Preparation And Liver Targeting Of RHDL-anti HBV Drug Complexes

Preparation and liver targeting of rHDL –anti HBV drug complexes Human high density lipoprotein(HDL) is a complex of lipids (largelyphosphatidylcholine ) and apoA-I ,which takes up delivering cholesterol from peripheraltissues back to the liver via apoA-I binding to the HDL receptor (SR-BI) on the liver cellmembrane . ApoA-I , more than 70% of total HDL proteins ,is a polypeptide with amolecular weight of 28.3kDa which containing 243 amino acid residues. Residues44-243 build up a contiguous series of highly homologous two 11-mer and eight 22-meramphipathic a-helices. ApoA-I plays an important role in stabilizing HDL particlestructure and binding to the HDL receptor (SR-BI). First , we obtained human apoA-I from blood palasma by DS.50-MnCl2 method andfrom waste blood FⅣ by cold Acetone- MnCl2 method, and also obtained recombinanthuman apoA-I(rh apoA-I) from pichia pastoris high expression product by coldAcetone - pI method . These apoA-I have activities of combining with SMMC-7721,HepG2 2.2.15,Wister rat liver cells with dot blotting and 125I analysis. Based on acyclovir and palmitoyl Chloride, prodrug acyclovir palmitate wassynthesized with a yield of 68% and the structure was confirmed by Uv-Vis, IR ,MS,1H NMR,13C NMR,D2O exchange ,BB-DEPT, H-1H COSY , H-1H TOCSY , 1 1HSQC and HMBC. Further recombinant high density lipoproteins(rHDL) –anti HBV drugs complexeswere prepared by ultrasonic method and sodium deoxycholate diaysis . The later cancontrol the complex size at about 20 nm. HepG2 2.2.15 cells test showed that human apoA-I and rh apoA-I / rHDL-nosiheptide complexes have the same anti-HBV activity. With the 50% inhibiton ofHBsAg , the nosiheptide concentration of rHDL- drug complexes was 20 fold less thanfree nosiheptide ,and 4 fold less than nosiheptide lipsomes. 5复旦大学博士学位论文 Also, human apoA-I and rh apoA-I / rHDL- acyclovir palmitate complexes have thesame anti-HBV activity. With the 50% inhibition of HBsAg , the acyclovir palmitateconcentration of rHDL- drug complexes was 200 fold less than free acyclovir, 40 foldless than acyclovir lipsomes, 20 fold less than free acyclovir palmitate and 20 fold lessthan free acyclovir palmitate lipsomes. Our studies indicated that human apoA-I and rh apoA-I / rHDL–drug complexes cantarget to liver with a high distributing percent of 76.9% and 71.2% in liver , 8.5% and10.2% in plasma , comparing to the free drugs 17.5% in liver and 53.3% in plasma .