Synthesis And As Sembly Of 7-Ethyl-10-Hydroxycamptothecin-Phospholipid

As one of the camptothecin-based antitumor drugs,7-Ethyl-10-hydroxycamptothecin (SN38) has a wide antitumor activity in vitro. However, its application in clinical treatment is extensively restricted by the poor solubility in pharmaceutically acceptable excipients. In order to improve the solubility of SN38, an amphiphilic SN38 conjugate was synthesized by conjugation with phospholipid. The synthetic route, assembly behavior, physical & chemical properties and in vitro antitumor activity of the conjugate were investigated. The main content and results of the theis are as follows:(1) Dual 7-ethyl-10-hydroxycamptothecin-tailed glycerylphosphorylcholine conjugate (Di-SN38-GC) was synthesized by SN38 and L-a-glycerophosphorylcholine (GPC) using succinic anhydride as a linker. HPLC, MS and NMR were applied to characterize the intermediate 7-ethyl-10-hydroxycamptothecin-20-O-hemisuccinate (20-O-SN38) and target product Di-SN38-GC. In addition, the optimization of the reaction condition involved with investigation of different esterification system, feed ratio, reaction time and temperature. The results showed that the purity of 20-O-SN38 and Di-SN38-GC was 98.62% and 98.76%, respectively. The structures of 20-O-SN38 and Di-SN38-GC were well confirmed by MS and NMR measurements. The optimization of preparing Di-SN38-GC was obtained according to the following conditions:using the CDI/DBU as catalysis system, two hours of reaction time and a feed ratio of n20-O-SN38:nCDI:nGPC:nDBU:=1:1.5:0.4:1 under 35?.(2) The Di-SN38-GC liposomes were prepared by applying conventional thin film dispersion method. To determine the equilibrium solubiliry of Di-SN38-GC conjugate in water, a standard curve model was established. Electric conductivity measurement was used to determine the critical aggregation concentration (CAC). The properties of conjugate liposomes were further evaluated by dynamic light scattering (DLS), transmission electron microscopy (TEM). The results showed that the equilibrium solubiliry of Di-SN38-GC conjugate in water was 426 ?g/mL, which was much higher than that of prodrug SN38 (7 ?g/mL). The self-assembly ability of amphiphilic Di-SN38-GC conjugate in water was well proved with a CAC of 53 ?g/mL. The average size of Di-SN38-GC liposomes was accorded with normal distribution with the hydrodynamic diameter about 159 ran, while the liposomes had Zeta potential about-21.6 mV. Apprently, the Di-SN38-GC liposomes carried such a negative surface charge which favors the formation of stable vesicular structure. The TEM image showed the rounded shape with a lipid bilayer, which accorded with the characteristic of general liposomes.(3) Cytotoxicity assay (MTT) was used to evaluate the in vitro anticancer activity of Di-SN38-GC conjugate and its liposomes. The results showed that the IC50 of the Di-SN38-GC on HepG-2, HeLa and HT-29 cancer cell lines were 35 ?g/mL,17 ?g/mL,26 ?g/mL respectively, indicating good anticancer activity of Di-SN38-GC. It was evident that Di-SN38-GC presented close toxicity in comparison with SN38. The phenomena above indicated that the conjugation with GPC had no bad effect on the activity of SN38.(4) The solid phase peptide synthesis (SPPS) method was applied to synthesize an anticoagulant drug Bivalirudin. The reaction conditions involved with different attachment of protected amino acids were investigated. The structures of Bivalirudin and its front decapeptide fragment were well confirmed by MS.In conclusion, SN38-phospholipid conjugates possess the ability of assembling into liposome-like nanospheres. This novel dosage form has high drug-loading and effective ancancer activity. Therefore, it may have potential for clinical application after extensive evaluation.