Study On Liposomal Formulations For Zidovudine Myristate

Highly active antiretroviral therapy (HAART) has led to a profound decrease in morbidity and mortality in infected people by suppressing HIV replication. However, the eradication of virus does not seen attainable with the present strategies of interventions which is due to two major obstacles: if resistant mutantions appear the virus will escape further treatment, and latent virus reservoirs exist which cannot be reached with the current treatment regimens. Therefore, the search for new promising agents against HIV from various sources and the development of new drug carrier systems which allow drugs targeting HIV reservoirs have become hot topics in the world.In this paper, zidovudine myristate (AZT-M), a potential double-barrelelled prodrug to AZT and myristic acid, was synthesized, and then encapsulated in three types of liposomes to fulfill targeted drug delivery, namely, (1) conventional liposomes, which could deliver AZT-M to cells of the mononuclear phagocyte system (MPS), an important reservoir of HIV; (2) active-targeting liposomes, by the modification with dextran sulfate derivatives (DS, which binds HIV-1 envelope glycoprotein gp120 with high affinity), which might specifically conjugate with HIV-infected cells and inhibit syncytium formation; (3) sterically stabilized active-targeting liposomes, by coating with poly (ethylene glycol) (PEG) polymer, which might be useful in targeting HIV infected deep tissue such as brain.This research work was granted by the National Natural Science Foundation of China with a key project number of 30371694.The contents in detail are as follows:After the synthesis, the physicochemical properties of AZT-M were investigated. AZT-M, a white powder with mp 56℃, is unsoluble in water, but very soluble in non-polar solvents such as chloroform. logP_app (n-octanol/phosphate buffer) for AZT-M was 5.30 at pH 7.4, larger than that of AZT (0.07). The very high lipophilicity might be helpful for AZT-M to be encapsulated in liposomes.The conventional liposomes containing AZT-M (AZT-ML) were prepared using a modified ethanol injection method followed by homogenization and filtration. The effects of homogenization pressure and number of cycles of Microfluidize on particle size distribution were investigated, and the factors in the formulations such as species of phospholipids and solvents, ratio of drug and lipid, and amount of cholesterol were tested. By optimizing the formulation and technique, AZT-ML with the mean particle size below 100 ran and the entrapment efficiency (EN) above 98% could be obtained. To improve the long term stability, AZT-ML were freeze-dried by the presence of trehalose, an excellent lyoprotectant. The mean particle size and EN of AZT-ML before and after lyophilization were almost the same. No significant physical instability or chemical degradation was observed in the lyophilized AZT-ML power after the storage of 12 months at 4℃.The novel derivatives of dextran sulfate, myristoyl dextran sulfate (DSM) and palmitoyl dextran sulfate (DSP), were synthesized. The effects of DSM/DSP concentration and the sequence of the addition on the characteristics of modified AZT-ML were investigated. Results showed that above 90％of the coating efficiency was acquired when polymer solution was added to the lipid solution before liposome formation, which was much higher than that when DSM/DSP solution was added to pre-formed AZT-ML. The particle size of modified AZT-ML was increased with the increasing concentration of polymer, suggesting the formation of coating layer on the surface of the liposoms. However, if the concentration of polymer was too high (2 mg/mL), aggregation of the liposomes may occur. The Zeta potential of modified AZT-ML was decreased as a result of the adsorption of DSM/DSP with negative charge. The effect of DSM/DSP on membrane permeability was studied by measuring the extent of calcein leakage from liposomes with the treatment of Triton X-100. Results indicated that DSM/DSP could improve the stability of liposomal bilayer membranes to some extent.To prolong the in vivo circulation time of DSM-AZTML, monomethoxy polyethyleneglycol succinyl cholesterol (CHS-PEG) was mixed with the pre-formed liposomes. The physical stability of PEG-DSM-AZT-ML was also increased with the increasing concentration of CHS-PEG..Stability of AZT-M in suspension and liposomes was investigated at various pH buffers or in the presence of animal plasma and tissue homogenates. The degradation of AZT-M in both suspension and liposomes followed a pseudo first-order reaction. Little degradation of AZT-M occurred at pH 4.0～7.0. When pH and temperature of the aqueous were raised, degradation rate of AZT-M increased accordingly. There was no significant difference between the degradation rate of AZT-M in suspension and liposomes at pH 4.0～7.4. However, at pH 9.0 and 80℃, the degradation rate of AZT-M in liposomes was slower than that in suspension. AZT-M underwent rapid degradation by esterases in plasma and tissues. The degradation rates of AZT-M among mice, rat and rabbit plasma were significantly different (mice＞rat＞＞rabbit), which suggested that there might exist species-dependent metabolism for AZT-M. Liposomes can protect the loaded AZT-M from esterases degradation in plasma and tissue homogenates. The half-lives of AZT-M in different liposomal formulations were longer than that in suspension (PEG-DSM-AZT-ML＞DSM-AZT-ML≈AZT-ML＞AZT-M).The pharmacokinetic profiles and tissue distribution of AZT after i.v. administration of different formulations of liposomal AZT-M in rats compared with AZT solution were investigated. AUC_0-∞ of AZT in AZT-ML, DSM-AZT-ML and PEG-DSM-AZT-ML were 1.6, 1.9 and 2.3-fold higher than that of AZT solution, respectively. Total body clearance (CL_tot) decreased from 16.6±2.3 mL/min (AZT solution) to 10.8±2.2 mL/min (AZT-ML), 8.6±1.4 mL/min (DSM-AZT-ML) and 7.1±1.1 mL/min (PEG-DSM-AZT-ML), respectively. Steady distribution volume (V_ss) declined from 1.2±0.3 L (AZT solution) to 0.8±0.2 L (AZT-ML), 0.8±0.1 L (DSM-AZT-ML) and 0.7±0.2 L (PEG-DSM-AZT-ML), respectively. Moreover, terminal half life (t_1/2)and mean resident time (MRT) of AZT in PEG-DSM-AZT-ML were significantly prolonged compared with those of AZT solution.Tissue distribution studies confirmed that AZT-ML were rapidly accumulated in organs of RES. The AZT levels 1 h after injection of AZT-ML were 6.9, 4.7 and 4.3 times as great as those of AZT solution in spleen, liver and lung respectively. Higher concentrations of AZT were observed in lung after dosing with DSM-AZT-ML than with other preparations. Compared with AZT solution, liposomal AZT-M led to significantly higher brain concentrations of AZT. The AZT concentrations in brain 1 h after administration of AZT-ML, DSM-AZT-ML and PEG-DSM-AZT-ML were 2.2, 2.0 and 6.1-fold than those of AZT solution respectively.Cytotoxicity of different formulations of free or liposomal AZT-M against MT-4 cells was tested by the trypan blue exclusion method for viability determination. Liposomal AZT-M significantly decreased the cytotoxicity compared with free AZT-M (PEG-DSM-AZT-ML＜DSM-AZT-ML＜AZT-ML＜AZT-M). Measurements of the Anti-HIV activity were based on the inhibition assay of SF33 virus-induced-CPE (Cytopathic effect). Results showed that the anti-HIV potency of free or liposomal AZT-M was comparable with that of AZT at the tested concentrations.