Pluronic胶束给药系统的构建及与肠道P-糖蛋白药泵作用研究
|
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P_(450)(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。
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.
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。
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.
引文
1. Hongming C, Robert L.Oral particulate delivey:status and future trend.Advanced[J]. Drug Delivery Reiview,1998,34(2-3):339-350
2. Amidon G, Lennernas H, Shah V, et al. A theoretical basis for a bioph- armaceutical drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability[J]. Pharm. Res,1995, 12(3):413–420
3. Zhang Y,Benet LZ. The gut as a barrier to drug absorption:combined role of cytochrome P450A and P-g1ycoprotein.Clin Pharmcokinet,200l,40(3):159-163
4. Chen CJ, Chin JE, Ueda K, et al. Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistance human cells[J]. Cell, 1986,47(3): 381–389
5. Ambudkar SV, Kimchi-Sarfaty C, Sauna ZE, et al. Pglycoprotein: from genomics to mechanism[J]. Oncogene ,2003,22(47):7468–7485
6. Akiyama SI, Cornwell MM, Kuwano M, et al. Most drugs that reverse multidrug resistance also inhibit photoaffinity labelling of P-glycoprotein by vinblastine analog. Mol. Pharmacol. 1988. 33(2): 144–147
7. Terao T, Hisanaga E, Sai Y,et al. Active secretion of drugs from the small intestinal epithelium in rats by P-glycoprotein functioning as an absorption barrier[J]. J. Pharm .Pharmacol, 1996; 48(10): 1083-1089
8.田治科,胡富强,丁建潮,等.西罗莫司固体脂质纳米粒的制备及在大鼠体内的相对生物利用度[J].中国医药工业杂志;2006, 37(9):610-613.
9.郑中华,张以忠,方青,Pgp动力学研究进展[J],吉林医药学院学报,2006,7(2):1-5
10. Bansal T, Akhtar N, Jaggi M , et al. Novel formulation approaches for optimising delivery of anticancer drugs based on P-glycoprotein modulation[J]. Drug Discovery Today, 2009,14(21-22): 1067-1074
11. Sachs B K. Lipid excipients Peceol and Gelucire 44/14 decrease P-glycoproteinmediated efflux of rhodamine 123 partially due to modifyingP-glycoprotein protein expression within Caco-2 cells[J]. J. Pharm. Pharm,Sci. 2007,10(3): 319–331
12. Dabholkar RD, Sawant RM., Mongayt D A. Polyethyleneglycol–phosphatidy -lethanolamine conjugate (PEG–PE)-based mixed micelles: Some properties, loading with paclitaxel, and modulation of P-glycoprotein-mediated efflux[J]. Int. J. Pharm, 2006,315(1-2): 148-157
13.田庆锷,张慧,李玛琳等.P一糖蛋白与多药耐药的关系[J]。药品评价,2005, 2(3):225-226
14.陈东,丁平田,邓意辉等.聚合物胶束用于口服给药的研究进展[J].药学学报, 2010, 45 (5): 560?564
15. Reif S, Kingreen D, Kloft C, et al. Bioequivalence investigation of high-dose etoposide and etoposide phosphate in lymphoma patients[J]. Cancer Chemother Pharmacol, 2001,48(2):134-140
16. Cheol-Hee C, Joon-Ho K, Sang-H yun K.Reversal of P-glycoprotein- mediated MDR by 5,7,3',4',5'-pentamethoxyflavone and SAR[J]. Biochem Biophy Res commomm,2004, 320(3):672-678
17. Lipinski CJ. Poor aqueous solubility– an industry wide problem in drug discovery[J], Am. Pharm Rev. 2002, 582–585
18. Kim RB, Wandel C, Leake B, et al. Interrelationship between substrates and inhibitors of human CYP3A and P-glycoprotein[J]. Pharm Res. 1999,16(3): 408–414
19.吴迪,李高,黄建耿,等.更昔洛韦的小肠吸收与P-糖蛋白药泵作用的关系研究.中国药师.2004; 7(6):408-411
20. Xiuhua Ren, Luqin Si, Gao Li, et al. Pharmaceutical Excipients Inhibit Cytochrome P450 Activity in Cell Free Systems and After Systemic Administration[J]. Eur. J. Pharm. Biopharm. 2008,70(1:) 279-288
21. Kataoka K, Harada A, Nagasaki Y, Block copolymer micelles for drug delivery:design, characterization and biological significance[J], Adv. Drug Deliv. Rev. 2001, 47(6) :113–131
22. Savic R, Luo L, Eisenberg AD. et al, Micellar nanocontainers distribute to defined cytoplasmic organelles[J], Science ,2003,300(4): 615–618
23. Gaucher G, Dufresne M, San V, et al. Block copolymer micelles: preparation, characterization and application in drug deliver y [J]. J. Control. Res,2005(2),109 : 169–188
24. Shen D,Lu L J,Li Y,Advance on amphiphilic block copolymer micelles used in drug carrier[J]. Acta Academiae Medicinae CPAF,2008,17(3):237-240
25. Agata AE, Tianyi MK, Katherine SJ , Enhancement of carboplatin toxicity by Pluronic block copolymers[J].J. Control Release 106 (2005) 188– 197
26. Alakhov V, Moskaleva E., Bathakova EV, et al. Hypersensitization of multidrug resistant human ovarian carcinoma cells by Pluronic P85 block copolymer[J], Bioconjug. Chem. 1996 7 (2) 209–216
27. Kabanov AV., Batraova EV, Alahov VY, Pluronic block copolymers for overcoming drug resistance in cancer [J], Adv. Drug Deliv. Rev. 54 (5) (2002) 759–779.
28. Vecnnce A, Li S. Mandeville R, Kabanov AV,et al. Hypersensitizing effect of Pluronic L61 on cytotoxic activity, transport, and subcellular distribution of doxorubicin inmultiple drug-resistant cells[J], Cancer Res,1996,56 (16) :3626–3629
29. Mathot F, Beijsterveldt LV, Preat V, et al. Intestinal uptake and biodistribution of novel polymeric micelles after oral administration, J. Control. Release[J], 2006,111(1-2) : 47–55
30. Mahesh D C , Yogesh P, Jayanth P.Susceptibility of nanoparticle-encapsulated paclitaxel to P-glycoprotein-mediated drug efflux[J]. Int.J.Pharm.2006,320 (1-2) 150–156
31. Ranaldi G, Consalvo R, Sambuy Y, et al. Permeability characteristics of parental and clonal human intestinal Caco-2 cell lines differentiated in serum-supplemented and serum-free media[J]. Toxicology in Vitro, 2003,17(5):761-767
32.齐云,王敏,蔡润兰. Caco-2细胞模型在口服药物转运机制研究中应用的进展[J].中国药学杂志,2009 44 (17):1281-1284
33.张生芳,李娟亚.药物渗透与吸收评价方法的研究进展.太传统医药[J]. 2010, 6(5):129-131
34. Batrakova E, Lee S, Li S, et al. Fundamental relationships between the composition of Pluronic block copolymers and their hypersensitization effect in MDR cancer cells[J]. Pharm. Res, 1999,16 (9) :1373– 1379
35. Agata AE, Tlanyi MK, Katherrine SJ , Enhancement of carboplatin toxicity by Pluronic block copolymers[J].J. Controll Release,2005, 106 (1-2) 188– 197
36. Elena V, Batrakova E ,Huai YH, et al. Effect of Pluronic P85 Unimers and Micelles on Drug Permeability in Polarizerd BBMEC and caco-2 Cells [ J ] .Pharma Research,1998,15(10): 1525-1532
37. Hasseltt PM, Janssens GE, Slot TK, et al. The influence of bile acids on the oral bioavailability of vitamin K encapsulated in polymeric micelles.[J].J. Controll Release,2009,133 (2): 161–168
38. Jason Z, John J, Mandeep B , et al. Enhanced cellular accumulation of a P-glycoprotein substrate,rhodamine-123, by caco-2 cells using low molecular weight methoxypolyethylene glycol-block-polycaprolactone diblock copolymers[J]. Eur J.Pharm Bio, 2002,54 (3) :299–309
39.王永忠.紫杉醇嵌段共聚物胶束给药系统克服肿瘤多药耐药性的研究:[学位论文],上海复旦大学,2007
40.董宇,张英丰,杨庆等。黄连提取物在大鼠肠外翻实验中的吸收研究[J].中国中药杂志.2008,33(9):1056-1061
41. Pierri E, Avgoustakis K, Poly(lactide)-poly(ethylene glycol) micelles as acarrier for griseofulvin[J], J. Biomed. Mater. Res. Part A,2005, 75 (3) :639–647
42. Zao JX,Jiang LQ,Qiu Y,Solubilization of Polycyclic Aromatic Hydrocarbons in F127 and P123 Aqueous Micellar Solutions [J]. Chem. J. Chinese Universities.2001,22(4):165-170
43. Li HY. Micellization behavior of Pluronic block copolymers in aqueous solution and its application on drug carrier [D] . Taiyuan :Taiyuan University of Technology,2007
44.徐运杰,方热军.外翻肠囊法的应用研究[J],饲料研究,2009,12(2):9-12
45. Tamihide M, Eiji K, Sachiyo Y, et al.Determination of P-Glycoprotein ATPase Activity Using Luciferase Biol. Pharm[J]. Bull, 2006,29(3) :560-564
46. Frédéric M, des RA. Ari?n A, et al.Transport mechanisms of mmePEG750P -(CL-co-TMC) polymeric micelles across the intestinal barrier[J].J.Controll Release.2007 124 (3): 134–143
47. Zastre J, Jackson J, Burt H, Evidence for modulation of P-glycoprotein–mediated efflux by methoxypolyethylene glycol-block-polycaprolactone amphiphilic diblock copolymers[J]. Pharm Res. 2004,21(8):1489-1496
48. Elena V B, Shu L, Li YL .Effect of Pluronic P85 on ATPase Activity of Drug Efflux Transporters[J], Pharm Res. 2004 ,21(12): 2226–2233
49. Gao X, Wang T, Wu J,et al. Quantum dots for tracking cellular transport of lectin-functionalized nanoparticles,Biochem. Biophys[J]. Res. Commun,2008,77 (1) : 35–40
50. Ma Z, Lim LY, Uptake of chitosan and associated insulin in Caco-2 cell monolayers: a comparison between chitosan molecules and chitosan nanoparticles[J]. Pharm. Res,2003,20 (11): 1812–1819
51. Roger E, Lagarce F, Garcion E, et al.Lipid nanocarriers improve paclitaxel transport throughout human intestinal epithelial cells by using vesicle-mediated transcytosis[J].J Controlled Release, 2009, 140 (2) 174–181
52. Yoo HS, Park TG, Biodegradable nanoparticles containing protein–fatty acid complexes for oral delivery of salmon calcitonin[J]. J. Pharm Sci,2004,93 (2): 488–495
53. Sahay G Alakhova D Y, Kabanov AV, Endocytosis of nanomedicines[J]. J .Controll Release ,2010,145 (3):182–195
54. Cai CX ,Chen JW.Overexperssion of Caveolin-1 Induces Alter Ation of Multidrug Resistance in Hs578T Breast Adenocarcinoma Cells[J].Int. J. Cancer.2004, 111(4): 522–529
55. Sahay G, Batrakova EV, Kabanov AV. Different Internalization Pathways of PolymericMicelles and Unimers and Their Effects on Vesicular Transport[J].Bioconjugate Chem. 2008, 19(10): 2023–2029
56. Garrigues A., Escargueil AE., Orlowski S, The multidrug transporter, P-glycoprotein , actively mediates cholesterol redistribution in the cell membrane[J].Proc. Natl. Acad. Sci. U.S.A. 2002 ,99(16):10347–10352.
57. Shao K, Huang RQ, Li JF .Angiopep-2 modified PE-PEG based polymeric micelles for amphotericin B delivery targeted to the brain[J] ,J.Controll Release ,2010,147 (1) :118–126
58. Bernard VE, Michiel VS, Raf M, et al. Itraconazole/TPGS/Aerosil 200 solid dispersions Characterization, physical stability and in vivo performance[J]. Eur J Pharm Sci,2009, 38 (3) 270–278
59. Wei CN, Bing G, HaoW, et al.Development and evaluation of novel itraconazole-loaded intravenous nanoparticles[J].Int J Pharm. 2008,362 (1-2): 133–140
60. Yi Yw, Yoon HJ, Kim BO.A mixed polymeric micellar formulation of itraconazole:Characteristics, toxicity and pharmacokinetics[J]. J Controll Releas -e ,2007,117 (1): 59–67
61. Wei YG, Jasmine T, Dave A. et al.High bioavailability from nebulized Itraconazole nanoparticle dispersions with biocompatible stabilizers[J].Int J Pharm,2008,361 (1-2):177–188
62. Hong JY , Kim JK , Song YK et al.A new self-emulsifying formulation of itraconazole with improved dissolution and oral absorption[J].J Controll Release ,2006,110 (2) 332– 338
63.刘艳杰,项荣武.星点设计效应面法在药学试验设计中的应用[J].中国现代应用药学杂志2007,24(6):455-457
64.吴伟,崔光华,陆彬.实验设计中多指标的优化:星点设计和总评“归一值”的应用[J].中国药学杂志2000,35 (8):530-533
65. Zhang XH, Jackson JK., Burt HM. Determination of surfactant critical micelle Research article concentration by a novel fluorescence depolarization technique[J].J. Biochem.Biophys Methods. 1996, 31 (3-4): 145-150
66.李海燕.Pluronic两亲嵌段共聚物的胶束化行为及其作为药物载体的初步应用:[学位论文],太原理工大学
67.陆冬云,温浩,刘会洲等.球形嵌段共聚物胶束的温度效应.物理化学学报,2004,20(l):38-42
68. Sang CL,Chulhee K,Iek CK,et al. Polylnerie micelles of Poly(2-ethyl-2-oxazoline)- bloek-Poly(εcaProlactone) copolymer as a carrier for Paclitaxel[J] .Control Rel,2003, 89(5):437-446
69. Vinayak PS, Damon S, Jean L .Enhancement of oral bioavailability of poorly water-soluble drugs by poly(ethylene glycol)-block-poly(alkyl acrylate-co-methacrylic acid) self-assemblies[J].J Controll Release ,2005.104 (2): 289–300
70. Zerrin S, Nilufer Y, Tamer B. Investigation of pluronic and PEG-PE micelles as carriers of meso-tetraphenyl porphine for oral administration[J] . Int J Pharm, 2007, 332( 1-2): 161-167
71. Gaucher G , Satturwar P, Jones MC,Polymeric micelles for oral drug delivery[J].Eur J Pharma and Biopharm,2010. 76 (2) :147–158
72. Christiansen A, Backensfeld T, Weitschies W. Effects of non-ionic surfactants on in vitro triglyceride digestion and their susceptibility to digestion by pancreatic enzymes[J] .Eur J Pharm Sci, 2010, 41 (2): 376-382
73.黄建耿.药用辅料抑制肠道P-糖蛋白药泵作用研究:[学位论文],华中科技大学,2008
74.韩丽妹.基于逆转肿瘤多药耐药性的共聚物胶束给药体系的研究:[学位论文],复旦大学,2006
75.向苗,温敏性聚合物胶束的制备及性质研究:[学位论文],兰州大学,2008
76. Kumar V, Wang L, Riebe M, et al,Formulation and Stability of itraconazole and odanacatib nanoparticles: governing physical parameters [ J ] .Molecular Pharmaceutics.2009,6(4 ):1118-1124
77. Hu YQ,Zheng LY,Qian CQ.Small intestinal absorbtion study of phone red as an ionic drug[J].J China Pharm Univ1996,27(1):355-359
78. Rogera E, Lagarceb F, Garcionb E, et al.Reciprocal competition between lipid nanocapsules and P-gp for paclitaxel transport across Caco-2 cells[J]. Eur J Pharm Sci,2010. 40 (5) :422–429
79. van Hasselt PM, Janssens GE, Slot TK, et al The influence of bile acids on the oral bioavailability of vitamin K encapsulated in polymeric micelle s [J].J Controll Release,2009, 133 (2): 161–168
1. Sezgin Z, Yuksel N, Baykara T, et al. Investigation of pluronic and PEG-PE micelles as carriers of meso-tetraphenyl porphine for oral administration[J]. Int. J. Pharm. 2007,332(3): 161-167.
2. Lavelle EC, et al.Targeted delivery of drug to gastrointestinal tract[J].Critical Revies in Therapeutic Drug Carrirer Systems. 2001,18(4):341-386.
3. Mira F.F, Mariana C, Fran?oise M, et al. Polymeric micelles for oral drug delivery:Why and how[J]. Pure Appl. Chem, 2004, 76(1):1321–1335.
4. Benet, L. Z. , Izumi,T., Zhang, Y., Silverman, J. A., Wacher, V. J., et al.Intestinal MDR transport proteins and P-450 enzymes as barriers to oral drug delivery[J]. J. Controlled Release. 1999,62(9): 25-31.
5. Hirunpanich, V. , Murakoso, K. , Sato, H. , et al. Inhibitory effect of docosahexaen -oic acid (DHA) on the intestinal metabolism of midazolam: In vitro and in vivo studies in rats[J]. Int. J. Pharm.,2008,351(7): 133-143.
6. Okada J, Cohen S ,R Langer, et al. In vitro evaluation of polymerized liposomes as an oral drug delivery system[J]. Pharm Res 1995, 12(8):. 576-582.
7. Kataoka K, Harada A, Nagasaki Y, et al.Block copolymer micelles for drug delive ry:design, characterization and biological significance[J]. Adv. Drug Deliv. Rev. 2001, 47(5) :113–131.
8. Savic R, Luo L, Eisenberg A, D. et al, Micellar nanocontainers distribute to defined cytoplasmic organelles[J].Science ,2003,300(4): 615–618.
9. Gaucher G, Dufresne M, San V, et al, Block copolymer micelles: preparation, characterization and application in drug delivery[J], J. Control. Rel. 2005,109(7) : 169–188.
10. Lev Bromberg, et al. Polymeric micelles in oral chemotherapy[J]. J. Controlled Release, 2008,128(1) : 99-112.
11. DeMario M, Ratain M, et al. Oral chemotherapy: rationale and future directions[J]. J.Clin. Oncol.1998, 16(7): 2557–2567.
12. Anderson J, Van Itallie C, et al.Tight junctions and the molecular basis for regulation ofparacellular permeability[J]. Am. J. Physiol. 1995,269(5), 467–475.
13. Gaurav S, Alakhova Y, Alexander V, et al.Endocytosis of nanomedicines[J]. J Controll Release 2010,145(6) :182–195.
14. Norris D., Puri N, Sinko P, et al.The effect of physical barriers and properties on the oral absorption of particulates[J], Adv. Drug Deliv. Rev. 1998,34(7) : 135–154.
15. Mathot F, Schanck A, Bambeke F A. et al, Passive diffusion of polymeric surfactants across lipid bilayers[J] J.Control. Release ,2007,120(1): 79–87.
16. Sezgin Z,. Yuksel N, Baykara T, et al.Investigation of pluronic and PEG-PE micelles as carriers of meso-tetraphenyl porphine for oral administration[J] Int. J.Pharm,2007,332(2): 161–167.
17. Francis M, Cristea M, Yang Y, et al, Engineering polysaccharidebased polymeric micelles to enhance permeability of cyclosporin A across Caco-2 cells[J]. Pharm. Res,2005,22(4) : 209–219.
18. J. Zastre, J. Jackson, H. Burt, et al.Evidence for modulation of P-glycoprotein -mediated efflux by methoxypolyethylene glycol-block-Polycaprolactone amphi -philic diblock copolymers[J].Pharm. Res.2004, 21(4):1489–1497.
19. Collnot EM., Baldes C, Wempe M F, et al, Influence of vitamin E TPGS poly(ethylene glycol) chain length on apical efflux transporters in Caco-2 cell monolayers[J], J. Control.Release,2006, 111(5): 35–40.
20. Ould O L., Noppe M, Langlois X., et al, Self-assembling PEG-p(CL-co-TMC) copolymers for oral delivery of poorly water-soluble drugs: a case study with risperidone[J].J. Control. Release 2005,102(3): 657–668.
21. Norris DA., Puri N, Sinko PJ, et al.The effect of physical barriers and properties on the oral absorption of particulates[J]. Adv. Drug Deliv. Rev,1998,34(2): 135–154.
22. Mathot F, Beijsterveldt LV, Preat V, et al, Intestinal uptake and biodistribution of novel polymeric micelles after oral administration[J]. J. Control. Release, 2006,111(6) : 47–55.
23. Mathot F, Rieux A, Arien A, et al,Transport mechanisms of mmePEG(750)P (CL-co-TMC) polymeric micelles across the intestinal barrier[J]. J. Control.Release ,2007,124(3):134–143.23.
24. Hasselt PM, Janssens GE, Slot TK, et al,The influence of bile acids on the oral bioavailability of vitamin K encapsulated in polymeric micelles[J]. J. Control. Release 2009,133 (7): 161–168.
25. Conner SD, Schmid SL, et al.Regulated portals of entry into the cell[J]. Nature ,2003,422(9):37–44.
26. Simons K, Toomre D, et al.Lipid rafts and signal transduction[J]. Nature Review,Molecular Cell Biology 2000,1 (5):31–39.
27. Carver LA, Schnitze JE, et al.Caveolae: mining little caves for new cancer targets [J].Nature Review Cancer 2003,3(3): 571–581.
28. Bronich TK, Keifer PA., Shlyakhtenko LS, et al.Polymer micelle with cross-link -ed ionic core[J]. Journal of the American Chemical Society,2005,127(2):8236–8237.
29. Vogel U, Sandvig K, Deurs BV, et al.Expression of caveolin-1 and polarized formation of invaginated caveolae in Caco-2 and MDCK II cells[J]. J Cell Sci,1998, 111(8): 825–832.
30. Sahay G, Kim JO,Kabanov AV, et al,The exploitation of differential endocytic pathways in normal and tumor cells in the selective targeting of nanoparticulate chemotherapeutic agents[J]. Biomaterials, 2010,31(3): 923–933.
31. Jenkins PG., Howard K.A. , Blackball NW, et al. Microparticulate absorption from the rat intestine[J]. J. Control. Release,1994,29(7):339-350.
32. Jani P, Halbert G W, Langridge J, et al, Nanosphere and microsphere uptake via Peyer's patches: observation of the rate of uptake in the rat after a single oral dose[J]. J. Pharm. Pharmacol,1989,41(3):809–812.
33. Desai M P., Labhasetwar V, Amidon G L, et al, Characterization of nanoparticle uptake by endothelial cells[J] .Pharm. Res, 1996,13(3):1838–1845.
34. Lai SK., Hida K., Chen C., et al, Characterization of the intracellular dynamics of a non-degradative pathway accessed by polymer nanoparticles[J]. J. Controll Release ,2008125(1):107–111.
35. Eldridge J. H., Hammond C. J., Meulbroek A. et al, J. Controll Release ,1990,11(7): 205–214 .
36. Ma Z, Lim LY, et al.Uptake of chitosan and associated insulin in caco-2 cell monolayers: a comparison between chitosan molecules and chitosan nanopart -icles[J]. Pharmaceutical Research,2003 20(4): 1812–1819.
37. Huang M,. Ma Z, Khor E, et al, Uptake of FITC-chitosan nanoparticles by A549 cells[J]. Pharmaceutical Research ,2002,19(1): 1488–1494.
38. Hillery, P.U. Jani AT. et al.Florence, Comparative quantitative study of lymphoid and non-lymphoid uptake of 50 nm polystyrene particles[J]. J. Drug Targeting ,1994,2 (8):151–156.
39. Mathiowitz E, Jacob J, Jong Y, G et al,Biologically Erodable Microspheres as Potential Oral Drug Delivery Systems[J]. Nature,1997, 386(5): 410-414.
40. Kriwet B, Walter E,. Kissel T . et al.Synthesis of bioadhesive poly(acrylic acid) nano- and microparticles using an invers emulsion polymerization method for the entrapment of hydrophilic drug candidates[J].J. Controll Release ,1998, 56(4), 149–158 .
41. Harush-Frenkel O, Debotton N, Benita S, et al, Targeting of nanoparticles to the clathrin-mediated endocytic pathway[J]. Biochemical and Biophysical Research Communications ,2007,353(7):26–32.
42. Harush-Frenkel O, Rozentur E,. Benita S, et al,, Surface charge of Nanoparticles determines their endocytic and transcytotic pathway in polarized MDCK cells[J]. Biomacromolecules,2008,9(6):435–443.
43. Rejman J, Bragonzi A, Conese M, et al.Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes[J]. Molecular Therapy,2005,12(4): 468–474.
44. Zhang LW, Monteiro-Riviere NA, et al.Mechanisms of quantum dot nanoparticle cellular uptake[J].Toxicological Sciences,2009 110(7):138–155.
45. LüJM, Wang X, Marin M C, et al,,Currentadvances in research and clinical applications of PLGA-based nanotechnology[J].Expert Review of Molecular Diagnostics,2009, 9(9):325–341.
46. Lahtinen U, Honsho M., Parton RG, et al,Involvement of caveolin-2 in caveolar biogenesis in MDCK cells[J]. Federation of the Societies of Biochemistry and Molecular Biology Letters,2003, 538 (4): 85–88.
47. Panyam J, Labhasetwar V, et al. Dynamics of endocytosis and exocytosis of Poly(D, Llactide-co-Glycolide) nanoparticles in vascular smooth muscle cells[J]. Pharmaceutical Research,2003, 20(4): 212–220.
48. Qaddoumi MG, Ueda H, Yang J, et al,The characteristics and mechanisms of uptake of PLGA nanoparticles in rabbit conjunctival epithelial cell layers[J]. Pharmaceutical Research, 2004,21(3): 641–648.
49. Vasir JK, Labhasetwar V, et al. Quantification of the force of anoparticle-cellmembrane interactions and its influence on intracellular trafficking of nanoparticles[J]. Biomaterials,2008, 29(6) : 4244–4252.
50. Sahay G,.Kim JO,.Kabanov A.V, et al,The exploitation of differential endocytic pathways in normal and tumor cells in the selective targeting of nanoparticulate chemotherapeutic agents[J]. Biomaterials,2010, 31(4): 923–933.
51. Hotter D,Dressman JB, et al.Influence of physicochemical properties on dissolution of drugs in gastrointestinal tract[J]. Advanced Drug Delivery Reviews,2001,46(8):75-87.
52. Russel-Jones GJ, et al.The potential use of receptor-mediated endocytosis for oral drug delivery[J].Adv. Drug Deliv. Rev,2001, 46(4): 59–73.
53. Russel-Jones GJ, et al. Use of targeting agents to increase uptake and localization of drugs to the intestinal epithelium[J]. J. Drug Target,2004,12(3):113–123.
54. Mahmud A,. Xiong XB, Montazeri H et al, Polymeric micelles for drug targeting[J]. J. Drug Target,2007,15(3): 553–584.
55. Torchilin VP, et al. Targeted polymeric micelles for delivery of poorly soluble drugs[J]. Cell. Mol. Life Sci,2004, 61(8): 2549–2559.
56. Vlerken LE., Vyas TK, Amiji MM., et al.Poly(ethylene glycol)-modified nanocarriers for tumor-targeted and intracellular delivery[J]. Pharm. Res,2007, 24(7): 1405–1414.
57. Daugherty AL, Mrsny RJ, et al.Transcellular uptake mechanisms of the intestinal epithelial barrier[J]. Part one, PSTT,1999, 2 (3): 144–151.
58. Francis MF,. Cristea M, Winnik FM., et al.Exploiting the vitamin B-12 pathway to enhance oral drug delivery via polymeric micelles[J]. Biomacromolecules, 2005,6(2):2462–2467.
59. Anderle P, Huang Y, Sadee W,. et al. Intestinal membrane transport of drugs and nutriments: genomics of membrane transporters using expression microarrays[J].Eur. J. Pharm. Sci,2004,21(1) :17–24.
60. Hansen JE, Weisbart RH, Nishimura RN, et al. Antibody mediated transduction of therapeutic proteins into living cells[J]. Sci. World,2005, 5(6): 782–788.
61. Ambudkar SV, Kimchi-Sarfaty C, Sauna ZE., et al, Pglycoprotein: from genomics to mechanism[J]. Oncogene ,2003,22(3):7468–7485.
62. Higgins CF, et al.Molecular basis of multidrug resistance mediated by Pglycoprotein[J]. Curr. Opin. Biotechnol, 1991,2(1): 278–281.
63. Lavie Y, Fiucci G, Liscovitch M, et al. Up-regulation of caveolae and caveolar constituents in multidrug-resistant cancer cells[J]. J. Biol. Chem, 1998,273(8): 32380–32383.
64. Minato S, Iwanaga K, Kakemi M., et al.. Application of polyethyleneglycol (PEG)-modified liposomes for oral vaccine: effect of lipid dose on systemic and mucosal immunity[J]. J. Control Rel, 2003. 89(9):189–197.
65.郑中华,张以忠,方青, et al.Pgp动力学研究进展[J].吉林医药学院学报,2006,7(4):1-5.
66. Ho RH, Kim RB, et al.Transporters and drug therapy: implications for drug disposition and disease[J]. Perspect. Clin. Pharmacol, 2005 , (1)260–277.
67. Wang Q, Bhardwaj RK, Herrera-Ruiz D, et al. Expression of multiple drug resistance conferring proteins in normal Chinese and Caucasian small and large intestinal tissue samples[J]. Mol. Pharm,2004, 1(3): 447–454.
68. Peng K, Cluzeaud F, Bens M, et al.Tissue and cell distribution of the multidrug resistanceassociated protein (MRP) in mouse intestine and kidney[J]. J.Histochem.Cytochem,197,47(5): 757–768.
69. Zimmermann C, Gutmann H, Hruz P, Gutzwiller JP, et al.Mapping of multidrug resistance gene 1 and multidrug resistance-associated protein isoform 1 to 5 mRNA expression along the human intestinal tract[J]. Drug Metab. Dispos,2005 ,33(7): 219–224.
70. Scheffer GL, Maliepart M, Pijnenborg AC, et al. Breast cancer resistance protein is localized at the plasma membrane in mitoxantrone- and topotecan-resistant cell lines[J]. Cancer Res,2000,60(3): 2589–2593.
71. Eisenblatter T, Galla H, et al.A new multidrug resistance protein at the blood–brain barrier[J]. Biochem. Biophys. Res. Commun. 2002,293(5): 1273–1278.
72. Collnot EV, Baldes C, Wempe MF., et al. Mechanism of inhibition of P-glycoprotein mediated efflux by vitamin E TPGS: influence of ATPase activity and membrane fluidity[J]. Mol. Pharm,2007,4(7):465–474.
73. Dintaman JM., Silverman JA., et al. Inhibition of P-glycoprotein by D-tocopheryl polyethylene glycol 1000 succinate (TPGS) [J]. Pharm. Res. 1999, 16(3):1550–1556.
74. Zhao H.Z., Tan EC, Yung LY,et al. Potential use of cholescalciferol polyethylene glycol succinate as a novel pharmaceutical additive[J]. J. Biomed. Mater. Res. Part A,2008 ,84(8):954–964.
75. Batrakova EV, WF Li, Miller D.W, et al.Mechanism of sensitization of MDR cancer cells by Pluronic block copolymers: selective energy depletion[J]. Br. J. Cancer ,2001,85(2): 1987–1997.
76. Batrakova EV, Li S, Alakhov VY, et al.Sensitization of cells overexpressing multidrug-resistant proteins by pluronic P85[J]. Pharm. Res.2003,20(2) : 1581–1590.
77. Batrakova EV, Li S, Alakhov VY, et al .Effect of pluronic P85 on ATPase activity of drug efflux transporters[J].Pharm. Res,2004,21(5):2226–2233.
78. Zhu S, Huang R, Hong M, et al .Effects of polyoxyethylene (40) stearate on the activity of P-glycoprotein and cytochrome P450[J]. Eur. J. Pharm. Sci,2009,37(4): 573–580.
79. Zastre J, Jackson J, Bajwa M, et al Enhanced cellular accumulation of a P-glycoproteinsubstrate, rhodamine-123, by Caco-2 cells using low molecular weight methoxypolyethylene glycol-blockpolycaprolactone diblock copolymers[J]. Eur. J. Pharm. Biopharm,2002,54(6) :299–309.
2. Amidon G, Lennernas H, Shah V, et al. A theoretical basis for a bioph- armaceutical drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability[J]. Pharm. Res,1995, 12(3):413–420
3. Zhang Y,Benet LZ. The gut as a barrier to drug absorption:combined role of cytochrome P450A and P-g1ycoprotein.Clin Pharmcokinet,200l,40(3):159-163
4. Chen CJ, Chin JE, Ueda K, et al. Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistance human cells[J]. Cell, 1986,47(3): 381–389
5. Ambudkar SV, Kimchi-Sarfaty C, Sauna ZE, et al. Pglycoprotein: from genomics to mechanism[J]. Oncogene ,2003,22(47):7468–7485
6. Akiyama SI, Cornwell MM, Kuwano M, et al. Most drugs that reverse multidrug resistance also inhibit photoaffinity labelling of P-glycoprotein by vinblastine analog. Mol. Pharmacol. 1988. 33(2): 144–147
7. Terao T, Hisanaga E, Sai Y,et al. Active secretion of drugs from the small intestinal epithelium in rats by P-glycoprotein functioning as an absorption barrier[J]. J. Pharm .Pharmacol, 1996; 48(10): 1083-1089
8.田治科,胡富强,丁建潮,等.西罗莫司固体脂质纳米粒的制备及在大鼠体内的相对生物利用度[J].中国医药工业杂志;2006, 37(9):610-613.
9.郑中华,张以忠,方青,Pgp动力学研究进展[J],吉林医药学院学报,2006,7(2):1-5
10. Bansal T, Akhtar N, Jaggi M , et al. Novel formulation approaches for optimising delivery of anticancer drugs based on P-glycoprotein modulation[J]. Drug Discovery Today, 2009,14(21-22): 1067-1074
11. Sachs B K. Lipid excipients Peceol and Gelucire 44/14 decrease P-glycoproteinmediated efflux of rhodamine 123 partially due to modifyingP-glycoprotein protein expression within Caco-2 cells[J]. J. Pharm. Pharm,Sci. 2007,10(3): 319–331
12. Dabholkar RD, Sawant RM., Mongayt D A. Polyethyleneglycol–phosphatidy -lethanolamine conjugate (PEG–PE)-based mixed micelles: Some properties, loading with paclitaxel, and modulation of P-glycoprotein-mediated efflux[J]. Int. J. Pharm, 2006,315(1-2): 148-157
13.田庆锷,张慧,李玛琳等.P一糖蛋白与多药耐药的关系[J]。药品评价,2005, 2(3):225-226
14.陈东,丁平田,邓意辉等.聚合物胶束用于口服给药的研究进展[J].药学学报, 2010, 45 (5): 560?564
15. Reif S, Kingreen D, Kloft C, et al. Bioequivalence investigation of high-dose etoposide and etoposide phosphate in lymphoma patients[J]. Cancer Chemother Pharmacol, 2001,48(2):134-140
16. Cheol-Hee C, Joon-Ho K, Sang-H yun K.Reversal of P-glycoprotein- mediated MDR by 5,7,3',4',5'-pentamethoxyflavone and SAR[J]. Biochem Biophy Res commomm,2004, 320(3):672-678
17. Lipinski CJ. Poor aqueous solubility– an industry wide problem in drug discovery[J], Am. Pharm Rev. 2002, 582–585
18. Kim RB, Wandel C, Leake B, et al. Interrelationship between substrates and inhibitors of human CYP3A and P-glycoprotein[J]. Pharm Res. 1999,16(3): 408–414
19.吴迪,李高,黄建耿,等.更昔洛韦的小肠吸收与P-糖蛋白药泵作用的关系研究.中国药师.2004; 7(6):408-411
20. Xiuhua Ren, Luqin Si, Gao Li, et al. Pharmaceutical Excipients Inhibit Cytochrome P450 Activity in Cell Free Systems and After Systemic Administration[J]. Eur. J. Pharm. Biopharm. 2008,70(1:) 279-288
21. Kataoka K, Harada A, Nagasaki Y, Block copolymer micelles for drug delivery:design, characterization and biological significance[J], Adv. Drug Deliv. Rev. 2001, 47(6) :113–131
22. Savic R, Luo L, Eisenberg AD. et al, Micellar nanocontainers distribute to defined cytoplasmic organelles[J], Science ,2003,300(4): 615–618
23. Gaucher G, Dufresne M, San V, et al. Block copolymer micelles: preparation, characterization and application in drug deliver y [J]. J. Control. Res,2005(2),109 : 169–188
24. Shen D,Lu L J,Li Y,Advance on amphiphilic block copolymer micelles used in drug carrier[J]. Acta Academiae Medicinae CPAF,2008,17(3):237-240
25. Agata AE, Tianyi MK, Katherine SJ , Enhancement of carboplatin toxicity by Pluronic block copolymers[J].J. Control Release 106 (2005) 188– 197
26. Alakhov V, Moskaleva E., Bathakova EV, et al. Hypersensitization of multidrug resistant human ovarian carcinoma cells by Pluronic P85 block copolymer[J], Bioconjug. Chem. 1996 7 (2) 209–216
27. Kabanov AV., Batraova EV, Alahov VY, Pluronic block copolymers for overcoming drug resistance in cancer [J], Adv. Drug Deliv. Rev. 54 (5) (2002) 759–779.
28. Vecnnce A, Li S. Mandeville R, Kabanov AV,et al. Hypersensitizing effect of Pluronic L61 on cytotoxic activity, transport, and subcellular distribution of doxorubicin inmultiple drug-resistant cells[J], Cancer Res,1996,56 (16) :3626–3629
29. Mathot F, Beijsterveldt LV, Preat V, et al. Intestinal uptake and biodistribution of novel polymeric micelles after oral administration, J. Control. Release[J], 2006,111(1-2) : 47–55
30. Mahesh D C , Yogesh P, Jayanth P.Susceptibility of nanoparticle-encapsulated paclitaxel to P-glycoprotein-mediated drug efflux[J]. Int.J.Pharm.2006,320 (1-2) 150–156
31. Ranaldi G, Consalvo R, Sambuy Y, et al. Permeability characteristics of parental and clonal human intestinal Caco-2 cell lines differentiated in serum-supplemented and serum-free media[J]. Toxicology in Vitro, 2003,17(5):761-767
32.齐云,王敏,蔡润兰. Caco-2细胞模型在口服药物转运机制研究中应用的进展[J].中国药学杂志,2009 44 (17):1281-1284
33.张生芳,李娟亚.药物渗透与吸收评价方法的研究进展.太传统医药[J]. 2010, 6(5):129-131
34. Batrakova E, Lee S, Li S, et al. Fundamental relationships between the composition of Pluronic block copolymers and their hypersensitization effect in MDR cancer cells[J]. Pharm. Res, 1999,16 (9) :1373– 1379
35. Agata AE, Tlanyi MK, Katherrine SJ , Enhancement of carboplatin toxicity by Pluronic block copolymers[J].J. Controll Release,2005, 106 (1-2) 188– 197
36. Elena V, Batrakova E ,Huai YH, et al. Effect of Pluronic P85 Unimers and Micelles on Drug Permeability in Polarizerd BBMEC and caco-2 Cells [ J ] .Pharma Research,1998,15(10): 1525-1532
37. Hasseltt PM, Janssens GE, Slot TK, et al. The influence of bile acids on the oral bioavailability of vitamin K encapsulated in polymeric micelles.[J].J. Controll Release,2009,133 (2): 161–168
38. Jason Z, John J, Mandeep B , et al. Enhanced cellular accumulation of a P-glycoprotein substrate,rhodamine-123, by caco-2 cells using low molecular weight methoxypolyethylene glycol-block-polycaprolactone diblock copolymers[J]. Eur J.Pharm Bio, 2002,54 (3) :299–309
39.王永忠.紫杉醇嵌段共聚物胶束给药系统克服肿瘤多药耐药性的研究:[学位论文],上海复旦大学,2007
40.董宇,张英丰,杨庆等。黄连提取物在大鼠肠外翻实验中的吸收研究[J].中国中药杂志.2008,33(9):1056-1061
41. Pierri E, Avgoustakis K, Poly(lactide)-poly(ethylene glycol) micelles as acarrier for griseofulvin[J], J. Biomed. Mater. Res. Part A,2005, 75 (3) :639–647
42. Zao JX,Jiang LQ,Qiu Y,Solubilization of Polycyclic Aromatic Hydrocarbons in F127 and P123 Aqueous Micellar Solutions [J]. Chem. J. Chinese Universities.2001,22(4):165-170
43. Li HY. Micellization behavior of Pluronic block copolymers in aqueous solution and its application on drug carrier [D] . Taiyuan :Taiyuan University of Technology,2007
44.徐运杰,方热军.外翻肠囊法的应用研究[J],饲料研究,2009,12(2):9-12
45. Tamihide M, Eiji K, Sachiyo Y, et al.Determination of P-Glycoprotein ATPase Activity Using Luciferase Biol. Pharm[J]. Bull, 2006,29(3) :560-564
46. Frédéric M, des RA. Ari?n A, et al.Transport mechanisms of mmePEG750P -(CL-co-TMC) polymeric micelles across the intestinal barrier[J].J.Controll Release.2007 124 (3): 134–143
47. Zastre J, Jackson J, Burt H, Evidence for modulation of P-glycoprotein–mediated efflux by methoxypolyethylene glycol-block-polycaprolactone amphiphilic diblock copolymers[J]. Pharm Res. 2004,21(8):1489-1496
48. Elena V B, Shu L, Li YL .Effect of Pluronic P85 on ATPase Activity of Drug Efflux Transporters[J], Pharm Res. 2004 ,21(12): 2226–2233
49. Gao X, Wang T, Wu J,et al. Quantum dots for tracking cellular transport of lectin-functionalized nanoparticles,Biochem. Biophys[J]. Res. Commun,2008,77 (1) : 35–40
50. Ma Z, Lim LY, Uptake of chitosan and associated insulin in Caco-2 cell monolayers: a comparison between chitosan molecules and chitosan nanoparticles[J]. Pharm. Res,2003,20 (11): 1812–1819
51. Roger E, Lagarce F, Garcion E, et al.Lipid nanocarriers improve paclitaxel transport throughout human intestinal epithelial cells by using vesicle-mediated transcytosis[J].J Controlled Release, 2009, 140 (2) 174–181
52. Yoo HS, Park TG, Biodegradable nanoparticles containing protein–fatty acid complexes for oral delivery of salmon calcitonin[J]. J. Pharm Sci,2004,93 (2): 488–495
53. Sahay G Alakhova D Y, Kabanov AV, Endocytosis of nanomedicines[J]. J .Controll Release ,2010,145 (3):182–195
54. Cai CX ,Chen JW.Overexperssion of Caveolin-1 Induces Alter Ation of Multidrug Resistance in Hs578T Breast Adenocarcinoma Cells[J].Int. J. Cancer.2004, 111(4): 522–529
55. Sahay G, Batrakova EV, Kabanov AV. Different Internalization Pathways of PolymericMicelles and Unimers and Their Effects on Vesicular Transport[J].Bioconjugate Chem. 2008, 19(10): 2023–2029
56. Garrigues A., Escargueil AE., Orlowski S, The multidrug transporter, P-glycoprotein , actively mediates cholesterol redistribution in the cell membrane[J].Proc. Natl. Acad. Sci. U.S.A. 2002 ,99(16):10347–10352.
57. Shao K, Huang RQ, Li JF .Angiopep-2 modified PE-PEG based polymeric micelles for amphotericin B delivery targeted to the brain[J] ,J.Controll Release ,2010,147 (1) :118–126
58. Bernard VE, Michiel VS, Raf M, et al. Itraconazole/TPGS/Aerosil 200 solid dispersions Characterization, physical stability and in vivo performance[J]. Eur J Pharm Sci,2009, 38 (3) 270–278
59. Wei CN, Bing G, HaoW, et al.Development and evaluation of novel itraconazole-loaded intravenous nanoparticles[J].Int J Pharm. 2008,362 (1-2): 133–140
60. Yi Yw, Yoon HJ, Kim BO.A mixed polymeric micellar formulation of itraconazole:Characteristics, toxicity and pharmacokinetics[J]. J Controll Releas -e ,2007,117 (1): 59–67
61. Wei YG, Jasmine T, Dave A. et al.High bioavailability from nebulized Itraconazole nanoparticle dispersions with biocompatible stabilizers[J].Int J Pharm,2008,361 (1-2):177–188
62. Hong JY , Kim JK , Song YK et al.A new self-emulsifying formulation of itraconazole with improved dissolution and oral absorption[J].J Controll Release ,2006,110 (2) 332– 338
63.刘艳杰,项荣武.星点设计效应面法在药学试验设计中的应用[J].中国现代应用药学杂志2007,24(6):455-457
64.吴伟,崔光华,陆彬.实验设计中多指标的优化:星点设计和总评“归一值”的应用[J].中国药学杂志2000,35 (8):530-533
65. Zhang XH, Jackson JK., Burt HM. Determination of surfactant critical micelle Research article concentration by a novel fluorescence depolarization technique[J].J. Biochem.Biophys Methods. 1996, 31 (3-4): 145-150
66.李海燕.Pluronic两亲嵌段共聚物的胶束化行为及其作为药物载体的初步应用:[学位论文],太原理工大学
67.陆冬云,温浩,刘会洲等.球形嵌段共聚物胶束的温度效应.物理化学学报,2004,20(l):38-42
68. Sang CL,Chulhee K,Iek CK,et al. Polylnerie micelles of Poly(2-ethyl-2-oxazoline)- bloek-Poly(εcaProlactone) copolymer as a carrier for Paclitaxel[J] .Control Rel,2003, 89(5):437-446
69. Vinayak PS, Damon S, Jean L .Enhancement of oral bioavailability of poorly water-soluble drugs by poly(ethylene glycol)-block-poly(alkyl acrylate-co-methacrylic acid) self-assemblies[J].J Controll Release ,2005.104 (2): 289–300
70. Zerrin S, Nilufer Y, Tamer B. Investigation of pluronic and PEG-PE micelles as carriers of meso-tetraphenyl porphine for oral administration[J] . Int J Pharm, 2007, 332( 1-2): 161-167
71. Gaucher G , Satturwar P, Jones MC,Polymeric micelles for oral drug delivery[J].Eur J Pharma and Biopharm,2010. 76 (2) :147–158
72. Christiansen A, Backensfeld T, Weitschies W. Effects of non-ionic surfactants on in vitro triglyceride digestion and their susceptibility to digestion by pancreatic enzymes[J] .Eur J Pharm Sci, 2010, 41 (2): 376-382
73.黄建耿.药用辅料抑制肠道P-糖蛋白药泵作用研究:[学位论文],华中科技大学,2008
74.韩丽妹.基于逆转肿瘤多药耐药性的共聚物胶束给药体系的研究:[学位论文],复旦大学,2006
75.向苗,温敏性聚合物胶束的制备及性质研究:[学位论文],兰州大学,2008
76. Kumar V, Wang L, Riebe M, et al,Formulation and Stability of itraconazole and odanacatib nanoparticles: governing physical parameters [ J ] .Molecular Pharmaceutics.2009,6(4 ):1118-1124
77. Hu YQ,Zheng LY,Qian CQ.Small intestinal absorbtion study of phone red as an ionic drug[J].J China Pharm Univ1996,27(1):355-359
78. Rogera E, Lagarceb F, Garcionb E, et al.Reciprocal competition between lipid nanocapsules and P-gp for paclitaxel transport across Caco-2 cells[J]. Eur J Pharm Sci,2010. 40 (5) :422–429
79. van Hasselt PM, Janssens GE, Slot TK, et al The influence of bile acids on the oral bioavailability of vitamin K encapsulated in polymeric micelle s [J].J Controll Release,2009, 133 (2): 161–168
1. Sezgin Z, Yuksel N, Baykara T, et al. Investigation of pluronic and PEG-PE micelles as carriers of meso-tetraphenyl porphine for oral administration[J]. Int. J. Pharm. 2007,332(3): 161-167.
2. Lavelle EC, et al.Targeted delivery of drug to gastrointestinal tract[J].Critical Revies in Therapeutic Drug Carrirer Systems. 2001,18(4):341-386.
3. Mira F.F, Mariana C, Fran?oise M, et al. Polymeric micelles for oral drug delivery:Why and how[J]. Pure Appl. Chem, 2004, 76(1):1321–1335.
4. Benet, L. Z. , Izumi,T., Zhang, Y., Silverman, J. A., Wacher, V. J., et al.Intestinal MDR transport proteins and P-450 enzymes as barriers to oral drug delivery[J]. J. Controlled Release. 1999,62(9): 25-31.
5. Hirunpanich, V. , Murakoso, K. , Sato, H. , et al. Inhibitory effect of docosahexaen -oic acid (DHA) on the intestinal metabolism of midazolam: In vitro and in vivo studies in rats[J]. Int. J. Pharm.,2008,351(7): 133-143.
6. Okada J, Cohen S ,R Langer, et al. In vitro evaluation of polymerized liposomes as an oral drug delivery system[J]. Pharm Res 1995, 12(8):. 576-582.
7. Kataoka K, Harada A, Nagasaki Y, et al.Block copolymer micelles for drug delive ry:design, characterization and biological significance[J]. Adv. Drug Deliv. Rev. 2001, 47(5) :113–131.
8. Savic R, Luo L, Eisenberg A, D. et al, Micellar nanocontainers distribute to defined cytoplasmic organelles[J].Science ,2003,300(4): 615–618.
9. Gaucher G, Dufresne M, San V, et al, Block copolymer micelles: preparation, characterization and application in drug delivery[J], J. Control. Rel. 2005,109(7) : 169–188.
10. Lev Bromberg, et al. Polymeric micelles in oral chemotherapy[J]. J. Controlled Release, 2008,128(1) : 99-112.
11. DeMario M, Ratain M, et al. Oral chemotherapy: rationale and future directions[J]. J.Clin. Oncol.1998, 16(7): 2557–2567.
12. Anderson J, Van Itallie C, et al.Tight junctions and the molecular basis for regulation ofparacellular permeability[J]. Am. J. Physiol. 1995,269(5), 467–475.
13. Gaurav S, Alakhova Y, Alexander V, et al.Endocytosis of nanomedicines[J]. J Controll Release 2010,145(6) :182–195.
14. Norris D., Puri N, Sinko P, et al.The effect of physical barriers and properties on the oral absorption of particulates[J], Adv. Drug Deliv. Rev. 1998,34(7) : 135–154.
15. Mathot F, Schanck A, Bambeke F A. et al, Passive diffusion of polymeric surfactants across lipid bilayers[J] J.Control. Release ,2007,120(1): 79–87.
16. Sezgin Z,. Yuksel N, Baykara T, et al.Investigation of pluronic and PEG-PE micelles as carriers of meso-tetraphenyl porphine for oral administration[J] Int. J.Pharm,2007,332(2): 161–167.
17. Francis M, Cristea M, Yang Y, et al, Engineering polysaccharidebased polymeric micelles to enhance permeability of cyclosporin A across Caco-2 cells[J]. Pharm. Res,2005,22(4) : 209–219.
18. J. Zastre, J. Jackson, H. Burt, et al.Evidence for modulation of P-glycoprotein -mediated efflux by methoxypolyethylene glycol-block-Polycaprolactone amphi -philic diblock copolymers[J].Pharm. Res.2004, 21(4):1489–1497.
19. Collnot EM., Baldes C, Wempe M F, et al, Influence of vitamin E TPGS poly(ethylene glycol) chain length on apical efflux transporters in Caco-2 cell monolayers[J], J. Control.Release,2006, 111(5): 35–40.
20. Ould O L., Noppe M, Langlois X., et al, Self-assembling PEG-p(CL-co-TMC) copolymers for oral delivery of poorly water-soluble drugs: a case study with risperidone[J].J. Control. Release 2005,102(3): 657–668.
21. Norris DA., Puri N, Sinko PJ, et al.The effect of physical barriers and properties on the oral absorption of particulates[J]. Adv. Drug Deliv. Rev,1998,34(2): 135–154.
22. Mathot F, Beijsterveldt LV, Preat V, et al, Intestinal uptake and biodistribution of novel polymeric micelles after oral administration[J]. J. Control. Release, 2006,111(6) : 47–55.
23. Mathot F, Rieux A, Arien A, et al,Transport mechanisms of mmePEG(750)P (CL-co-TMC) polymeric micelles across the intestinal barrier[J]. J. Control.Release ,2007,124(3):134–143.23.
24. Hasselt PM, Janssens GE, Slot TK, et al,The influence of bile acids on the oral bioavailability of vitamin K encapsulated in polymeric micelles[J]. J. Control. Release 2009,133 (7): 161–168.
25. Conner SD, Schmid SL, et al.Regulated portals of entry into the cell[J]. Nature ,2003,422(9):37–44.
26. Simons K, Toomre D, et al.Lipid rafts and signal transduction[J]. Nature Review,Molecular Cell Biology 2000,1 (5):31–39.
27. Carver LA, Schnitze JE, et al.Caveolae: mining little caves for new cancer targets [J].Nature Review Cancer 2003,3(3): 571–581.
28. Bronich TK, Keifer PA., Shlyakhtenko LS, et al.Polymer micelle with cross-link -ed ionic core[J]. Journal of the American Chemical Society,2005,127(2):8236–8237.
29. Vogel U, Sandvig K, Deurs BV, et al.Expression of caveolin-1 and polarized formation of invaginated caveolae in Caco-2 and MDCK II cells[J]. J Cell Sci,1998, 111(8): 825–832.
30. Sahay G, Kim JO,Kabanov AV, et al,The exploitation of differential endocytic pathways in normal and tumor cells in the selective targeting of nanoparticulate chemotherapeutic agents[J]. Biomaterials, 2010,31(3): 923–933.
31. Jenkins PG., Howard K.A. , Blackball NW, et al. Microparticulate absorption from the rat intestine[J]. J. Control. Release,1994,29(7):339-350.
32. Jani P, Halbert G W, Langridge J, et al, Nanosphere and microsphere uptake via Peyer's patches: observation of the rate of uptake in the rat after a single oral dose[J]. J. Pharm. Pharmacol,1989,41(3):809–812.
33. Desai M P., Labhasetwar V, Amidon G L, et al, Characterization of nanoparticle uptake by endothelial cells[J] .Pharm. Res, 1996,13(3):1838–1845.
34. Lai SK., Hida K., Chen C., et al, Characterization of the intracellular dynamics of a non-degradative pathway accessed by polymer nanoparticles[J]. J. Controll Release ,2008125(1):107–111.
35. Eldridge J. H., Hammond C. J., Meulbroek A. et al, J. Controll Release ,1990,11(7): 205–214 .
36. Ma Z, Lim LY, et al.Uptake of chitosan and associated insulin in caco-2 cell monolayers: a comparison between chitosan molecules and chitosan nanopart -icles[J]. Pharmaceutical Research,2003 20(4): 1812–1819.
37. Huang M,. Ma Z, Khor E, et al, Uptake of FITC-chitosan nanoparticles by A549 cells[J]. Pharmaceutical Research ,2002,19(1): 1488–1494.
38. Hillery, P.U. Jani AT. et al.Florence, Comparative quantitative study of lymphoid and non-lymphoid uptake of 50 nm polystyrene particles[J]. J. Drug Targeting ,1994,2 (8):151–156.
39. Mathiowitz E, Jacob J, Jong Y, G et al,Biologically Erodable Microspheres as Potential Oral Drug Delivery Systems[J]. Nature,1997, 386(5): 410-414.
40. Kriwet B, Walter E,. Kissel T . et al.Synthesis of bioadhesive poly(acrylic acid) nano- and microparticles using an invers emulsion polymerization method for the entrapment of hydrophilic drug candidates[J].J. Controll Release ,1998, 56(4), 149–158 .
41. Harush-Frenkel O, Debotton N, Benita S, et al, Targeting of nanoparticles to the clathrin-mediated endocytic pathway[J]. Biochemical and Biophysical Research Communications ,2007,353(7):26–32.
42. Harush-Frenkel O, Rozentur E,. Benita S, et al,, Surface charge of Nanoparticles determines their endocytic and transcytotic pathway in polarized MDCK cells[J]. Biomacromolecules,2008,9(6):435–443.
43. Rejman J, Bragonzi A, Conese M, et al.Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes[J]. Molecular Therapy,2005,12(4): 468–474.
44. Zhang LW, Monteiro-Riviere NA, et al.Mechanisms of quantum dot nanoparticle cellular uptake[J].Toxicological Sciences,2009 110(7):138–155.
45. LüJM, Wang X, Marin M C, et al,,Currentadvances in research and clinical applications of PLGA-based nanotechnology[J].Expert Review of Molecular Diagnostics,2009, 9(9):325–341.
46. Lahtinen U, Honsho M., Parton RG, et al,Involvement of caveolin-2 in caveolar biogenesis in MDCK cells[J]. Federation of the Societies of Biochemistry and Molecular Biology Letters,2003, 538 (4): 85–88.
47. Panyam J, Labhasetwar V, et al. Dynamics of endocytosis and exocytosis of Poly(D, Llactide-co-Glycolide) nanoparticles in vascular smooth muscle cells[J]. Pharmaceutical Research,2003, 20(4): 212–220.
48. Qaddoumi MG, Ueda H, Yang J, et al,The characteristics and mechanisms of uptake of PLGA nanoparticles in rabbit conjunctival epithelial cell layers[J]. Pharmaceutical Research, 2004,21(3): 641–648.
49. Vasir JK, Labhasetwar V, et al. Quantification of the force of anoparticle-cellmembrane interactions and its influence on intracellular trafficking of nanoparticles[J]. Biomaterials,2008, 29(6) : 4244–4252.
50. Sahay G,.Kim JO,.Kabanov A.V, et al,The exploitation of differential endocytic pathways in normal and tumor cells in the selective targeting of nanoparticulate chemotherapeutic agents[J]. Biomaterials,2010, 31(4): 923–933.
51. Hotter D,Dressman JB, et al.Influence of physicochemical properties on dissolution of drugs in gastrointestinal tract[J]. Advanced Drug Delivery Reviews,2001,46(8):75-87.
52. Russel-Jones GJ, et al.The potential use of receptor-mediated endocytosis for oral drug delivery[J].Adv. Drug Deliv. Rev,2001, 46(4): 59–73.
53. Russel-Jones GJ, et al. Use of targeting agents to increase uptake and localization of drugs to the intestinal epithelium[J]. J. Drug Target,2004,12(3):113–123.
54. Mahmud A,. Xiong XB, Montazeri H et al, Polymeric micelles for drug targeting[J]. J. Drug Target,2007,15(3): 553–584.
55. Torchilin VP, et al. Targeted polymeric micelles for delivery of poorly soluble drugs[J]. Cell. Mol. Life Sci,2004, 61(8): 2549–2559.
56. Vlerken LE., Vyas TK, Amiji MM., et al.Poly(ethylene glycol)-modified nanocarriers for tumor-targeted and intracellular delivery[J]. Pharm. Res,2007, 24(7): 1405–1414.
57. Daugherty AL, Mrsny RJ, et al.Transcellular uptake mechanisms of the intestinal epithelial barrier[J]. Part one, PSTT,1999, 2 (3): 144–151.
58. Francis MF,. Cristea M, Winnik FM., et al.Exploiting the vitamin B-12 pathway to enhance oral drug delivery via polymeric micelles[J]. Biomacromolecules, 2005,6(2):2462–2467.
59. Anderle P, Huang Y, Sadee W,. et al. Intestinal membrane transport of drugs and nutriments: genomics of membrane transporters using expression microarrays[J].Eur. J. Pharm. Sci,2004,21(1) :17–24.
60. Hansen JE, Weisbart RH, Nishimura RN, et al. Antibody mediated transduction of therapeutic proteins into living cells[J]. Sci. World,2005, 5(6): 782–788.
61. Ambudkar SV, Kimchi-Sarfaty C, Sauna ZE., et al, Pglycoprotein: from genomics to mechanism[J]. Oncogene ,2003,22(3):7468–7485.
62. Higgins CF, et al.Molecular basis of multidrug resistance mediated by Pglycoprotein[J]. Curr. Opin. Biotechnol, 1991,2(1): 278–281.
63. Lavie Y, Fiucci G, Liscovitch M, et al. Up-regulation of caveolae and caveolar constituents in multidrug-resistant cancer cells[J]. J. Biol. Chem, 1998,273(8): 32380–32383.
64. Minato S, Iwanaga K, Kakemi M., et al.. Application of polyethyleneglycol (PEG)-modified liposomes for oral vaccine: effect of lipid dose on systemic and mucosal immunity[J]. J. Control Rel, 2003. 89(9):189–197.
65.郑中华,张以忠,方青, et al.Pgp动力学研究进展[J].吉林医药学院学报,2006,7(4):1-5.
66. Ho RH, Kim RB, et al.Transporters and drug therapy: implications for drug disposition and disease[J]. Perspect. Clin. Pharmacol, 2005 , (1)260–277.
67. Wang Q, Bhardwaj RK, Herrera-Ruiz D, et al. Expression of multiple drug resistance conferring proteins in normal Chinese and Caucasian small and large intestinal tissue samples[J]. Mol. Pharm,2004, 1(3): 447–454.
68. Peng K, Cluzeaud F, Bens M, et al.Tissue and cell distribution of the multidrug resistanceassociated protein (MRP) in mouse intestine and kidney[J]. J.Histochem.Cytochem,197,47(5): 757–768.
69. Zimmermann C, Gutmann H, Hruz P, Gutzwiller JP, et al.Mapping of multidrug resistance gene 1 and multidrug resistance-associated protein isoform 1 to 5 mRNA expression along the human intestinal tract[J]. Drug Metab. Dispos,2005 ,33(7): 219–224.
70. Scheffer GL, Maliepart M, Pijnenborg AC, et al. Breast cancer resistance protein is localized at the plasma membrane in mitoxantrone- and topotecan-resistant cell lines[J]. Cancer Res,2000,60(3): 2589–2593.
71. Eisenblatter T, Galla H, et al.A new multidrug resistance protein at the blood–brain barrier[J]. Biochem. Biophys. Res. Commun. 2002,293(5): 1273–1278.
72. Collnot EV, Baldes C, Wempe MF., et al. Mechanism of inhibition of P-glycoprotein mediated efflux by vitamin E TPGS: influence of ATPase activity and membrane fluidity[J]. Mol. Pharm,2007,4(7):465–474.
73. Dintaman JM., Silverman JA., et al. Inhibition of P-glycoprotein by D-tocopheryl polyethylene glycol 1000 succinate (TPGS) [J]. Pharm. Res. 1999, 16(3):1550–1556.
74. Zhao H.Z., Tan EC, Yung LY,et al. Potential use of cholescalciferol polyethylene glycol succinate as a novel pharmaceutical additive[J]. J. Biomed. Mater. Res. Part A,2008 ,84(8):954–964.
75. Batrakova EV, WF Li, Miller D.W, et al.Mechanism of sensitization of MDR cancer cells by Pluronic block copolymers: selective energy depletion[J]. Br. J. Cancer ,2001,85(2): 1987–1997.
76. Batrakova EV, Li S, Alakhov VY, et al.Sensitization of cells overexpressing multidrug-resistant proteins by pluronic P85[J]. Pharm. Res.2003,20(2) : 1581–1590.
77. Batrakova EV, Li S, Alakhov VY, et al .Effect of pluronic P85 on ATPase activity of drug efflux transporters[J].Pharm. Res,2004,21(5):2226–2233.
78. Zhu S, Huang R, Hong M, et al .Effects of polyoxyethylene (40) stearate on the activity of P-glycoprotein and cytochrome P450[J]. Eur. J. Pharm. Sci,2009,37(4): 573–580.
79. Zastre J, Jackson J, Bajwa M, et al Enhanced cellular accumulation of a P-glycoproteinsubstrate, rhodamine-123, by Caco-2 cells using low molecular weight methoxypolyethylene glycol-blockpolycaprolactone diblock copolymers[J]. Eur. J. Pharm. Biopharm,2002,54(6) :299–309.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |