离子载体A23187介导pH梯度法制备重酒石酸长春瑞滨长循环脂质体

目的:采用A23187制备重酒石酸长春瑞滨长循环脂质体,优化了处方工艺,并考察了含量、包封率、药脂比和体外释放等检测指标。方法:采用A23187介导的pH梯度法制备了重酒石酸长春瑞滨脂质体;用HPLC法检测了脂质体中重酒石酸长春瑞滨的含量和脂质(HSPC)的含量,考察了药脂比;采用阳离子交换树脂分离脂质体和游离药物,HPLC法检测包封率;以4 mmol.L-1NH4Cl-PBS(pH 7.4)为体外释放介质考察了脂质体的体外释放行为。结果:重酒石酸长春瑞滨脂质体包封率为96.1%,药脂比为1∶5(w/w);高药脂比有利于延长药物体外释放的时间。结论:采用A23187介导的pH梯度法制备重酒石酸长春瑞滨脂质体工艺可行、载药量大、包封率高;所建立体外释放的检测方法快速、准确。     

重酒石酸长春瑞滨脂质微球注射液的制备

目的研制重酒石酸长春瑞滨脂质微球注射液。方法通过高压均质使乳粒产生高速碰撞和空穴作用,降低乳粒直径,并控制高压均质的压力和次数来提高稳定性。结果重酒石酸长春瑞滨脂质微球注射液的平均粒径为200 nm,包封率为85%,具有良好的稳定性。结论经过制剂稳定性实验及动物安全性实验,表明采用上述制备工艺制备的脂质微球注射液具有一定的稳定性和安全性。     

重酒石酸长春瑞滨注射液致过敏性休克

1例62岁男性患者因左肺小细胞癌行化疗治疗。给予重酒石酸长春瑞滨注射液40 mg入0.9%氯化钠注射液100 mL静滴,用药结束后,患者出现头晕、出冷汗、血压升高,逐渐出现背部紧缩感、呼吸急促、血压下降、躯干部皮疹、面部潮红。给予吸氧、非那根、肾上腺素、多巴胺、地塞米松等治疗后症状逐渐好转。     

HPLC法测定重酒石酸长春瑞滨脂质体注射液中的溶血卵磷脂

目的建立重酒石酸长春瑞滨脂质体注射液中溶血卵磷脂1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine.(P-lyso-PC)和1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine(S-lyso-PC)含量测定的HPLC法。方法色谱柱:Waters XTerra MS C18柱(4.6 mm×150 mm,3.5μm),流动相:甲醇-0.02 mo.lL-1的草酸溶液(体积比为85∶15),流速:1.0 mL.min-1,柱温:35℃,示差折光检测器,进样量:100μL。结果重酒石酸长春瑞滨不干扰溶血卵磷脂的检测,P-lyso-PC质量浓度在0.04～0.40 g.L-1内与峰面积呈良好线性关系(r=0.999 7);S-lyso-PC质量浓度在0.04～0.40 g.L-1内与峰面积呈良好线性关系(r=0.999 3),平均回收率分别为98.5%(n=9)和99.1%(n=9)。结论本方法适用于重酒石酸长春瑞滨脂质体注射液中的溶血卵磷脂的质量控制。     

重酒石酸长春瑞滨脂质体包封率测定方法比较

目的制备重酒石酸长春瑞滨脂质体,比较葡聚糖凝胶微柱离心法和阳离子交换树脂离心法测定重酒石酸长春瑞滨脂质体包封率的差异。方法pH梯度法制备重酒石酸长春瑞滨脂质体,分别以葡聚糖凝胶Sephadex G-50和阳离子交换树脂装柱,离心分离脂质体和游离药物,采用HPLC法测定药物含量,计算包封率,并用SPSS软件进行t检验。结果两种方法测定不同载药量的脂质体包封率结果均无显著性差异(P>0.05)。结论葡聚糖凝胶微柱离心法和阳离子交换树脂离心法均可用来测定重酒石酸长春瑞滨脂质体包封率,优选阳离子交换树脂离心法。     

重酒石酸长春瑞滨注射用乳剂及其市售注射液在肿瘤患者中的药动学比较

目的比较重酒石酸长春瑞滨注射用乳剂与市售注射液在肿瘤患者的药动学。方法在符合入选标准的肿瘤患者中,采用随机平行对照单次给药的方法进行药动学试验,给药剂量30 mg·m-2,采用LC-MS/MS法测定血浆样品中长春瑞滨的浓度,计算主要药动学参数并进行统计分析。结果重酒石酸长春瑞滨注射用乳剂和市售注射液在肿瘤患者中的主要药动学参数分别为:t1/2(16.78±5.24)和(20.03±24.72)h,CL(22.75±5.81)和(17.71±2.88)L·h-1·m-2,Vc(541.04±223.36)和(486.03±567.17)L·m-2,AUC0-t(1 297.09±308.73)和(1 629.31±309.41)μg·L-1·h,AUC0-∞(1 388.37±318.54)和(1 736.23±299.83)μg·L-1·h,ρmax(1 426.25±397.56)和(2 187.50±828.04)μg·L-1,tmax(0.22±0.04)和(0.29±0.18)h,MRT0-t(6.95±1.08)和(4.94±1.31)h;两制剂AUC0-t、t1/2、tmax、ρmax和Vc均无显著差异(P>0.05)。结论重酒石酸长春瑞滨注射用乳剂和市售注射液相比,在肿瘤患者中具有相似的药动学特征。     

Reconsideration of drug release from temperature-sensitive liposomes

The liposomal phase transition temperature was monitored in unstirred suspensions using a differential scanning calorimeter. The main and pre-transition temperatures under conditions of stirring were measured by the change in 90 degrees light scattering using a fluorescence spectrophotometer. Both methods show the same main transition temperature either with or without stirring. Temperature sensitive liposomes were made of DPPC (dipalmitoylphosphatidylcholine), DMPC (dimylisitoylphosphatidylcholine) or DSPC (distearoylphosphatidylcholine). The calcein release profile from the liposomes depends on the stirring time of the liposome suspension at the main transition temperature. For 1 h incubation, the leakage profile with and without stirring is similar. It had been hypothesized that temperature sensitive liposomes released drug at the main-transition temperature. However, calcein leakage from liposomes is observed also at the pre-transition temperature. Thus, a liposomal encapsulated drug will likely leak from DPPC liposomes at body temperature (37 degrees C), even if the liposomes were designed to have a higher main transition temperature.     

Characterization of impurities in semi-synthetic vinorelbine bitartrate by HPLC-MS with mass spectrometric shift technique

A simple and sensitive method of high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESI-MS) was developed to separate and identify impurities in semi-synthetic vinorelbine bitartrate sample. The analytical HPLC was carried out on a reversed-phase C8 column using 0.02 M ammonium formicate buffer (pH 4.2) and methanol (46:54, v/v) as mobile phase at a flow rate of 0.8 ml/min at room temperature and a UV detection at 267 nm. The on-line HPLC/ESI-MS/MS was performed using ion trap analyzer in positive ion mode. Applying mass spectrometric shift technique to HPLC/ESI-MS/MS analysis, four impurities were identified as 18'-O-demethylvinorelbine (impurity-1), 6'-N-methylvinorelbine (impurity-2), 23-O-demethylvinoreline (impurity-3) and 17-bromovinorelbine (impurity-4), respectively, in investigated vinorelbine bitartrate sample. The four impurities, in which the impurity-1 was not reported as the semi-synthetic process impurity for vinorelbine bitartrate elsewhere, were isolated by preparative high-performance liquid chromatography. Their structures were further confirmed by means of 1D and 2D NMR spectra. Structural elucidation by spectral data was discussed.     

Hyperthermia-induced antitumor activity of thermosensitive polymer modified temperature-sensitive liposomes

Temperature-sensitive liposomes (TS-liposomes) have been studied for chemotherapeutic purposes to enhance the release of anticancer drugs at tumor sites. In this study, we prepared poly(N-isopropylacrylamide-co-acrylamide) (PNIPAM-AAM) and polyethylene glycol (PEG)-modified TS-liposomes (PETS-liposomes). PETS-liposomes significantly increased in vitro drug release in serum compared with PEG-fixed or PNIPAM-AAM-modified liposomes. Furthermore, incorporation of both PNIPAM-AAM and PEG into PETS-liposomes enhanced the stabilities of liposomes in serum by inhibiting protein adsorption. In addition, to investigate the therapeutic efficacy of doxorubicin (DOX)-loaded PETS-liposomes, the in vivo antitumor activity of liposomes in combination with hyperthermia was evaluated in a B16F10 melanoma tumor-bearing mouse model. PETS-liposomes showed much higher levels of tumor growth inhibition than PEG-fixed or PNIPAM-AAM-modified TS-liposomes. Moreover, the antitumor activity of PETS-liposomes was enhanced significantly when they were administered in combination with hyperthermia. PETS-liposomes were found to be highly efficacious carriers for the in vivo delivery of anticancer drugs, and to have potential anticancer applications in combination with hyperthermia.     

Efficacy and mechanism of antiresistant vinorelbine liposomes in treating resistant breast cancer cells

Among the comprehensive treatment strategies of breast cancer, chemotherapy plays a crucial role in eliminating cancer cells. However, multidrug resistance is one of the major obstacles to successful treatment. The objectives of this study were to construct an antiresistant vinorelbine liposomes and to demonstrate the efficacy and mechanism for treating resistant breast cancer cells. The study was performed using breast cancer MCF-7/adr cells. The antiresistant vinorelbine liposomes were modified with tamoxifen and dequalinium. The average particle size of antiresistant vinorelbine liposomes were approximately 100 nm, and they showed a robust overall anticancer efficacy by direct killing and by apoptosis induction. The mechanisms were associated with increased cellular uptake, decreased ABCB1 and ABCC10 transporters, and targeting to mitochondria. The apoptosis signaling pathways were ralated to activiated caspases (8, 9, 3), induced release of cytochrome c from mitochondria, activated Bax, inhibited Mcl-1, and generation of ROS. The new antiresistant vinorelbine liposomes could be a useful formulation deserving further development, and the present study provides a potential strategy for circumventing drug resistance in breast cancer.     

星点设计-效应面法优化异长春花碱脂质体的处方

目的采用星点设计-效应面法优化异长春花碱脂质体的处方。方法以磷脂/药(质量比)、胆固醇/磷脂(摩尔比)、水化温度为考察因素,以脂质体的包封率和脂质体的平均粒径为指标,采用二次多项式方程描述考察指标和影响因素之间的数学关系,根据此数学模型描绘效应面,选择最佳处方,并进行预测分析。结果所建立的考察指标和影响因素之间的数学模型具有较高的可信度。以优化后的处方制备样品,各指标实测值与预测值偏差较小。结论所建立的模型预测性良好,制备的脂质体符合设计要求。     

局部温热并埋封阿霉素微脂粒治疗乳腺癌的研究

目的　观察局部温热并埋封热敏性微脂粒治疗乳腺癌的作用。方法　采用局部温热并埋封热敏性阿霉素微脂粒 (HT Ts LiP ADM )治疗乳腺癌 40例 ,以环磷酰胺、阿霉素、5 氟尿嘧啶 (5 FU)联合方案治疗乳腺癌 2 0例作对照 (CAF组 )。结果　 1疗程后 ,HT Ts LiP ADM组完全缓解 +部分缓解 (CR +PR) 3 4例 ,好转 (MR)4例 ,稳定 (SD) 2例 ,总有效率 85 % ,未见病变进展。CAF组CR +PR 8例、MR 6例 ,SD 4例 ,总有效率 40 % ,2例病变进展。两组比较HT Ts LiP ADM组疗效明显提高 (P <0 0 1) ,其毒性反应、恶心呕吐、心脏毒性及骨髓抑制明显减少。结论　局部加热并埋封阿霉素微脂粒治疗乳腺癌有协同作用     

培美曲塞-磷脂偶联物修饰的靶向性舒尼替尼与长春瑞滨脂质体的研制及其在体外对耐药性乳腺癌的抑制效应

乳腺癌是女性最易罹患的疾病,而肿瘤多药耐药性通常是化疗失败的主要原因。本研究以培美曲塞（PMT）和DSPE-PEG2000-NH2为原料合成了新的靶向性偶联物DSPE—PEG2000．PMT,并将其修饰到脂质体表面,制备了同时包封有舒尼替尼与长春瑞滨的靶向性脂质体,以增强化疗药物对多药耐药性乳腺癌的治疗效果。经过质谱分析证实,合成的靶向性载体材料DSPE．PEG2000-PMT与目标产物相符。建立了可同时检测舒尼替尼和长春瑞滨含量的高效液相色谱分析方法,检测波长为215nm,柱温30℃,流动相为乙腈-0．05MKH2P04（pH3．5）-三乙胺（35：65：0．3,v／v／v）。舒尼替尼和长春瑞滨的最低检测浓度分别为25ng／mL和5ng／mL,最低定量浓度均为0．25μg／mL。两药在0．5-25．0μg／mL范围内线性良好。各脂质体包封率均大于90％,粒径均-（～90nm）,Zeta电位略显负电性。在体外耐药乳腺癌MCF-7／Adr细胞中评价了靶向性舒尼替尼与长春瑞滨脂质体的抗增殖效应。结果显示,同对照组相比,靶向性舒尼替尼与长春瑞滨脂质体对MCF-7／Adr细胞具有最强的抑制增殖效应。以靶向性香豆素脂质体为荧光探针,考察了靶向性脂质体在耐药乳腺癌MCF-7／Adr细胞中的靶向性,同非靶向性制剂相比,靶向脂质体在耐药性癌细胞中摄取最多。因此,制备的靶向性舒尼替尼与长春瑞滨脂质体是-种新的靶向制剂,能够被耐药乳腺癌细胞靶向性摄取,可在体外显著抑制耐药性乳腺癌生长,从而为耐药乳腺癌的化学治疗提供了-种新的策略。     

Development of ligand-targeted liposomes for cancer therapy

The continued evolution of targeted liposomal therapeutics has resulted in new agents with remarkable antitumour efficacy and relatively mild toxicity profiles. A careful selection of the ligand is necessary to reduce immunogenicity, retain extended circulation lifetimes, target tumour-specific cell surface epitopes, and induce internalisation and subsequent release of the therapeutic substance from the liposome. Methods for assembling targeted liposomes, including a novel micellar insertion technology, for incorporation of targeting molecules that efficiently transforms a non-targeted liposomal therapeutic to a targeted one, greatly assist the translation of targeted liposome technology into the clinic. Targeting strategies with liposomes directed at solid tumours and vascular targets are discussed. The authors believe the development of ligand-targeted liposomes is now in the advanced stage and offers unique and important advantages among other targeted therapies. Anti-HER2 immunoliposomal doxorubicin is awaiting Phase I clinical trials, the results of which should provide new insights into the promise of ligand-targeted liposomal therapies.     

阳性脂质体介导基因转染及其研究进展*

基因治疗是一种很有前景的治疗模式,而阳性脂质体介导的基因转染是目前基因治疗的研究热点之一。现综述近5年来有关阳性脂质体的文献,介绍了阳性脂质体的基本组成,并从生物学、理化性质及制剂学等几个方面介绍了影响阳性脂质体/DNA复合物转染效率的主要因素,最后从新的阳性脂质成分及阳性脂质体或阳性脂质体/DNA复合物的表面修饰等方面介绍了近年来有关改善阳性脂质体介导基因转染的研究进展。     

Development of ligand-targeted liposomes for cancer therapy

The continued evolution of targeted liposomal therapeutics has resulted in new agents with remarkable antitumour efficacy and relatively mild toxicity profiles. A careful selection of the ligand is necessary to reduce immunogenicity, retain extended circulation lifetimes, target tumour-specific cell surface epitopes, and induce internalisation and subsequent release of the therapeutic substance from the liposome. Methods for assembling targeted liposomes, including a novel micellar insertion technology, for incorporation of targeting molecules that efficiently transforms a non-targeted liposomal therapeutic to a targeted one, greatly assist the translation of targeted liposome technology into the clinic. Targeting strategies with liposomes directed at solid tumours and vascular targets are discussed. The authors believe the development of ligand-targeted liposomes is now in the advanced stage and offers unique and important advantages among other targeted therapies. Anti-HER2 immunoliposomal doxorubicin is awaiting Phase I clinical trials, the results of which should provide new insights into the promise of ligand-targeted liposomal therapies.     

Physicochemical and pharmacokinetic characteristics of cationic liposomes

After intravenous administration of plasmid DNA (pDNA)/cationic liposome complexes, the gene expression is predominantly observed in the lung due to the physicochemical properties of the liposome complexes and the physiology of the lung. To determine the physicochemical properties and distribution behavior of cationic liposomes for lung-selective drug and/or gene delivery systems, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA)/cholesterol and 1,2-dioleoyl-3-trimethyl-ammoniopropane (DOTAP)/cholesterol liposomes were studied. The particle sizes of DOTMA/cholesterol and DOTAP/cholesterol liposomes were very similar: 126 and 128 nm, respectively. Furthermore, the zeta potentials of these two liposomes were 51 and 66 mV, respectively. After intravenous injection into mice, both cationic liposomes were rapidly eliminated from the blood circulation and preferentially recovered in the lung. Interestingly, the highest lung accumulation was observed at 1 min, and then, decreased gradually. The distribution characteristics of DOTMA/cholesterol and DOTAP/cholesterol liposomes were almost identical due to the similarities in their physicochemical properties. These results demonstrated that DOTMA/cholesterol and DOTAP/cholesterol liposomes, which possess a positive charge, are promising carriers for lung-selective drug and/or gene delivery systems.     

Preparation and characteristics of dichloromethylene diphosphonate-containing liposomes

Dichloromethylene diphosphonate (DMDP), encapsulated in liposomes and administered intravenously in mice, will eliminate all macrophages in spleen and liver. DMDP can be incorporated in liposomes to a maximum of 15 mM, if less than 12 mM is encapsulated not all macrophages will be eliminated (since the animals could not be injected with more liposomes). To determine DMDP content of the liposomes before in vivo administration, an in vitro test system was developed. This method is based on the competition for calcium binding by either DMDP or murexide. Murexide is a metallochromatic indicator which gives a distinct wavelength shift after binding of calcium. The decrease in absorbance at 510 nm of the murexide-calcium complex, due to the addition of DMDP, was used as a reliable (s.d. 5 per cent) value for measurement of DMDP concentrations. The possible use of this system in the study of calcium binding and transport over artificial biomembranes is discussed.     

Development and pharmacokinetics of nimodipine-loaded liposomes

In order to improve the water solubility of nimodipine and prolong the time of the drug in the circulation, nimodipine-loaded liposomes with a small size and high entrapment efficiency were prepared by a method that was easy to scale up (the modified ethanol injection method). The nimodipine liposome dispersions were characterized with respect to particle size distribution, zeta potential and entrapment efficiency. Liposomal nimodipine and nimodipine solution were intravenously administered to mice as a single dose of 4 mg kg-1. The pharmacokinetic parameters of nimodipine changed significantly when encapsulated in liposomes. The clearance of nimodipine encapsulated in liposomes was reduced and the elimination half-life was prolonged. The ratios of the area under the curve values of nimodipine liposomes to nimodipine solution were 1.78 and 1.90 in plasma and cerebral tissue, respectively. The drug concentration in cerebral tissue and in plasma showed a good linear correlation, which showed that liposomes could efficiently deliver nimodipine into brain tissue. These findings suggest that intravenous administration of liposomal nimodipine produces higher and more stable plasma and cerebral drug concentrations compared with nimodipine solution. In conclusion, liposomal nimodipine is a promising alternative to the solution preparation.