Directed Self-assembled Homogenous Nanosuspensions Improve The Oral Bioavailability Of Insoluble Probucol

Nanosuspension, which is stabilized by surfactants, is a millimicron colloidal dispersion system of pure particles of drug prepared by the technology of grinding or crystallizing. Recently, researchers have found that nanosuspensions can significantly increase the aqueous solubility and oral bioavailability of insoluble drugs to achieve the pharmacological activity.Probucol was effective in reducing the levels of cholesterol and low-density-lipoprotein in the blood due to its various biological activities including anti-atherosclerosis, anti-oxygen, cholesterol-lowering, preventing restenosis after percutaneous transluminal coronary angioplasty and regression of xanthomas, etc. Moreover, in the treatment of hyperlipoidemia and atherosclerosis, probucol shows good therapeutic effect and fewer side-effects. However, probucol is practically insoluble in water, which resulted in its very low absorption in the gastrointestinal tract with the oral bioavailability of less than 10%. As a result, the therapeutic effects and clinical application of probucol is greatly limited.In the present study, the probucol loaded nanosuspension was developed by directed self-assembly technology to improve its oral absorption. The physicochemical properties of self-assembled probucol loaded nanosuspensions (SPN) were characterized. Then, the cellular uptake in the enterocytes and in vivo pharmacokinetic behavior of SPN were evaluated to verify the effectiveness in enhancing the oral delivery of insoluble probucol. In particular, the possible mechanism of SPN on the markedly improved oral bioavailability of probucol was investigated. In addition, the gastrointestinal safety of SPN was preliminary assessed in rats after oral administration.SPN was prepared by the self-assembly technology. The included ingredients and preparation parameters were optimized by means of single-factor test, such as the ratios of surfactant and drug, hydration temperatures and drug concentrations. Various SPN were optimized and their physicochemical properties were evaluated. The measured results indicated that SPN were nanometeric homogenous spherical particles with narrow size distribution. Almost no significant changes of SPN size occurred after freeze-drying or during storage for 5 days at room temperature, which confirmed the good stability. The X-ray diffractograms indicated that probucol could present as crystalline nature in SPN.A sensitive and convenient HPLC-UV external standard method was established for determining the concentration of probucol in biological samples, which was successfully applied to evaluate the cellular uptake of SPN in Caco-2 cell monolayer and the pharmacokinetic behavior in rats. The experimental results showed that uptake of SPN increased significantly with the incubation time, but that of probucol solution was not obviously changed with time. Compared to the cellular uptake of probucol solution in the Caco-2 cell monolayer, the cellular uptake of various SPN were enhanced by 1.16,1.82,1.59, 5.31 and 6.95 folds, respectively. Most importantly, the peak concentration of probucol after oral administration of various SPN were enhanced by 3.82,4.96,8.20,10.57 and 15.17 folds, respectively. Similarly, the relative bioavailability of various SPN were evidently improved by 3.00,5.37,4.13,10.18 and 13.14 folds. These results suggested that SPN could greatly improve the oral bioavailability of insoluble probucol.Moreover, the possible mechanism of remarkably increased oral absorption of probucol by SPN was investigated by the in vitro and in vivo characterizations. SPN is a core-shell nanostructured carrier with good stability under different pH values and diluted conditions. The aqueous solubility of probucol was surprisingly increased at least 400,000 folds higher than free PBC in water. The cellular uptake of SPN in Caco-2 cell monolayer decreased about 90% at 4℃when comparing with that at 37℃. Moreover, the cellular uptake of SPN decreased about 50% with the treatment of chlorpromazine, but not significantly changed in the presence of filipin III. Therefore, SPN could be internalized into the Caco-2 cells via the clathrin-mediated endocytic pathway. Meanwhile, the F-actin filaments in the Caco-2 cell monolayer were obviously changed after the treatment of SPN, which implicated that the cell membrane fluidity was significantly increased by SPN. The transmission electron microscopy images showed that SPN could uptake into the enterocytes lining of the small intestine after oral administration. Furthermore, the intestinal transport of SPN was clarified in a cycloheximide treated rat model. The measured results indicated that the oral absorption of SPN was greatly reduced about 90% after the inhibition of lymphatic transport pathway, which implied that SPN was mainly absorbed into the systemic circulation through lymphatic transport pathway.Additionally, the gastrointestinal safety of SPN after oral administration was evaluated in rats. No obvious inflammatory response was observed in the intestine after oral administration of SPN. Meanwhile, the protein content and diamine oxidase activity in the intestine juice was not evidently changed. These results confirmed the safety of the orally administrated SPN.In summary, SPN is a core-shell monodispersed nano-delivery system with good physiological compatibility after oral administration. The absorption of insoluble probucol in Caco-2 cell monolayers and rats was markedly improved by the self-assembled nanocarrier. Therefore, SPN was an effective approach for enhancing the oral bioavailability of insoluble probucol.