Preparation And Study Of Long Circulation Adriamycin Liposome

Objective: Adriamycin is anthracycline. As an antitumor drug, ADM has good therapeutic effect. Since being marketed, ADM has been used with other drugs as standard therapy of many kind of tumors. But ADM has heart toxicity which anthracyclines have commonly, and the disadvantage has limited clinical application of ADM for a long time. That also is a focus of chemical reconstitution and new formulation R&D. To ADM′s two characters—good therapeutic effect and significant heart toxicity, we try to research and development long circulation adriamycin liposome, utilizing the liposome 's long circulation character to bring ADM's good therapeutic effect into full use, utilizing the liposome 's character of changing target to reduce ADM′s heart toxicity. Long circulation adriamycin liposome can both reduce toxicity and enhance therapeutic effect and be used conveniently—prolong dosing interval.MethodLong circulation adriamycin liposome determination method : According to adriamycin and phospholipid character, acidized isopropanol was used as solvent, and ultraviolet spectrophotometry was used to determine drug content of long circulation adriamycin liposome. The standard curve of the method was A=0.168C+0.00,method recovery was 100～102% , intra-day precision test result was RSD<1% , inter-day precision test result was RSD<1%. Sephadex gel chromatography was used to seperate and determine loading efficiency and in vitro release rate of long circulation adriamycin liposome. According to preparation characters, suitable sephadex gel,column length,mobile phase and flow rate were used to get good seperating effect, column recovery was 99～101%, recovery of drug in blank liposome was 99～100%. 90Plus Particle Size Analyzer was used to determine particle size and size distribution of long circulation adriamycin liposome .Long circulation adriamycin liposome preparation: According to adriamycin characters, film method was used to prepare blank liposome, and ammonium ion gradient method was used to entrap adriamycin. According to long circulation adriamycin liposome formula and technique, 10 factors were selected to conduct single factor test. The results indicated: organic solvent, rotation temperature and rotation speed only affected the speed of preparing film, not affected the quality of the film, and entrapment time did not affect loading efficiency. According to phospholipid characters, the technique conditions were determined: solvent was ethanol isopropanol (1:3), rotation temperature was 55?C and rotation speed was 110rpm, entrapment time was 30min. The proportion of DSPE had no influence on loading efficiency and in vitro drug release rate, and the proportion was 0.25:1. But ultrasound time had influence on preparing blank liposome, according to phospholipid characters and preparation process, the time was determined 4 hours. And investigated the 4 unsure factors : ADM to phospholipid ratio, cholesterol to phospholipid ratio, concentration of ammonium sulfate and entrapment temperature, further. 4 factors and 3 levels orthogonal experiment was designed, the experiment result: ADM to phospholipid ratio was 1:6, cholesterol to phospholipid ratio was 1:3.5 , concentration of ammonium sulfate was 175mmol/L, entrapment temperature was 70?C—4 optimal conditions with other 6 conditions composed the complete long circulation adriamycin liposome formulation and preparation conditions. The optimal result was used to prepare 3 batchs long circulation adriamycin liposome.Long circulation adriamycin liposome quality and stability test: According to long circulation adriamycin liposome characters, appearance, PH, particle size and distribution, entrapment efficiency, in vitro drug release rate, drug content were investigation indexs, test results: long circulation adriamycin liposome was red translucent liquid, PH 6.5, particle size was 120nm. Size was normal distribution and size distribution range was 40nm, loading efficiency was above 95% , in vitro drug release rate of 1 hour and 4 hour were 40%±10% and 60%±10%, respectively. Drug content was 2mg/ml±0.1mg/ml . Long term test was under 4?C, 6 months later, and every index maintained stable. Accelerative test was under 25?C, 3 months later, long circulation adriamycin liposome maintained red, loading efficiency and in vitro drug release rate had no changes. The result indicated: long circulation adriamycin liposome was stable .Long circulation adriamycin liposome pharmacokinetic test: According to data, characters of adriamycin and plasma and tissue, internal standard method was used to set up high performance liquid phase assay to determine adriamycin content in plasma and tissue of mouse, daunorubicin was used as internal standard. The standard curve of high performance liquid phase assay was Y=0.1808X+0.036, r=0.9766. The recoveries of 0.125μg/ml,10μg/ml,200μg/ml were above 95%. RSD of inter-day and intra-day was below 5%. In order to investigate the long circulation effect of long circulation adriamycin liposome, 12 Wistar mouse (body weight 180-220g) were equally divided into two groups randomly, long circulation adriamycin liposome and adriamycin hydrochloride injection were given respectively, at 0, 0.08,0.25,0.5,1,2,4,8,12,24 and 48 hour, blood was collected. The samples were determined with high performance liquid phase assay, the result of adriamycin hydrochloride injection group was that AUC (h·μg/mL) was 61.985, and t1/2α(h) was 1.577, and t1/2β(h) was 28.55, and CL(mg/kg/h)/(μg/mL) was 0.081. The result of long circulation adriamycin liposome group was that AUC (h·μg/mL) was 4856.414, and t1/2α(h) was 6.667, and t1/2β(h) was116.705, and CL(mg/kg/h)/(μg/mL) was 0.001. The result indicated that pegylated carriers significantly increased the exposure of adriamycin in the blood, and prolonged the half-life of adriamycin in comparison with that of free adriamycin. In order to investigate the reducing heart toxicity effect of long circulation adriamycin liposome, 72 Kunming mouse (body weight 18-21g) were equally divided into two groups randomly , long circulation adriamycin liposome and adriamycin hydrochloride injection were given respectively, at 0.08, 1, 2, 4, 6, 24 and 48 hour, the rat were sacrificed, and the heart, liver, kidney, spleen, and lung were immediately collected and treated. The samples were determined with high performance liquid phase assay, the result of adriamycin hydrochloride injection group was that AUC (h·μg/mL) was 411. The result of long circulation adriamycin liposome group was 282. The result indicated the lower concentration of adriamycin in heart significantly leaded to a lower toxicity of adriamycin .ResultsThe results indicated that long circulation adriamycin liposome had good stability, and had good effects of long circulation and reducing heart toxicity .