| Cytarabine (cytosine arabinoside, Ara-C) is an effective chemotherapeutic agent for the treatment of acute myelogenous leukaemia and lymphocytic leukaemias. Cytarabine is an S-phase-specific drug. Because this drug kills cells only when they are synthesizing DNA, prolonged exposure of cells to therapeutic concentrations is critical to achieve maximum cytotoxic activity. However, the activity of cytarabine is decreased by its rapid deamination to the biologically inactive metabolite uracil arabinoside. Cytarabine is indicated for the intrathecal treatment of lymphomatous meningitis, and prophylaxis and treatment of leukemia. Cytarabine depot preparation could prolong the half time in cerebrospinal fluid and relieve adverse event. Encapsulation of drugs into multivesicular liposomes (DepoFoam(?)) offers a novel approach to sustained-release drug delivery. DepoFoam-encapsulation has been shown to result in sustained-release lasting over several days to weeks after non-vascular administration. DepoFoam(?) particles are distinguished structurally from unilamellar vesicles, multilamellar vesicles, and neosomes in that each particle comprises a set of closely packed non-concentric vesicles, like honeycomb. Multivesicular liposomes containing cytarabine (MVL-Cyt) was studied including formulation and processing, and was evaluated about pharmaceutical property and initial stability; Taking cytarabine solution as a reference preparation, the pharmacokinetics of MVL-Cyt in rats was also performed by HPLC.An HPLC method was established for the assay of cytarabine. The analysis method was precise, simple and reliable for the quality control of cytarabine. Liposomes and free drug were separated with lower speed centrifugation. The content of cytarabine was determined by HPLC, and to calculate encapsulation efficiency. Recovery was from 95 % to 105 %. This method is accurate, simple and reproducibility is well.Multiple emulsion method was applied to prepare the MVL-Cyt, emphasizing the research on formulations and procedure and taking encapsulation efficiency and morphology as target. The influence of ingredients including L-lysine (type, concentration), PC, DPPG, CH, organic solvent and pH of inner aqueous phase, out aqueous phase were investigated by single factor or orthogonal design. The preparation process parameter including the flow rate of nitrogen gas, the temperature of water-bath, the intensity of emulsifying were optimized by single factor. When lysine was free base lysine, PC, DPPG, CH at concentration of 10,2,7.5 mg·mL-1 , pH of inner aqueous phase was 1.0, mixed solvent were chloroform and diethyl ether (1:1,v/v), flowing nitrogen gas at a flow rate of 0.3 m3·h-1, in a shaking water bath at 37℃, the first mixing intensity was 14000 r·min-1 for 8 minutes, multivesicular liposomes containing cytarabine prepared had higher encapsulation efficiency and morphology in micrograph was well.Chloroform and diethyl ether mixed solvent of DOPC (1, 2-dioleoyl-sn-glycero-3-phosphoc -holine), DPPG (1, 2-dipalmitoyl-sn-glycero-3-phosphoglycerol), CH (cholesterol), and TO (1, 2, 3-trioleoyl-sn-glycerol) at concentrations was added to a container. An aqueous solution containing cytarabine at a concentration of 30 mg·mL-1 in 0.1 mol·L-1 hydrochloric acid was layered on top of the organic solution in the container. A water-in-oil emulsion was prepared from the two immiscible solutions on a mixer running at 14000 r·min-1 for 8 min. An aqueous solution containing 4 % glucose and 40 mmol·L-1 L-lysine was added to the water-in-oil emulsion in the teat glass capped. The teat glass was then attached to the head of a vortex machine and shaken for 10 s resulting in a water-in-oil-in-water double emulsion. After a dilution of the double emulsion in the glucose and lysine solution, organic solvent were removed by flowing nitrogen gas over the double emulsion placed in a shaking water bath at 37℃. The resultant multivesicular lipid particles were washed repeating three times by the steps of first adding a solution of 0.9 % sodium chloride, followed by centrifugation at 600(?)g and discarding the supernatant.To research pharmaceutical quality of MVL-Cyt preparation, appearance, morphology, encapsulation efficiency, the mean diameter and particle size distribution, the release behavior in vitro were investigated. The MVL-Cyt preparation was white or off-white suspension. The light micrograph at 400 magnification showed that particles were showing the smooth, spherical, and multivesicular nature of MVLs particles, and there was no debris. The mean particle size of MVLs was 19.49μm and span was 0.91. Encapsulation percent could be much higher, and free drug was lower than ten percent. The cytarabine of MVLs released sixty percent for two weeks in blank human plasma without burst release. The MVL-Cyt preparation could be stable to consist of contents, encapsulation percent, appearance, pH, particle size for one month in 4℃environment.The HPLC method was adopted for the determination of the concentration of cytarabine in plasma. This method was feasible, and endogenous material and internal standard acyclovir did not interfere with determination of cytarabine. The pharmacokinetic characters of MVL-Cyt in rats were monitored by HPLC by comparison with cytarabine solution at the same time. The pharmacokinetics parameters were analyzed with DAS, and statistical moment parameters were more reasonable. AUC, MRT and Cmax of MVL-Cyt and cytarabine solution were (43.89±9.82) and (104.18±9.54)μg·h·mL-1, (3.22±0.50) and (1.95±0.26) h, (8.79±2.10) and (37.64±3.00)μg·mL-1 respectively. The relative bioavailability was only 40.98 %. The MVL-Cyt preparation could not sustain release for two weeks in rats. Because the remained particles of MVLs were still retention at the injection site; and the concentration of cytarabine in plasma could not determine by mean of cytarabine decreased by its rapid deamination to the metabolite. The MVL-Cyt preparation could probably relieve side effect. |
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