Studies On The Intestinal Absorptive Mechanism And Self-Microemulsif Ying Drug Delivery System Of 9-Nitrocamptothecin

9-nitrocamptothecin is a novel analog of camptothecin, which had indicated that the antitumor activity for many cancer cells, especially for advanced pancreatic cancer. The main aims of this research were to study the physicochemical properties and the intestinal absorption mechanisms of 9-nitrocamptothecin, and investigate the factors that affect the absorption of 9-nitrocamptothecin for preparing a novel oral drug delivery system. Self-microemulsifying drug delivery systems (SMEDDS) of 9-nitrocampthecin were developed. The uptake and transport of 9-nitrocamptothecin SMEDDS in Caco-2 cells was investigated. The molecular pharmaceutical properties of SMEDDS were evaluated. Finally, the studies on pharmacokinetics and tissue distribution of different SMEDDSs of 9-nitrocamptothecin were determined.Resolution of the lactone and carboxylate forms of 9-nitrocamptothecin was accomplished by reverse-phase HPLC. 9-nitrocamptothecin is sparingly solubility in water. The oil-water partition coefficient of 9-nitrocamptothecin was the pH-dependent. With increasing of pH, the coefficient became decreasing. The hydrolysis and lactonization reactions followed pseudo-first-order kinetics in the approach to equilibrium. The hydrolysis rate was dependent on pH, with the closed form (lactone) dominant at low pH and the open form dominant at physiological pH or above. The hydrolysis rate of 9-nitrocamptothecin was affected by temperature. The Ea at different pH were 5.039, 12.476, 10.094, 10.021, 9.647, 11.35KJ/mol, respectively.The intestinal transport of 9-nitrocamptothecin was performed by applying everted intestinal sacs method. The absorption of 9-nitrocamptothecin was passive diffusion and had no significant difference in different gut regions (P>0.05). Adding verapamil in the everted gut sacs, the absorption of 9-nitrocamptothecin was significantly enhanced (P<0.05), especially more markedly in lower small intestine. The accumulative transport flux of 9-nitrocamptothecin was increasing by two-fold in 90min added 50 μg/ml verapamil.The Caco-2 cell model was used to study the uptake, transport and efflux kinetics of 9-nitrocamptothecin. The effect of surfactants, such as Cremophor EL, Labrasol and Transcutol P, on the transport of drug was investigated. The results indicted: (1) The uptake of 9-nitrocamptothecin is passive diffusion as the dominating process. The uptake of 9-nitrocamptothecin is positively correlated to uptake time, and negatively correlated to pH and temperature. The inhibitors, cyclosporine A and verapamil,significantly enhanced the uptake amount of 9-nitrocamptothecin (.P<0.05) .(2) The absorptive transport of 9-nitrocamptothecin was pH dependent and the transport was enhanced at weakly acidic pH on the apical side. No concentration dependence and saturation were observed for the absorptive transport of 9-nitrocamptothecin, while secretory transport were concentration dependent and saturable process, Km was (49.8±1.2) uM, Vmaxwas (38.28±0.8) ng/cm2/min, respectively. (3) The transport of 9-nitrocamptothecin across Caco-2 cell monolayers was directional. Papp of Basolateral to Apical was much more than that of Apical to Basolateral (2.6-6.9 folds). In the presence of verapamil and CsA, potent inhibitors of P-glycoprotein or MRP2, the Papp BL-Ap/Papp ap-bl ratio was decreased from 3.4 to 1.4 and 1.3, respectively. (4) The efflux of 9-nitrocampothecin was fitted to apparent two-order process. The m0 [(148.0±2.18)] pmol-cm'2) and the efflux rate (41.1 pmol-cm2-min*') on Apical side were higher than the m0 [(121.3±6.9) pmol-cm'2] (PO.05) and the efflux rate (29.2 pmol-cm2-min"1) on Basolateral side (PO.01). (5) Surfactants could enhance the absorption of 9-nitrocampthothecin, and decrease the efflux. Overall, the current study suggests that pH and efflux transporters are capable of mediating the absorption and efflux of 9-nitrocamptothecin, and they may play significant roles in limiting the oral absorption of 9-nitrocamptothecin.In the study of preformulation, the equilibrium solubility of 9-nitrocampothecin in different oil, surfactants and cosurfactants was investigated and pseudo-ternary phase diagrams were constructed to evaluate the formulation effect. An optimized formulation consisted Maisine 35-1: Miglycol 812 N (1:1) as oil phase, Cremopher EL: Labrasol (4:1) as surfactants, and Transcutol P as co-solvent. With the amount of oil and Km as the independent variables, the optimized formulation was determined by the central composite design-response surface methodology. The response variables were particle size, Zeta potential, solubility, and cloud point, the cumulative percentage of 9-nitrocamptothecin in 5 min or 60 min. The optimized formulation of 9-nitrocamptothecin was quickly and conveniently obtained by the central composite design-response surface methodology, i.e. oil: surfactants: cosolvent=30%:47%:23%. For the positively charged SMEDDS of 9-nitrocamptothecin, oleylamine (1%, w/w) was additionally added. For the super-saturable SMEDDS of 9-nitrocamptothecin, HPMC (0.5% E5LV, w/w) was additionally added.The effect of dilution on the particle size and zeta potential of 9-nitrocamptothecin in Caco-2 cells was investigated. The results indicated that dilution had hardly effecton the particle size of microemulsion, while the zeta potential were increased with dilution. When the negatively charged, positively charged and HPMC SMEDDS were diluted with water in the ratio of 1:10, the size of microemulsions were (24.7±7.9), (25.0±8.3) and (32.8±7.5) nm, respectively. The SMEDDS of 9-nitrocamptothecin could decrease the hydrolysis rate to a certain extent, increase the stability of 9-nitrocamptothecin in buffer solution.The results of the uptake and transport of 9-nitrocamptothecin SMEDDS in Caco-2 cells indicated: (1) Self-microemulsifying drug formulations of 9-nitrocamptothecin could significantly improve the uptake of 9-nitrocamptothecin (PO.05), except for the positively charged SMEDDS at the dilution of 1/50.(2) SMEDDS of 9-nitrocamptothecin could enhance the transport across Caco-2 cell monolayers. (3) The effect of pH on the transport of 9-nitrocamptothecin SMEDDS formulations was markedly decreased compared with the control. The ratio (2.19-3.68) of Papp of SMEDDS formulation between different pH was lower than that (6.02) of control.Effect of the negatively charged and positively charged self-microemulsifying drug delivery systems on the P-glycoprotein and cellular tight junction complex was to been investigated at molecular cell level. The results indicated: negatively charged or positively charged SMEDDS at different dilution could significantly affect the integrity of Caco-2 cell monolayers (PO.05), which depended on the dilution times. The cell accumulation of RH123, a substrate of P-glycoprotein, were significantly enhanced by two SMEDDSs in different dilutions (1.6-6.4 folds).The improvement of positively charged SMEDDS on the accumulation was higher than that of negatively charged SMEDDS in the same dilution. Immunoflurescent and Western-blotting results indicated the mechanism of opening of tight junctions by SMEDDS was changed the distribution of ZO-1 and actin. In addition, to change the cell membrane fluidity inhibited the efflux activity of P-glycoprotein. MTT results showed the positively charged SMEDDS in lower dilution presented certain cytotoxicity.The pharmacokinetic parameters of 9-nitrocamptothecin solution, micelle, negatively charged, positively charged and HPMC SMEDDS were investigated after oral administration to rats at a single dose of 6mg/kg. The results were as follows: Cmax was (93.83±72.79), (143.25±29.91), (176.42±93.43), ( 167.38±52.12 ) and (333.23±96.26) ng/ml, respectively; AUC was (127.62±51.6), (239.78±137.60), (331.91±161.04), (595.04±42.00) and (412.21±136.04) ng-mr'-h, respectively; MRT was (1.6±0.9 ), (1.3±0.4), (1.2±0.6), (4.3±2.6) and (2.6±0.6) h, respectively.