Studies On Nanosuspension Drug Delivery Systems Of Hydroxycamptothecin

In this study, Nanosuspesions was selected as a new drug delivery system, and Hydroxycamptothecin (HCPT) was used as a researched object. The formula, the process of preparation and physico-chemical properties of Nanosuspensions for intravenous and oral administration were systematically investigated, and the in vivo pharmacokinetics, tissue distribution, in vitro anticancer activity of intravenous Nanosuspension (HCPT-Nano) were studied. In addition, the uptake and transport across Caco-2 cells, in vivo pharmacokinetics of two oral Nanosuspensions (Nano-1 and Nano-2) were evaluated.Firstly, A HPLC method was developed to determine the concentration of HCPT in vitro. Subsequently, the saturated solubility of HCPT in many kinds of solvents and phosphate buffered solutions with different pH values were determined. The results proved that HCPT was both poor hydrophilic and lipophilic, and the solubility in water was influenced largely by pH values. And analyzing the percentages of lactone and carboxylate in different pH phosphate buffered solutions were also carried out. It showed there was a pH-dependent equilibrium between lactone form and carboxylate form.After formula screening and technology investigation, Nanosuspension for injection was manufactured using poloxamer 188 (F68) and beans phospholipid (SP) as stabilities using microsuspension-high pressure homogenization method. The freeze-drying formulations and conditions were optimized. At last, the formulation with 7.5%(w/v) maltose and 2.5%(w/v) mannitol was selected as the optimal formulation, and the final freeze-drying process was established.In this article, the pharmaceutical characters of HCPT-Nano were investigated. The nanoparticles, being observed by transmission electron microscopy (TEM),were claviform or columna in shape. The particle size was measured by laser diffraction, and the mean diameter before and after lyophilization was 131±2.08nm and 14±1.00nm respectively;Zeta potential of HCPT-Nano was between-20～-30mV;DSC and X-ray diffraction revealed that HCPT was amorphous in nanoparticles.The dissolution test was carried out using membrane filtering method. The result demonstrated the dissolution rate was markedly enhanced in the nanosuspensions, and nearly 100% of the drug dissolved within 15 min, as opposed to only 53% for the coarse HCPT suspensions. For studying the stability of HCPT active lactone form in HCPT-Nano in comparison to the HCPT solution, the lactone form of HCPT at different pH values was determined using HPLC.We found that less than 30% of HCPT in solution was left after the 24h incubation at physiological pH values (pH 7.0-7.4).However, nearly 90% of HCPT in HCPT-Nano was preserved in the lactone form even at the least stable pH (8.0), clearly demonstrating that the nanosuspension delivery system could effectively protect the active lactone form of HCPT.In this paper, we established the HPLC-FD analysis method of HCPT in the biological specimens, and studied pharmacokinetic behaviors of two intravenous formulations (Solution and HCPT-Nano) in rats. The results showed that the AUC(0-∞), MRT(0-∞) and ti/2 were obviously different between HCPT-Nano and Solution. HCPT-Nano markedly dropped off drug’s distribution in plasma, and prolonged slightly the drug circulation time.The mice tissue distribution of the tow formulations were examined after intravenous administration. The drug concentrations in the plasma, heart, liver, spleen, lung, kidney and brain were determined at different time. The results suggested that the HCPT-Nano changed the tissue distribution character remarkably compared with drug solution. HCPT-Nano had a higher drug concentration in liver and spleen, and showed some typical passive targeting features.The cytotoxicity of HCPT solution and HCPT-Nano to the MCF-7/Adr and PC-3 were compared by MTT. The results showed that HCPT-Nano had stronger anti-tumor activity in both two cell lines, especially to the PC-3,and the strength of activity depended on the time and dose, which meaned HCPT-Nano could improve the cytotoxicity of the drug.Selecting Hypromellose (HPMC E50) and water soluble Vitamins E (TPGS) as stabilities, we prepared two oral Nanosuspensions (including Nano-1 and Nano-2) by the same method above mentioned. The results of particle size analysis showed that the mean diameters before and after lyophilization were 135±5.51 nm and 173±22.03 nm for Nano-1,138±11.72 nm and 175±12.74 nm for Nano-2 respectively. Zeta potentials of Nano-1 and Nano-2 both were below-20 mV. DSC and X-ray diffraction revealed that HCPT existed in the form of crystal for Nano-1 and Nano-2.The results of supersaturation dissolution test demonstrated that oral Nanosuspensions could maintain high supersaturation level for a long time.The Caco-2 cell model was used to study the uptake and transport of HCPT solution and Nano-1 and Nano-2.The results showed that the absorption process of HCPT was possibly affected by P-gp, and the surfactants including Cremphor EL, TPGS and P-gp inhibitors could increase the uptake and transport of HCPT across Caco-2 cells. The test also demonstrated that Nanosuspensions could improve the uptake and transport of HCPT, and P-gp inhibitors could further enhance the uptake and transport of HCPT in Nano-2. We studied pharmacokinetic of the four oral formulations (Solution, Microsuspension, Nano-1 and Nano-2) in rats in the same method. The study suggested there was no significant difference between the two Nanosuspensions groups and the Solution group in AUC(0-∞)and Cmax, which indicated the bioavailability of the nanosuspension was similar to the Solution after oral administration. By contrast, the AUC(0-∞) and Cmax of the two Nanosuspensions groups was higher marketly than the Microsuspension group. So comparing with Microsuspension, Nanosuspension could improve the oral bioavailability of HCPT notably.