Study Of The In Vitro And In Vivo Pharmaceutical Behaviors Of AmphotericinB Freeze-Drying Liposome

Objective: AmphotericinB is a macrocyclic, polyene, antifungal antibiotic produced from a strain of Streptomyces nodosus. AmphotericinB (AmB) is the drug of choice for systemic fungal infection. The structure of amphotericin B is shown below: Amphotericin B, the active ingredient of AmBisome, acts by binding to the sterol component of a cell membrane leading to alterations in cell permeability and cell death. While amphotericin B has a higher affinity for the ergosterol component of the fungal cell membrane, it can also bind to the cholesterol component of the mammalian cell leading to cytotoxicity.Liposomes are well recognized drug delivery vehicles that have been shown to enhance the therapeutic activity of a number of drugs. Liposomes have the ability to passively accumulate in tissues. This process can result in the accumulation of significantly greater amounts of drug in tissues than can be achieved by the administration of free drug. Drugs could boost therapeutic index and reduce or avoid general side and toxic effect. To consider the above aspects, amphotericinB being used as model drug, freeze-drying liposome was prepared to orient release of the drug and cut down toxicity to achieve the effect of prolonged action.Methods: Preparation technique and prescription of amphotericin B freeze-drying liposomes was initially determined on the basis of literature and preliminary test, and influential factors were determined by single factor investigation.Four methods such as film dispersion method, reverse-phase method, active loading method, injection method were used to produce liposomes, and sephadex column was used to separate liposomes with free drugs. The encapsulated ratio of the liposomes was determined, which showed that the film dispersion method was the best preparation method.Encapsulated ratio,the shape,appearance and particle size distribution were studied as index, the preparation techoligy of liposomes was optimized by single factor inwestigation, and inspected lipids ratio, the lipids and cholesterol ratio, drug concentration and the lipids and freeze-drying protectant ratio were studied as influential factors. Consequently, L9(34) orthogonal design was made to screen the dosage of adjuvant. The optimized prescription was decided by range analysis to the encapsulated ratio determination result.Respectively mapping amphotericinB liposome and solution in vitro release curve, and use of zero-order, first order, Higuchi and Weibull equation for fit, calculation the coefficient correlation. Taking size of liposome, appearance, colour, oxidation index as a index to inspected amphotericinB liposome physical and chemical stability.A simple HPLC method with ultraviolet detection was developed for the determination of amphotericinB in rat plasma and mice tissue, Diamonsil C18 column (200 mm x 4.6 mm I.D., 5 microm) with UV detector at 385 nm. The mobile phase was acetonitrile-water- EDTA-2Na(0.005mol/L)=40:60(v/v). Flow rate 1ml/min, the column temperature 37℃. The pharma-cokinetic parameters were calculated by 3P87 program. Investigation biodistribution of rats, evaluate targeting in tissue. Result: Film dispersion method of amphotericinB liposomes preparation is efficient with higher entrapment efficience. The optimal prescription is lipid 450mg, cholesterol 150mg, sucrose 90mg, mannitol 150mg, drug 50mg. The entrapment efficiency were 74.3%, and pH was 6.75. The size of the resulting liposomes was 173.5nm. Particle size requirements consistent with targeting.The amphotericinB release behavior from amphotericinB liposome and solution in vitro was in accord with weibull and the equation was as follows: ln[-ln(1-Q)]=1.395lnt-4.146, ln[-ln(1-Q)]=0.6352lnt-3.091, T50 of amphotericinB solution is 3.96h, and T50 of amphotericinB liposome is 14.82h.The physical stability of amphotericinB liposome was shown as follows: The freeze-drying liposomes were well-stacked with good appearance which stored at 4℃, protected from light for a month. This indicated that liposomes needed to be stored under cold conditions and protected from light.The pharmacokinetic behavior of amphotericinB and its liposome were as follows: in comparison with amphotericinB water solution, the liposome's AUC was in-creased notable and the liposome CI (clearance rate) was decreased significantly. Compared to amphotericinB water solution, the liposomes half-life time prolonged from 6.11h to 8.25h. Liposomes have the ability to passively accumulate in tissues, can result in the accumulation of significantly greater amounts of cytotoxic drug in tumor tissues than can be achieved by the administration of free drug. AUC was significantly increased in plasma, liver, speeld and lung in contrast to that of control group, so it can orient release the drug and cut down toxicity to achieve the effect of prolonged action.Conclusion: Phspholipid and cholesterol were used as membrane stuff, amphotericinB liposomes were made by film dispersion method. This preparation technology was simple and efficient. The quality of liposome is controllable and it can remarkably enhances the medicine curative effect and reduces the poisonous effect.