Studies On Itraconazole-Loaded Liposomes Coated By Carboxymethyl Chitosan

Objective:In recent years, the incidence of opportunistic fungal infection (especially in the lungs, urinary tract and intestinal tract) rised sharply as the increasing of immune diseases such as AIDS, cancer, autoimmune diseases and diabetes. The fungal infections did great harm to human health. Itraconazole (ITZ) is a triazole antifungal agent against a broad spectrum of fungal species and it is widely clinically used for a variety of serious fungal infections since 1988. The clinical application of itraconazole is mostly the imports SporanoxR capsules and injection. ITZ has a poor water solubility and it is difficult to maintain an effective therapeutic concentration after orally administered because of the unstable drug absorption and blood concentration. SporanoxR injection uses a total prescription of 40%of HP-β-CD as inclusion material to increase the drug solubility. Because HP-β-CD is mainly removed through kidney, it may lead to toxicity because of the accumulation and may be potentially carcinogenic. Therefore, the development of a low toxicity and efficient new intravenous itraconazole formulations becomes the focus of research.Liposome is a new intravenous drug delivery system uses phospholipid and cholesterol as carrier material. Phospholipids and cholesterol are endogenous substances and non-toxic to human, they have been approved as medicinal materials by FDA, and there is liposomal amphotericin B used in clinic as antifungal agents. Carboxymethyl chitosan (CMC) is a water-soluble chitosan derivative which has characters such as biodegradability, biocompatibility, low toxicity and so on. We use the CMC to form a dense layer of hydrophilic protective film on the liposome's surface for the modification, in order to increase the stability of liposomes and extend the liposome's circulation time in vivo through enhancing the exclusion of plasma protein.ITZ was selected as the model drug and PC and CHOL were used as the carrier material in this experiment. The ITZ loaded liposomes were prepared by film dispersion method and then modified with CMC by the adsorption of positive and negative charge. This article was composed of the following four parts:study of preparation and physical/chemical properties of ordinary liposomes and modified liposomes; in vitro antifungal activity of liposomes; preliminary safety evaluation and in vivo pharmacokinetics and tissue distribution studies.1. Preparation and physical/chemical properties of ordinary liposomes Filtration was applied to separate the common liposomes and free drugs. UV spectrophotometry method was established to determine the itraconazole concentration and the method was studied; preparation method of itraconazole loaded liposomes was selected using entrapment efficiency as index; the optimal formulation was obtained by orthogonal experimental design studies; DSC method was used to identify the formation of liposomal crystalline phase; the morphology of liposomes was observed by transmission electric microscope (TEM), particle size and Zeta potential were measured by laser particle size analyzer and surface potential analyzer, respectively, pH value of the liposome suspension was measured by pH meter.The results showed that UV spectrophotometry method established to determine the itraconazole concentration in vitro is easy, convenient and correct; filtration applied to separate the free drug and liposomes performed good accuracy and accorded with the requirement of separation; a higher liposomal encapsulation efficiency can be obtained by the film dispersion method; the optimized procedure was as follows:the ultrasonic power is 500w, and ultrasonic time is 15s, the optimized prescription was as follows: lecithin/cholesterol (12:1, g/g), drug/lipids (1:20, g/g), hydration medium/organic solvent(1:4, v/v); The particle size of unmodified liposomes was (170.1±15)nm, the Zeta potential was (-9.39±0.75) mV and pH value was 7.38±0.07. Reproducible experimental results of three batches showed that, ITZ loaded liposomes have a reasonable formulation composition and stable process.2. Preparation and physical/chemical properties of modified liposomes Filtration was applied to separate the modified liposomes and free drugs. UV spectrophotometry method was established to determine the itraconazole concentration and the method was studied; Ordinary liposome was then coated by carboxymethyl chitosan, and the effect on the modification results of amount of octadecylamine and the concentration of CMC were investigated; the morphology of modified liposomes was observed by transmission electric microscope (TEM). Particle size and Zeta potential were measured by laser particle size analyzer and surface potential analyzer,respectively. pH value of the liposome suspension was measured by pH meter. The results showed that UV spectrophotometry method established to determine the itraconazole concentration in vitro is easy, convenient and correct; filtration applied to separate the free drug and modified liposomes performed good accuracy and accorded with the requirement of separation; the amount of 2.5% octadecylamine and 0.2% of carboxymethyl chitosan were best to liposomes modified; the size of liposomes modified by carboxymethyl chitosan was larger than that without modification, and the average diameter was (349.3±18)nm, Zeta potential was (-35.71±0.62) mV and pH value was 7.94±0.13. Reproducible experimental results of three batches showed that, CMC modified liposomes have a reasonable formulation composition and stable process.3. In vitro antifungal activity and preliminary safety evaluation Antifungal activity of ITZ loaded liposome coated with CMC was tested using Candida albicans by microdilution method in accordance with the fungi susceptibility testing program (M27-A program) established by Clinical Standardization Committee U.S. (NCCLS); in vitro hemolysis and blood vessels irritation were tested. The results showed that itraconazole loaded liposomes maintained a strong antifungal activity in vitro and it was not inhibited by the encapsulation of drug; the agents were safe to use without hemolysis and agglutination of blood cells and there was no irritation during the intravenous injection and the recovery period.4. In vivo pharmacokinetics and tissue distribution studies. HPLC method was established to investigate itraconazole concentrations of plasma and tissue in mice; In vivo pharmacokinetics and tissue distribution in mice were studied after injected with two kinds of liposomes and injection; organs' targeting efficiency was evaluated by the relative uptake rate (Re), the relative targeting efficiency (Te) and Ce as the index. The results showed that endogenous substances did not interfere with itraconazole determination on the conditions of selected chromatographic; the extraction recovery, method recovery of drug in blood and tissues in mice, the linearity, RSDs of intra-day and inter-day, and the accuracy all satisfied with the need of analysis for biological detection; compared with the injection, MRT in plasma of ordinary and modified liposomes extended from 4.706h to 13.479h and 17.432h, respectively; AUC were increased from 6.427mg/L*h to 13.479mg/L*h and 17.432mg/L*h; drug distribution of common liposomes in the liver and spleen were increased, the AUC increased from 139.150μg/g*h and 48.821μg/g*h to 192.007μg/g*h and 230.059μg/g*h, respectively; compared with common liposomes, drug distribution of modified liposomes in the liver, and spleen decreased, and the AUC was 134.284μg/g*h and 180.285μg/g*h, respectively; drug distribution in the lungs significantly increased, compared with the solution group, AUC of the common liposomes increased from 102.955μg/g*h to 222.309μ.g/g*h; the relative uptake rate of the modified liposomes targeted to the lung increased from 2.159 to 4.243, and the targeting efficiency increased from 27.31%to 49.71%; the distribution of drugs in heart and kidney reduced, compared with the injection, Te of the heart of common liposomes and modified liposomes reduced from 13.54%to 7.42%and 5.96%, Te of kidney decreased from 31.14%to 11.09% and 5.32%, respectively; Cmax was significantly lower, and all of the above were conducive to reducing the toxic side effects of these sites. Conclusion:In this study itraconazole was successfully encapsulated into carboxymethyl chitosan-modified liposomes, and the preparation is simple, reproducible, and has a high encapsulation efficiency. The physical and chemical properties meet the injection requirements. Antifungal activity in vitro was not reduced after packaged in liposomes, and the preliminary safety study results showed no hemolysis and irritating after intravenous injection. Compared with the contrast agents itraconazole loaded CMC modified liposomes extended the drug circulation time and drug accumulation on sites which are susceptibility to fungal increased, such as the lung; drug accumulation in heart and kidney reduced and the side effects could be decreased. It provided a reliable theoretical basis for clinical application.