Construction Of Pluronic Micelle Drug Delivery System And Its Effect On Intestinal P-glycoprotein

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.