| Objective: Fluconazole is a triazole antifungal drug which was developed by Pfizer Company in 1980. Its mechanism action is contribution to dyssynthesis of ergosterin in fungal cell by inhibition of fungus cytochrome P-450, so it achieves the effect of inhibition and killing of fungus, and has the advantage of wide antimicrobial spectrum. Its half-life in blood plasma is about 30 hours, and concentration in cerebrospinal fluid is correspondent to 80~90% of concentration in blood plasma. In 1991 Pfizer Company raised the application of fluconazole in precaution of fungous infection in USA, and it already got authorization in Austria,Ireland,Italy and Switzerland to be used to prevent fungous infection in cancer patients. At present, fluconazole is gradually taken to be a safe and valid antifungal drug.Liposome is a kind of ultra-fine form globular carrier preparation which encapsulates the drug in the thin film formed by lipid bilayer. Liposome is analog to cellular structure, and has the characteristics and function of biological membrane. It has the ability of enveloping both water-solubility and lipo-solubility drug, and it is a kind of multi-functional targeted durg carrier which could cut down toxic and side effect, elevate bioavailability, prolong action and delay release. Liposomes for transdermal use can increase the drugs retained in the skin to the maximum, and reduce the drugs in the blood circulation to the minimum. Drugs in the liposomes carrier could penetrate stratum corneum, and formed medicine library between dermis and epidermis. It is a skin-targeted administration system of delayed and control release effect. Drugs could lastingly cure the topical damaged cells or tissues, boost therapeutic index and reduce or avoid general side and toxic effect because less amount of drugs enter greater circulation.Fluconazole being used as model drug, liposomal fluconazole gel was prepared to increase the intradermal hold-up and cut down toxicity to achieve the effect of prolonged action.Methods: Preparation technique and prescription of liposomal fluconazole gel was initially determined on the basis of literature and preliminary test, and influential factors were determined by single factor investigation. Suitable membrane stuff for liposomes was selected from several kinds of phospholipids. The impact of encapsulated ratio determination result by different assay methods and different sephadex column conditions was studied.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 as index, the preparation technology of liposomes was optimized by single factor investigation, and the hydration temperature and rotary speed of rotary evaporation 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.The accumulative release curve of fluconazole liposomes and fluconazole solution were mapped respectively and compared. Zero order,first order,Higuchi,and Weibull equation were used to fit the release process, and coefficient correlation were calculated and studied.The shape,appearance,particle size distribution,viscosity,and physical and chemical stability were studied. The HPLC method to determine the quality of fluconazole in liposomes and liposomal gel was constructed on the basis of literature and preliminary test.Carbopol was used as matrix to make liposomal fluconazole gel. Mice being used as animal model, the transdermal delivery test ex vivo were done to compare their release process which were fitted by zero order,first order,Higuchi and Weibull equation respectively,and the correlation coefficient was calculated and studied.Stress test, stability paramer and sedimentation ratio were done to investigate the stability of lipsomes and lipsomal gel. And the stability of fluconazole liposomes to hot pressing sterilization was also studied.Results: The optimized prescription of fluconazole liposomes was: 640mg phospholipids, 106mg cholesterol, 20mg PVP, and pH 7.0 PBS. Liposomes were made by film dispersion method and the encapsulation was determined by sephadex G-75 column. The diameter/height ratio, flow rate of the PBS solution and volume of PBS above the column all had effect to the separation result. The appearance of fluconazole liposomes was uniform ivory yellow suspension, with the encapsulated ratio 55.03% and mean diameter 249.8nm.The polydispersity was 0.281.The release process in vitro of both fluconazole solution and fluconazole liposomes fitted the Weibull equation. The equation were lnln[1/(1-Q)]=0.703lnt-0.4304 and lnln[1/(1-Q)]= 0.5043lnt-0.801, with the correlation coefficient R2=0.9813 and R2=0.9847 respectively. T50 and Td were 1.10h,1.84hand 2.37h,4.89h, respectively. The release process of fluconazole liposomes and solution before and after 2h were studied: the release process of liposomes before 2h was mainly diffusion process and fitted Higuchi equation, which was probably the free and topical drug release process. The release process after 2h fitted Weibull equation best.The liposomal gel made by carbopol 941 had suitable viscosity. Transdermal delivery test showed that the release process of liposomal and non-liposomal gel all fitted zero order,first order,and Weibull equation with R2>0.99. The drugs of liposomal gel retained in the skin was much more than that of non-liposomal gel (P<0.05).Stability test showed that liposomes and liposomal gel were not stable to high temperature. But the stability parameter of fluconazole liposomes was 0.066, and the fluconazole liposomes suspension was stable when laid up for a long period, only small amount of precipitation was formed after 3 months.Conclusion: Phospholipids and cholesterol were used as membrane stuff, fluconazole liposomes were made by film dispersion method. This preparation technology was simple and convenient. Liposomal gel made by carbopol matrix was stable, and the transdermal delivery test result showed that the liposomal gel had more drugs retained in skin than that of non-liposomal gel. Liposomal gel as fluconazole percutaneous absorption dosage form was expected to be a new applicable preparation. |
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