摘要
本文建立了六甲蜜胺原料药的体外HPLC分析方法,并确立了六甲蜜胺脂质体主药的测定方法,以及确立微柱离心-HPLC法测定六甲蜜胺脂质体包封率,同时也为其制剂的含量测定、理化性质及稳定性等性质的研究提供了可靠的分析方法。
采用乙醇注入法制备了六甲蜜胺普通脂质体,并以包封率为指标,对磷脂种类、磷脂用量、水相介质pH、胆脂比、乙醇的用量、制备温度、搅拌速度、投药量、缓冲液浓度等处方工艺进行了单因素考察,又通过正交设计优化处方,制备了包封率大于85%的六甲蜜胺普通脂质体。在此基础上,考察了壳聚糖包衣脂质体的制备工艺,制备成六甲蜜胺壳聚糖包衣脂质体。
采用逆向蒸发法制备了六甲蜜胺胆盐脂质体,以脱氧胆酸钠为模型胆盐,以包封率为指标,对总脂量、胆脂比、脱氧胆酸钠与总脂量的摩尔比等进行单因素考察,又通过制备钙黄绿素胆盐脂质体考察以上因素对胆盐脂质体在20 mmol·L~(-1)胆盐中稳定性的影响,处方优化后得到的六甲蜜胺胆盐脂质体抵抗胆盐破坏的能力较强。
考察了六甲蜜胺三种脂质体的体外性质,三种脂质体形状圆整,大小均一;在盐酸溶液中,壳聚糖脂质体的释放速率慢于普通脂质体;在胆盐溶液中,胆盐脂质体的释放速率慢于普通脂质体;在pH7.4的磷酸盐缓冲液中,三种脂质体的释放性质没有显著差异。根据外观、粒径、包封率、pH值、药物含量以及过氧化值对三种脂质体的体外稳定性进行了初步考察,这三种脂质体在低温避光下保存稳定性较好。
建立了HPLC法测定大鼠血浆中六甲蜜胺的含量,考察了六甲蜜胺溶液、六甲蜜胺普通脂质体、六甲蜜胺壳聚糖包衣脂质体以及六甲蜜胺胆盐脂质体在大鼠口服给药的体内药动学过程。四者的药动学过程均符合双隔室模型,六甲蜜胺壳聚糖包衣脂质体和胆盐脂质体的半衰期和相对生物利用度均较普通脂质体有所提高。
The HPLC analytical method for Hexamethylmelamine(HMM)in vitro was developed.The method of detecting the content of HMM and the encapsulation efficiency of HMM liposome was established,which also used in study of HMM physicochemical property and stability.The entrapment efficiency of HM liposome was determined by minicolum_n centrifuge-HPLC method.
The HMM liposome was prepared by ethanol injection method.The single factor investigation and the orthogonal design were adopted to obtain the optimized prescription,the entrapment efficiency of HMM liposome was above 85%.On the base of above,the prepared technology of liposome coated chitosan was investigated and HMM liposome coated by chitosan was prepared.
The HMM/DOC liposome was prepared by reverse-phase evaporation method.The single factor was investigated by taking sodium deoxycholate(DOC)as the model of bile salts and entrapment efficiency as the evaluation index.Liposomal membrane integrity was judged by bite salt-induced release of DOC-liposomes encapsulated calcein(Cal/DOC-liposomes).The optimized prescription of HMM/DOC liposome was stable in bile salts solution.
The physicochemical properties of HMM liposome,HMM liposome coated by chitosan and HMM/DOC liposome were studied.The there liposomes were all spheroidal and uniform.The releasing rate of HMM/DOC liposome was slower than HMM liposome in bile salts solution;the releasing rate of HMM liposome coated by chitosan was slower than HMM liposome in chlorhydric acid;there's no difference in releasing rate of three liposomes in phosphate solution of pH 7.4.The results of stability tests indicated that three liposomes should be stored in low temperature.
The detection method of HMM in plasma of rats was established.The pharmacokinetic processes in rats for the three liposomes and HMM solution,which were administrated orally, were accorded with two compartmental models.Compared with HMM liposome,the HMM liposome coated by chitosan and HMM/DOC liposome both had longer half-life and higher relative bioavailability.
引文
[1]C.Rhoda Lee and Diana Faulds.Altretamine:A review of its pharmacodynamic properties,and therapeutic potential in cancer chemotherapy.Drugs,1995,49(6):932-953.
[2]王建东。六甲蜜胺治疗卵巢癌的研究进展。国外医学肿瘤学分册,2001,28(5):387.
[3]Krys J.Miller,Renee M.McGovern and Matthew M.Ames.Effect of a hepatic activation system on the antiproliferative activity of hexamethylmelamine against human tumor cell lines.Cancer Chem Pharm,1985,15:49-53.
[4]Soloway AH,Brumbaugh RJ,Witiak DT.Carbinolamines and related structures:potential alkylating metabolites of clinically active anticancer drugs.J Theoret Biol 1983,102:361-73.
[5]Bonomi PD,Mladineo J,Morrin B,et al.Phase Ⅱ trial of hexamethylmelamine in ovarian carcinoma resistant to alkylating agents.Cancer Treat Rep 1979,63:137-8.
[6]Runhaar EA,Neijt JP,Holthuis JJM,et al.The bioavailability of three altretamine formulations.Pharm Weekbl Sci,1989,11:218-23.
[7]Klippert PJM,Hulshoff A,Mingels M-JJ,et al.Low oral metabolism of hexarnethylmelamine in the rat due to simultaneous hepatic and intestinal metabolism.Cancer Res,1983,43:3160-64.
[8]Enrico Garattini,Tina Colombo and Maria Grazia Donelli,et al.Distribution,metabolism,and irreversible binding of hexamethylamine in mice bearing ovarian carcinoma.Cancer Chern Pharm,1983,11:51-55.
[9]John K.Chan,Vera Loizzi and Alberto Manetta,et al.Oral altretamine used as salvage therapy in recurrent ovarian cancer.Gynecologic Oncology,2004,92:368-371.
[10]Maurie Markman,M.D.and John A.Blessing et al.Altretamine(Hexamethylmelamine)in platinum-resistant and platinum-refractory ovarian cancer:A Gynecologic Oncology Group Phase Ⅱ Trial.Gynecologic Oncology,1998,69:226-229.
[11]Halnsworth JD,Jones Ⅲ HW,Burnett LS,et al.The role of hexamethylmelamine in the combination chemotherapy of advanced ovarian cancer:a comparison of hexamethylmelamine,cyclophosphamide,doxorubicin and cisplatin(H-CAP)versus cyclophosphamide,doxorubicin,and cisplatin(CAP).Am J Clin Oncol,1990,13:410-15.
[12]Omura G,Blessing JA,Ehrlich CE,et al.A randomized trial of cyclophosphamide and doxorubicin with or without cisplatin in advanced ovarian carcinoma.Cancer,1986,57:1725-130.
[13]Wharton JT.Hexamethylmelamine(altretamine)activity as a single agent in previously untreated advanced ovarian cancer.Cancer Treat Rev,1991,18(Suppl.A):15-21.
[14]Bangham A.D.Negative staining of phospholipids and their structural modification by surface active agents as observed in the electron microscope.J.Mol.Biol,1964,8:660-668.
[15]Allen,T.M.and chonn,A.,Large unilamellar liposomes with low uptake into the reticuloendothelial system.FEBS Lett,1987,223:42-46.
[16]Cottel,L.,Ceruti,M.,Dosio,F.,Prom conventional to stealth liposomes:a new frontier in cancer chemotherapy.Tumori,2003,89(3):237-249.
[17]朱梅芳,金鹏。紫外分光光度法测定六甲蜜胺片的含量。江苏药学与临床研究,2002,10(2):16-17。
[18]王瑞芹,胡向青,刘江。六甲蜜胺片的溶出速度考察。中国医院药学杂志,2005,25(4):380-382。
[19]郑俊民主编.经皮给药新剂型.北京:人民卫生出版社,1997.173-178.
[20]吴翠栓.硝酸益康唑脂质体凝胶剂的研究.沈阳药科大学硕士学位论文.2000,6.P13.
[21]陆彬.药物新剂型与新技术[M].北京:人民卫生出版社,1998.128.
[22]王秀敏,邓英杰,郝艳丽等。微柱离心-气相色谱法测定β-榄香烯脂质体包封率。沈阳药科大学学报,2004,21(6):416-419。
[23]雷国峰,邓英杰,钟海军,郝爱军,张丽娜.超滤-HPLC法测定灯盏花素脂质体包封率,沈阳药科大学学报,2006,23(4):237-239。
[24]洪慧,龙晓英,李力任,等.葡聚糖微型凝胶柱测定辣椒碱柔性脂质体包封率的条件探讨[J].广东药学院学报,2005.21(2):120-123。
[25]Naeff,R.,Feasibility of topical liposome drags produced on an industrial scale.Adv.Drug Deliv.Rev.,1996,18:343-347.
[26]Wagner,A.,Vorauer-Uhl,K.,Kreismayr,G.,Katinger,H.The cross flow injection technique:an improvement of the ethanol injection method.J.Liposome Res.,2002,12:259 - 270.
[27]Lin,H.H.,Ko,S.M.,Hsu,L.R.,Tsai,Y.H.,The preparation of norfloxacin-loaded liposomes and its in vitro evaluation in pig's eye.J.Pharm.Pharmacol.,1996,48:801 - 805.
[28]Ports M,et al.Liposomes obtained by the ethanol injection method.Int J pharm.1993,95(1-3):51-57.
[29]Ingrid Henriksen,Gro Srnistad and Jan Karlsen,Interactions between liposomes and chitosan[J].International Journal of Pharmaceutics,1994,101:227-236.
[30]Toshinori Sato,Junzo Sunamoto.Site specific liposomes coated with polysaccharides.In:Gregoriadis G.ed.Liposome Technology.Vol.Ⅲ.2ed.Boca Raton,FL:CRC Press,1992:179-198.
[31]Demarger-Andre,S.and Domard,A..Chitosan behaviours in a dispersion of undecylenic acid.J.Carbohydr.Polvm,1993,22:117-126.
[32]Matthew M.Ames.Hexamethylmelamine:Pharmacology and mechanism of action.J Cancer Treatment Reviews,1991,18:3-14.
[33]I.Craig Henderson and Charles L.Shapiro.Hexamethylmelamine use in the treatment of metastatic breast cancer.J Cancer Treatment Reviews,1991,18:91-98.
[34]Philip S.Schein,Barbara Scheffler and William McCulloch.The role of hexamethylmelamine in the management of ovarian cancer.J Cancer Treatment Reviews,1991,18:67-75.
[35]Runhaar EA,Neijt JP,Holthuis JJM,et al.The bioavailability of three altretamine formulations.J Pharm Weekbl Sci 1989,11:218-23.
[36]Conacher M,Alexander J,Brewer JM.Oral immunisation with peptide and protein antigens by formulation in lipid vesicles incorporating bile salts(bilosomes)J Vaccine.2001,19:2965-74.
[37]Senior,K.Bilosomes:the answer to oral vaccine delivery? J Drug Discov Today.2001,6:1031-1032.
[38]Szoka FJ,Papahadjopoulos D.Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation..J Proc Natl Acad Sci U S A,1978,75:4194.
[39]Alfonso de la Maza and Jose Luis Parra.et al.Vesicle to micelle phase transitions involved in the interaction of sodium cholate with phosphatidylcholine liposomes.J Colloids and Surfaces A:Physicochemical and Engineering Aspects.1997,127:125-134.
[40]Schubert R,et al.Studies on the mechanism of bile-salt-induced liposomal membrane damage.J Digestion,1983,28:181.
[41]平其能主编,现代药剂学,北京。中国医药科技出版社,1998:613.
[42]陈奇主编,中药药理实验方法学,北京。人民卫生出版社,1985:941.
[43]Y Diebold and M Jarrin.Ocular drug delivery by liposome-chitosan nanoparticlecomplexes.Biomaterials J,2007,28:1553-64.
[44]D McPhail,L Tetley et al.Liposomes encapsulating polymeric chitosan based vesicles-a vesicle in vesicle system for drug delivery.Int.J Pharm,2000,200:73-86.
[45]董岩,张群正,李晓玲等。壳聚糖在药物载体中的应用进展。现代食品科技,2007,23(7):98-100。
[46]蒋刚彪,张余,刘慧等。壳聚糖作为药物载体的应用概况。新药与辅料,2007,18(13):1022-24。
[47]张修宇,邓英杰,赵春杰等。PVP包覆β-榄香烯口服脂质体大鼠体内药物动力学研究.中药材,2006,29(2):157-160。
[48]C Brindley and A Gescher.Studies of the mode of action of antitumor triazenes and triazines-Ⅲ.Metabolism studies on hexamethylmelamine.Biochem Pharm,1982,31(5):625-31.
[49]E Garattini and M Grazia.In vivo and in vitro irreversible binding of hexamethylmelamine to liver and ovarian tumor macromolecules of mice.Biochem Pharm,1981,30(10):1151-54.
[50]A Begleiter and J Grover.Uptake and metabolism of hexamethylmelamine and pentamethylmelamine by L5178Y lymphoblasts in vitro.Cancer Res,1980,40:4489-94..
[51]Paul J.A.Borm and Marie Jose J.Cellular and subcellular studies of the biotransformation of hexamethylmelamine in rat isolated hepatocytes and intestinal epithelial cells.Cancer Res,1984,44:2820-26.
[52]郭海燕,莫穗林。脂质体物理稳定性和包封率的影响因素。中国新药杂志,2004,13(6):498-501。
[53]Y Barenholz.Liposome application:problems and prospects.Cur Opinion:in Colloid & Interface Sci,2001,6:66-77.
[54]王玮,周建平,任逢晓。主动载药法制备双氯芬酸钠脂质体及其体外释放。中国药科大学学报,2007,38(4):323-326。
[55]魏农农,陆彬。结肠定位壳聚糖包衣氟尿嘧啶脂质体的制备、形态与体外释放。药学学报,2003,38(1):53-56。
[56]姚亚红,张立伟。黄芩素脂质体的制备及体外释放的研究。中医药学报,2006,34(3):31-33。
[57]张霞,尹宗宁,王怡鑫。纳豆激酶脂质体的制备及体外释放。中国药学杂志,2007,42(4):279-282。