Studies On Oleanolic Acid Nanosuspensions

Oleanolic acid is a widely distributed triterpene possessing many pharmacological activities. As an effective component of many Traditional Chinese Medicines, oleanolic acid is commonly used in the therapy of hepatitis in clinic. Because of its poorly solubility in water, the orally administration of oleanolic acid often results in low or erratic bioavailability, which limits its therapeutic application. One of the most powerful means in the development of oleanolic acid new dosage forms is to increase its solubility and drug dissolution velocity. For poorly soluble drugs, nanosuspensions (nanocrystals) are reported to be a promising approach in pharmaceutical research, which could lead to numerous advantages such as increased saturation solubility and drug dissolution velocity which result in improved bioavailability, reduced fed/fasted variability and inter-subject variability, as well as high drug loading compared to other nanoparticulate drug delivery systems. In this dissertation, oleanolic acid nanocrystals are prepared by two means. Thorough characterization of different oleanolic acid nanosuspensions has been performed. Small intestine absorption of oleanolic acid nanosuspensions was investigated. The hepatoprotective effect of oleanolic acid nanosuspensions on carbon tetrachloride induced liver injury has also been studied. Results obtained are encouraging, which will pull a new way for the development of novel orally-administrated formulations of oleanolic acid. (1) Oleanolic acid nanosuspensions were firstly prepared by nanoprecipitation method employing ethanol-water system. Different surfactants or surfactant at different concentrations used led to nanocrystals with different particle sizes, different polydispersities and different zeta potentials. The increased volume of organic phase led to decreased particle size at first, but the particle size was increased when the organic/aqueous ratio was above 0.5. (2) Oleanolic acid nanosuspensions were also prepared by high-pressure homogenization. In this process, the main parameter that determines the particle size is pressure: The increased pressure led to a decreased particle size; the increased homogenization cycles helped to decrease the polydispersity of nanocrystals; surfactants exhibited no influence on particle size, but they contributed to the stability of nanoparticulate systems. (3) Oleanolic acid nanocrystals obtained from nanoprecipitation are spherical or near-spherical in shape, while those obtained from homogenization are short and rob-like, which might come from the disruption of origined needle-like oleanolic acid crystals. XRD and DSC investigations indicated that oleanolic acid existed in amorphous state in nanocrystals obtained from nanoprecipitation, while the crystalline structure of coarse oleanolic acid had been kept in nanosuspensions prepared by high-pressure homogenization. Because of its small particle size in the range of nanometer, solubility of oleanolic acid was improved to 5~6 times than that of coarse drug powder. At the same time, drug dissolution velocity was also increased, as well as the cumulative drug dissolved. Due to its good mono-dispersity, Ostwald-ripening of nanoparticles was reduced to a certain extent, so nanosuspensions prepared by homogenization showed better stability than that obtained from precipitation method. (4) Drug absorption of oleanolic acid nanocrystals in small intestine was carried out by everted gut sac technique and in situ circulation methods. Results showed that, nanocrystals could increase the small intestine absorption of oleanolic acid. Reasons might lie in: the increased drug solubility and drug dissolution velocity, lymphoid uptake in Peyer's patches, non-lymphoid intestinal cell uptake, etc. No difference has been noticed between negatively and positively charged nanocrystals (P>0.05). The bio-adhesion of oleanolic acid was improved after nanonization (P<0.05 compared to coarse drug powder suspension, both negatively and positively charged nanocrystals). Because of the charge attraction, together with the bio-adhesion of chitosan, positively charged nanocrystals exhibited stronger bio-adhesion (P<0.05 compared to negatively charged nanocrystals). (5) Positively charged oleanolic acid nanocrystals were obtained by surface modification of negatively charged nanocrystals. In the pharmacodynamic research, oleanolic acid in coarse powder showed obviously hepatoprotective activity (P<0.01 compared to CCl4 model group). Oleanolic acid nanocrystals, both negatively and positively charged, exhibited enhanced hepatoprotectiveactivities (both P<0.01 compared to coarse oleanolic acid). While the stronger hepatoprotective activity was observed in the group of positively charged oleanolic acid nanocrystals (P<0.05 compared to negatively charged nanocrystals). Histological examination gave the similar results. The hepatoprotective activity of oleanolic acid has been shown to be dose-dependant, so the enhanced hepatoprotective activity of oleanolic acid nanocrystals might come from the improved absorption of oleanolic acid nanocrystals. Though no difference has been observed in the intestinal absorption of the two nanoparticulate drug delivery systems, it is the enhanced bio-adhesion of positively charged nanocrystals that should be responsible for its enhanced hepatoprotective activity.