Preparation And Preliminary Study Of The Asialoglycoprotein Receptor-mediated Hepatic Targeting Of Acyclovir

Objective: In order to reduce the side effects of acyclovir (ACV) in the treatment of chronic hepatitis B and explore the possibility of obtaining a delivery to liver through asialoglycoprotein receptor (ASGPR), the drug was conjugated with lactosaminated poly-L-lysine (Lac-PLL) which selectively enters hcpatocyte by ASGPR. Methods: l.Lactosylation of the poly-L-lysine (Mw 40,000Da) was performed by reductive lactosamination in the presence of sodium cyanoborohydride. The conjugate Lac-PLL-ACV was prepared via the ACV imidazolides incubated with lactosaminated poly-L-lysine at 370C pH9.5. 2.The molecular weight of the conjugate was determined by sephadex G-lOO column calibrated with lysozyme( 1 4,400Da),egg white albumin(40,000Da)and bovine serum albumin(66,200Da).ACV content was determined by ultraviolet- visible spectrophotomery at 254nm(E=622).Lactose content was determined by enthronlsulphuric acid method using a lactose standard.3. Setting up method of high performance liquid chromatography(HPLC)to determine ACV concentration in plasma and organs. 4.The conjugate was incubated at 370Cin fresh human plasma( 1 mg/ml).At defferent intervals(O, 1 ,2,4hr) the sample was extracted by PEG-6000.The released drug in the supernatant was measured by HPLC and the stability of the conjugate was measured by the percent of the released drug. 5.Adult healthy mice,weighing 20 g,were equally randomized into 20 groups,which were injected i.v.ACV and Lac-PLL-ACV at the dose of 5mg/kg weight.At different times after i.v. injection mice were bled from the retro-orbital plexus and liver,spleen,kidney were rapidly removed and homogenized in phosphate buffer 0.lmol/L pH7.4. The protein was precipitated by 12% TCA.ACV concentration in plasma and organs were measured by HPLC respectively.6.Pharmacokinetic properties of ACV and the conjugate were studied by 3p87 program.7. Adult healthy mice,weighing 20 2g,were equally randomized into different groups ,10 mice each group.Different concentration of Lac-PLL-ACV and PLL, ACV solution (pH7.4)were administered to mice by i.v. route to study the acute toxicity.The volume injected was 0.lml/lOg weight. The lethal dose 50%( LD50)of the Lac- PLL-ACV and PLL, ACV used for preparing this conjugate were calculated. Results: 1. Lac-PLL-ACV has a molecular weight of 56,000Da and the drug/conjugate and lactose/conjugate molar ratio were 28 and 50 respectively. We found that conjugate was high soluble since it dissloved easily in water at 200mg/ml.2. ACV in plasma and tissues were determined using Beckman Gold system HPLC equipment with a ODS analytical column and chromatographed with the following conditions.Mobile phase:methyl and water in a ratio of 5:95(v/v);flow rate:1.Oml/min;detection:UV at 254nm,0.1 absorbance unit full scale(AUFS). (1)Retention time of ACV was about 7.Smin.(2)Plasma and homogenate of tissues had no disturbance to standard and showed good linearity in the concentration range of 0.1-20 I.t gIml(g) with r=0.99.(3)The detecting limit of ACV concentration in plasma and tissues were 2Ong/ml and SOng/g respectively.(4) Both the intraday precision and the interday precision were less than 6%.The average recovery was 100.35%. 3. When Lac-PLL- ACV was incubated at 370C in fresh human plasma ,the percentages of drug released from the conjugate were 3% after lhr and 6% after 4hr. 4.(1)After i.v. injection of free ACV ,the concentration of ACV was the highest in kidney and low in plasma,liver and spleen which were undetected after l2hr.A...