| Objective:Vinorelbine Ttartrate (VRT) can interact tumor cell metabolism and induce mitosis microtubules disaggregation by effecting microtubule protein of tumor cell, to stop Cell Growth in metaphase of cell division. It is effective on the treatment of NSCLC (No-small cell lung cancer), acute lymphoblastic leukemia, metastatic breast cancer, Hodgkin’s or non-Hodgkin’s lymphoma and soft tissue sarcoma, etc. At present, VRT is applied to clinical as two dosage forms including injections and oral soft capsules. Injection is unacceptable for patients because it causes severe pain at injection site and phlebitis. Otherwise, VRT causes severe bone marrow depression, and damages the blood and digestive system. So VRT is limited in clinical. Fortunately, studies indicate that liposome loaded chemotherapeutics, witch can target to tumors, are harmful less and more effective than chemotherapeutics. This work combines Long Circulation Liposome and Thermo sensitive specific target and aims to prepare VRT Long Circulation Thermo-sensitive Liposome (V-LTSL), to promote VRT’s clinical compliance, improve its pharmacokinetics characteristics in vivo and make V-LTSL release drug rapidly in heated tumor site. Thus, VRT can be harmfulless and more effective.Methods:Based literatures investigation, technological line was designed; With DPPC, DSPG and MSPC, blank SUV liposome was prepared by film dispersion-probe ultrasound method. Formula and preparation technology of blank SUV were optimized roundly according to diameter and stability. VRT was loaded into blank SUV by pH gradient method. Main factors affecting drug loading technology were investigated by haplo-factor and orthogonal test according to EE%. Lastly, the formula and preparation technology of VRT Thermo-sensitive liposome (V-TSL) were defined and validated. Antiphase ion pair HPLC was established to assay VRT and its related substance in V-TSL. Then, EE% assay method of V-TSL was found by taking gel micro-column separate liposome and VRT. Physico-chemical property of V-TSL, such as appearance, diameter, pH and phase-transition temperature, was investigated. Basely, the stability of V-TSL was preliminarily assessed. Based the formula and preparation technology of V-TSL, V-LTSL were prepared taking DSPE-PEG as stereo protective agent. Then, comparative studies on rat’s pharmacokinetics and tumor-bearing mice pharmacodynamic tests among VRT normal injection (V-I), VRT normal liposome and V-LTSL were carried out.Results:The formula of blank SUV was DPPC/DSPG/MSPC(w/w, 7.5:1:0.8), Preparation SOP of 5mL blank SUV was enacted.3 batches of blank SUV were prepared according to this SOP, their mean diameter were 95.5±1.1nm,92.9±1.5nm and 94.4±1.3nm, after 30d storage (4℃) their diameter raised 10-20nm. This formula and preparation technology was repeatable, bland SUV was stable and suitable for drug loading. V-TSL was formulated with 4mL blank SUV liposome(50 mg·mL-1) and 1mL VRT solution(8.0mg·mL-1), drug-lipid ratio was 1:25, hydration medium was citrate buffer solution(200mM, pH3.5-4.0), elution solvents was PBS(200mM, pH6.5-6.8), incubation temperature was 35℃and incubation time was 35min. Preparation SOP of 5mL V-TSL was enacted,3 batches of V-TSL were prepared according to this SOP, their EE% were 91.2±1.3%, 90.0±1.7% and 91.9±1.1%, their mean diameter were 115±2.1nm, 118±1.7nm and 121±1.9nm. Assay methods of content, related substance and EE% fit the bill of quantitative analysis. V-TSL was bluish semitransparent suspension, liposome was spheroid and its mean diameter was 116±3.5nm, pH was 6.55, phase-transition temperature was 41.8℃, the thermo sensitive release was apparent. The alteration of diameter and EE% of V-TSL was not apparent under room temperature lucifugal condition in 3d, and when diluted with 5% glucose solution and 9% NaCl solution the alteration of diameter and EE% of V-TSL was not apparent too. Diameter increased from 103.2nm to 212.5nm and EE% decreased from 92.9% to 48.5% under 4℃lucifugal condition in 2 months. Taking 2% lactose as cryoprotectant, Diameter increased from 108.2nm to 141.5nm and EE% decreased from 93.5% to 82.7% under -20℃lucifugal condition in 2 months. Plasma concentration-time curve of V-L was fit for the two-compartment model, and the curves of V-I and V-LTSL were fit for the three-compartment model. T1/2beta, AUC of V-LTSL were equal to 10.5 times,8.8 times V-I and 15.9 times,3.8 times V-L, and CL of V-I and V-L were equal to 8.9 times,3.8 times V-LTSL. Tumor growth curve of tumor-bearing mice treated by V-LTSL and thermotherapy was planus, tumor inhibition rate of V-LTSL was highest, and tumor tissue section shown that V-LTSL possessed the strongest potency against tumor cell.Conclusion:The formula and preparation technology of VRT thermo-sensitive liposome was stable and repeatable. V-TSL was bluish semitransparent suspension, liposome was spheroid and its mean diameter was 110- 125nm. Drug-loading rate of V-TSL exceeded 1.8 mg.mL-1, EE% exceeded 90%, content of related substance was less than 0.6%, pH was close to 7.0, phase transition temperature was 41.8℃, thermo sensitive release was apparent. It was stable under short-term placement and elution condition, and suitable for clinical application. Taking 2% lactose as cryoprotectant, V-TSL was stable under -20℃lucifugal condition in 2 months. V-LTSL can prolong circulation, increase AUC and decrease clearance of VRT in rats. Results of pharmacodynamic tests shown that, combining with thermotherapy, V-LTSL enhanced the potency and reduced the toxicity of VRT in tumor-bearing mice, based on this we can conclude that V-LTSL possesses thermo target characteristic. |
