理想的实验结果,但在整体动物实验中抑制作用往往减弱甚至消失。原因可能是复杂的胃肠道生理因素（介质的稀释、肠上皮细胞膜的通透性、内容物阻滞等）使药用辅料未能有效地接触或进入肠上皮细胞发挥抑制P-gp的功能。因此,增加具有阳性抑制作用的药用辅料与肠上皮细胞有效的接触和渗入,提高辅料在肠壁细胞内的浓度,更好地发挥“药泵”抑制作用以增加药物的透过率,可能是突破P-gp底物药物吸收瓶颈的重要手段。随着一些新的给药系统的快速发展,越来越多的研究证明,脂质体,微球,聚合物胶束、纳米粒等药物给药系统都可以有效的提高药物治疗作用,同时降低药物的副作用,而且组成这些纳米给药系统的载体材料很多本身已经被证明也有抑制P-gp药泵的作用。因此将两者联合使用来是否可以更好的促进P-gp底物药物吸收,改善药物的有效治疗已逐渐引起人们的注意。本文首先以P-gp底物-R-123为荧光探针,Pluronic聚合物为研究对象,采用Caco-2细胞模型和外翻肠囊模型筛选出对肠道P-gp药泵具有抑制作用的载体材料;继而考察其抑制P-gp的作用机制及载体材料胶束化前后对R-123在Caco-2细胞跨膜机制的影响;然后以伊曲康唑为模型药物,以抑制P-gp作用最强的P123为主要载体材料构建了伊曲康唑胶束给药系统,通过大鼠小肠单向灌流法和体内法验证胶束给药系统提高生物利用度的可能性,进一步探讨胶束给药系统促进药物吸收与P-gp药泵的关联。本实验采用MTT法,首先考察了Pluronic载体材料对Caco-2细胞的毒性作用。结果表明,除L61外,Pluronic F127,F68,P123和P85载体材料在实验浓度（0.0001%～5%）范围内细胞毒性较小,对细胞的生长基本没有影响。采用Caco-2细胞模型,进行细胞摄取和转运试验,以维拉帕米为阳性对照,利用HPLC法测定并计算R-123的细胞摄取量、跨膜转运参数表观渗透系数Papp与外排率ER值,来评价Pluronic载体材料对Caco-2细胞膜上P-gp外排功能的抑制作用。细胞摄取试验表明F127,F68,P85和P123对R-123的细胞摄取具有不同程度的促进作用,但是具有浓度依赖性,在接近或超过临界胶束浓度（CMC）后,细胞摄取达到最大值,然后随着Pluronic浓度的继续增大,促进作用反而逐渐减弱,提示其对P-gp药泵具有一定的抑制作用。细胞转运试验结果表明P123在胶束化前后（浓度分别为0.001%、1%）均可明显降低R-123的P₍app（（BL→AP））值（P<0.05）,显著降低ER（P<0.05）,与维拉帕米组的ER值无显著差别,提示其抑制P-gp作用强度与维拉帕米相当。而F127、F68和P85组,高浓度（1%）组均可明显降低R-123的P₍app（（BL→AP））值（P<0.05）,显著降低ER（P<0.05）,而低浓度组（0.001%）对R-123的转运无显著影响,这与药物摄取试验结果相一致,进一步验证Pluronic系列载体材料对P-gp的抑制作用,采用外翻肠囊法考察P123和F127对R-123小肠吸收的影响,结果表明125μg/mL维拉帕米、0.001%、0.1%、1%P123和0.001%、0.1F127均能在一定程度上提高R-123M→S透膜吸收,并可相应提高R-123的小肠吸收率;同时通过增溶试验表明P123和F127对水难溶性药物有更大的增溶能力,更适合作为胶束载体材料。细胞膜的流动性试验表明浓度为0.001%-1%P123可以增加细胞膜的流动性,而F127对细胞膜流动性基本没有影响,而且当浓度升至1%时,还具有减小Caco-2细胞流动性的趋势。P-gpATP酶活性试验结果表明P123和F127可以不同程度的降低P-gpATP酶活性,但随着载体材料浓度的增加,P-gpATP酶活性又有所恢复。说明载体材料可能通过这两种机制抑制P-gp药泵。P123胶束化前后对R-123跨膜转运机制的研究表明,P123胶束主要不是通过细胞旁路转运,P123胶束进入细胞应该涉及到网格蛋白介导的胞吞方式,而且具有能量依赖性,在一定程度上具有逃逸P-gp药泵的作用。选用对P-gp药泵有明显抑制作用的P123为载体材料制备ITZ-P123胶束,采用薄膜分散法在单因素考察的基础上,以包封率、载药量为指标,采用星点设计-效应面优化法优化处方,获得较优处方:P123为60mg,ITZ1mg,去离子水为17.5mL,温度为60℃。优化后胶束的载药量大约为1.02%;为了提高PluronicP123的增溶能力和对P-gp的抑制作用,通过增溶试验、MTT试验、Caco-2细胞摄取试验进行筛选,确定以P123/RH40=8:2,采用上述方法制备了ITZ-P123/RH40混合胶束,处方为:P123与RH40分别为80mg和20mg,ITZ1.7mg,去离子水为3.5mL,温度为66℃。优化后胶束的载药量大约为1.57%;体外释放度试验表明:ITZ-P123胶束和ITZ-P123/RH40胶束在人工胃液中72h内累积释放百分率分别为87.5±7.%和71.2±5.4%,而在人工肠液中则无法检测到;体外溶出试验表明:与市售Sporanox胶囊相比,在人工胃液和人工肠液中,两种胶束给药系统的累积溶出百分率都达到100%,15min之内基本全部溶出,不受pH影响。从单向灌流实验结果可见,与ITZ溶液相比,ITZ-P123胶束和ITZ-P123/RH40胶束可显著提高ITZ吸收速率常数（Ka）和小肠有效渗透系数（Peff）,（p<0.05）,说明胶束可以提高ITZ在小肠的吸收。但ITZ浓度为50μg/mL时的Ka和有keff均比5μg/mL组低,且Peff的差异比Ka更明显,因此表明ITZ胶束的吸收可能存在一定的饱和性。在ITZ-P123胶束和ITZ-P123/RH40胶束中加入维拉帕米后,对不同浓度的ITZ胶束的小肠吸收基本没有明显的影响,这说明载药的P123和P123/RH40形成的胶束,不受P-gp抑制剂的影响,可以通过逃逸P-gp的药泵外排作用增加P-gp底物的小肠吸收,这与前面Caco-2细胞模型实验结果相符。单剂量灌胃给予大鼠Sporanox胶囊、ITZ-P123胶束和ITZ-P123/RH40胶束后,结果显示,在禁食和正常饮食状态下,两种胶束制剂其主要药动学参数与Sporanox组具有明显差异。相对于Sporanox组,在禁食状态下,ITZ-P123胶束组AUC和Cmax明显提高;而ITZ-P123/RH40胶束组无论是否禁食,都可显著提高ITZ的AUC_0-∞和Cmax;此外两种胶束制剂也分别延长了ITZ在大鼠体内的MRT及t_1/2,这说明ITZ胶束不仅可以促进药物口服吸收,而且还可延长药物在体内的循环时间。综上所述,本文筛选的Pluronic系列载体材料具有不同程度的P-gp抑制作用,因而可以促进P-gp底物-R-123的跨膜吸收,但是试验结果也表明Pluronic载体材料在胶束化前后对药物跨膜吸收的影响机制是不同的,形成胶束后可以通过网格蛋白介导的胞吞方式吸收,将抑制作用最强的P123及RH40为主要载体材料构建了ITZ-P123胶束和ITZ-P123/RH40胶束给药系统,结果表明两种胶束给药系统在大鼠小肠的吸收不受P-gp药泵的影响,可以通过逃逸P-gp药泵的外排作用来提高药物的口服生物利用度,证明P123和RH40形成的胶束给药系统可以利用其自身结构及载体材料改变药物的生物膜通透性和跨膜方式,促进药物的跨膜转运,这为设计其小肠吸收与P-gp密切相关的药物的“高生物利用度”新型载体系统提供理论和实验依据,具有重要地研究价值和意义。更多还原

【Abstract】 Three major factors affecting oral absorption are dissolution rate, solubility, and permeability. Dissolution of drugs with high hydrophobicity is the rate-limiting process for their oral absorption. Therefore, increasing their solubility and dissolution could improve the oral bioavailability. In addition, recent studies showed that efflux pump effect of P-glycoprotein (P-gp) and drug metabolism by CYP enzymes located in intestinal epithelial membrane demonstrated significant effect on drug transmembrane transport and therefore influenced its bioavailability. P-gp energy dependently pumps absorbed drugs back to intestinal lumen, acting as a critical barrier for drug oral absorption. More recent literatures reported some pharmaceutical excipients common used as solubilizer and stabilizer exhibited inhibitory effect on P-gp, which provided fair promise to enhance oral bioavailability via inhibiting P-gp efflux pump. However, many excipients attenuate inhibitory effect in in vivo animal model despite that in vitro studies showed excellent results. This might be due to complex gastrointestinal physiologic factors including medium dilution, permeability of intestinal epithelial membrane, and content blocking, which resulted in insufficient contact with intestinal epithelial membrane. Thus, increasing percontiguum and penetration of pharmaceutical excipients with inhibitory effect lead to raise the concentration in intestinal cells and therefore increase drug transmissivity, which provide important strategies and attain great breakthrough for P-gp substrates. With rapid development of new drug delivery systems, liposomes, microspheres, polymer micelle, and nanoparticle were able to improve pharmacotherapy effect and diminish the side effects of drugs. Furthermore, the carrier materials of nanoformulation have been proven to be P-gp inhibitor. Collectively, combination of P-gp inhibition and nanotechnology could markedly promote the oral absorption of P-gp substrates.Early on, P-gp substrate, R-123, was chosen as fluorescent probe, and Caco-2 cell and everted intestinal sac models were used to screen carrier vehicles with inhibitory effect on intestinal P-gp. Moreover, the inhibitory and transmembrane mechanisms of these carrier vehicles in Caco-2 cell model. Furthermore, the pharmaceutical excipients with best P-gp inhibition effect, P123 and RH40, using itraconazole as model drug were applied to construct itraconazole micelle delivery system. In addition, rat intestinal single pass perfusion and in vivo animal models were used to test the bioavailability of itraconazole, in order to investigate the correlation between drug absorption and P-gp pump.In present study, cytotoxicity of Pluronics was tested using MTT method. In contrast to Pluronic L61, Pluronics F127, F68, P123, and P85 showed little cytotoxicity and had no influence on cellular growth.Cellular uptake and drug transport studies were performed in Caco-2 model using verapamil as positive control. HPLC was utilized to quantify R-123 and then cellular uptake and the apparent coefficient of permeability (Papp), as well as efflux ratio (ER) were calculated to evaluate the inhibitory effect of Pluronic on P-gp on Caco-2 cell monolayer. The results indicated that F127, F68, P85 and P123 possessed increased cellular uptake and showed concentration dependence. However, increasing Pluronic concentration up to CMC achieved highest cellular uptake and attenuate the inhibitory effect when the concentration was higher than CMC. In addition, cellular transport results demonstrated lower concentration of P123 (0.001%, 1%) markedly decreased Papp,BA and ER (p<0.05), but no significance was observed between P123 and verapamil. Additionally, high concentration (1%) of F127, F68, and P85 obviously reduced Papp,BA and ER (p<0.05), whereas low concentration (0.001%) of F127, F68, and P85 had no effect on drug transport. Therefore, these results are in good agreement with the findings from cellular uptake experiments, which indicated Pluronics had inhibitory effect on P-gp.The everted intestinal sac model was also applied to study the effect of P123 and F127 on intestinal absorption of R-123. The results showed that 125μg/mL verapamil, 0.001%, 0.1%, 1% P123, and 0.001%, 1% F127 could increase mucosal to submucosal transport of R-123. Pluronics enhance intestinal absorption of R-123 due to both solubilization of poor water soluble compounds and P-gp inhibition and therefore show the advantages for micelle carrier materials.The finding resulted from fluidity test of cell membrane showed that 0.001%-1% P123 could increase the cell membrane fluidity, whereas F127 had no effect on membrane fluidity. Furthermore, increasing the concentration of F127 up to 1% would decrease the fluidity of Caco-2 cell monolayers. In addition, both P123 and F127 were able to reduce the ATPase activity to a different extent. However, further increase of the concentration of P123 and F127 would result in recovery of ATPase activity, which implied that P-gp was inhibited through two or more than two different mechanisms. Also, P123 micelle demonstrated clathrin mediated endocytosis rather than paracellular transport from the effect of pre- and post-micellization of P123 on R-123 transport. Moreover, it showed energy dependency and escape from P-gp efflux pump effect.In order to investigate the effects of a drug delivery system constructed with excipient inhibitors on the P-gp drug pump, P123, which significantly inhibited P-gp drug pump, was selected as main formulation component. Central composite design-response surface methodology was used to optimize the preparation of ITZ-P123 micelles. ITZ-P123 micelles were prepared by film dispersion method and the optimized micelles formulation formed from the encapsulation efficiency and the drug loading capacity met the requirements of micelles preparation. Basic prescription for the ITZ-P123 micelles were: P123 was 60mg, ITZ was 1mg, deionized water was17.5mL, the temperature was 60℃. Optimized drug loading capacity was approximately 1.02%; To improve PluronicP123 solubilization and increase the inhibition effect of P-gp drug pump, solubilization test, MTT test, Caco-2 cell uptake test were used to optimize the preparation of ITZ-P123 micelles, and the basic prescription for the mixed micelles were: P123 was 80mg, Cremophor RH40was 20mg, ITZ was 1.7mg, deionized water was 3.5mL, the temperature was 66℃, optimized drug loading capacity was approximately 1.57%. In vitro release test showed that the percentage of cumulative release of ITZ-P123 micelles and ITZ-P123/RH40 were 87.5±7% and 71.2±5.4% in artificial gastric juice, while in the artificial intestinal juice the content of ITZ can not be detected. Compared with the commercial Sporanox capsules, the cumulative percentage of dissolution for two micellar drug delivery system were 100% in artificial gastric juice and artificial intestinal juice, ITZ were almost completely dissolved within 15min and were not significantly changed with pH value.The results of single pass perfusion test shown that ITZ-P123 micelles and ITZ-P123/RH40 micelles can significantly improve the Ka and keff values compared with the ITZ solution (p <0.05), which indicated that micelles can increase the absorption of ITZ in the small intestine. However, the Ka and Keff values of ITZ at concentration of 50μg/mL were lower than that of ITZ at concentration of 5μg/mL and Peff differences was more obvious than the Ka values. The comparison results of single pass perfusion of different MDZ formulations showed that the absorption of ITZ micelles may exist a certain saturation in small intestine. Furthermore, when added verapamil in ITZ-P123 micelles and ITZ-P123/RH40 micelles, no significant impacts on the absorption of ITZ in small intestine were observed in the ITZ micelles at different concentration, indicating that P-gp substrates can escape the efflux function when the substrates were contained in P123 and P123/RH40 micelles, and the excipient inhibitor-based formulation is a potential protective platform for increase oral bioavailability of sensitive drugs that are P-gp substrates, which is consistent with the results of in vitro Caco-2 cell model.We assessed the effects of the commercial Sporanox capsules、P123 micelles and P123/RH40 micelles on the pharmacokinetics of ITZ in rats following single-dose administration, the results showed that the main pharmacokinetic parameters of P123 micelles and P123/RH40 micelles were significantly different with that of the commercial formulation in fasting and normal diet. ITZ-P123 micelle group significantly improved AUC and Cmax of ITZ in fasting state, and ITZ-P123/RH40 micelle group, whether or not fasting, can significantly improve the AUC0-∞and Cmax of ITZ. Furthermore, two micellar formulations were also extended MRT and t1/2 of ITZ in rats, which shows that ITZ micelles not only can promote oral absorption of ITZ, but also extend ITZcirculation time in vivo.In summary, this screening carrier materials of pluronic have different capability of P-gp inhibition, which can contribute to P-gp substrate (R-123) transmembrane- uptake. But the experiments also show that the mechanism of carrier material on the substrate transmembrane- uptake is different before and after the prepration of Pluronic micelles. The substrate can be absorbed by clathrin-mediated endocytosis after the formation of micelles. This study shown that the absorption of substrate in two micelle delivery system is not influenced by P-gp in the rat intestine and can escape P-gp efflux function to improve substrate oral bioavailability. The excipient inhibitor-based formulation can promote cellular uptake of substrates by changing the membrane permeability, which can provide a theoretical basis for the design of the new formulation to improve the low bioavailability drug, and therefore has an important theoretical and practical significance.更多还原