Preparation and evaluation of norcantharidin-encapsulated liposomes modified with a novel CD19 monoclonal antibody 2E8

In this study, norcanthridin （NCTD）-encapsulated liposomes were modified with a novel murine anti-human CD19 monoclonal antibody 2E8 （2E8-NCTD-liposomes） and the targeting efficiency and specific cytotoxicity of 2E8-NCTD-liposomes to CD19^＋ leukemia cells were evaluated. BALB/c mice were injected with 2E8 hybridoma cells to obtain 2E8 monoclonal antibody （mAb）. NCTD-liposomes were prepared by using film dispersion method. 2E8 mAbs were linked to NCTD-liposomes using post-incorporation technology. Flow cytometry showed that the targeting efficiency of purified 2E8 mAbs on CD19＋ Nalm-6 cells was 99.93%. The purified 2E8 mAbs were conjugated with NCTD-liposomes to prepare 2E8-NCTD-liposomes whose targeting efficiency on CD19^＋ Nalm-6 was also 95.82%. The average size of 2E8-NCTD-liposomes was 118.32 nm in diameter. HPLC showed that the encapsulation efficiency of NCTD was 46.51%. When the molar ratio of 2E8/Mal-PEG2000-DSPE reached 1：50, we obtained the liposomes with 9 2E8 molecules per liposome. The targeting efficiency of 2E8-NCTD-liposomes on CD19^＋ leukemia cells was significantly higher than that on CD19-1eukemia cells. Similarly, the targeting efficiency of the immtmoliposomes was also higher than that of the NCTDAiposomes on CD 19^＋ leukemia cells. Those results were consistent with those observed by laser scanning confocal microscopy. 3-（4, 5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay demonstrated that 2E8-NCTD-liposomes specifically killed Nalm-6 cells in a dose- and time-dependent manner. The viability of Nalm-6 cells treated by 2E8-NCTD-liposomes was significantly lower than that of Molt-3 cells and it was also significantly lower than that of Nahn-6 cells treated with the same concentration of NCTD-liposomes or free NCTD. We are led to concluded that 2E8 antigen can serve as a specific targeting molecule of B lineage hematopoietic malignancies for liposome targeting, and 2E8-NCTD-liposomes can be used as a new and effective means for the treatment of B lineage hematopoietic malignancies.     

去甲斑蝥酸钠脂质微球注射液对荷人肺癌A549移植瘤裸鼠肿瘤生长的抑制作用

目的: 观察去甲斑蝥酸钠脂质微球注射液对荷人肺癌A549细胞移植瘤裸鼠模型肿瘤生长的抑制作用。方法:在BALB/c-nu裸鼠右前肢腋下接种A549细胞株建立人肺癌动物模型,移植11 d后采用均衡法随机分为5组,即去甲斑蝥酸钠脂质微球注射液高剂量组(5 mg/kg)、中剂量组(2.5 mg/kg)、低剂量组(1.25 mg/kg)以及阳性对照组(2.5 mg/kg去甲斑蝥酸钠注射液)和阴性对照组(等体积空白脂质微球注射液),每组7只裸鼠,给药体积为0.2 mL。采用尾静脉注射给药,各组动物于分组后第1、4、8、11、15、18、22天给药,共7次。于给药第7、14、24天检测并计算各组肿瘤体积(V_t)、相对体积(V_RT)、增殖率(T/C)等,给药后第24天检测并计算各组肿瘤质量和抑瘤率。结果:去甲斑蝥酸钠脂质微球注射液给药第7天后,各给药组肿瘤体积、相对体积均明显小于阴性对照组(P<0.05),并具有剂量-效应关系(r_{7 d}=0.994,r_{14 d}=0.993,r_{24 d}=0.996,P均<0.05);24 d后各剂量组动物肿瘤T/C均小于35%,其中2.5、5 mg/kg剂量组和阳性对照组T/C均小于20%;各给药组平均肿瘤质量明显低于阴性对照组(P<0.01),肿瘤生长抑制率均大于50%。结论:去甲斑蝥酸钠脂质微球注射液具有抑制人肺癌A549细胞生长,降低人肺癌A549细胞移植瘤增殖率的作用。     

去甲斑蝥素对人乳腺癌血管生成的抑制作用

背景与目的:去甲斑蝥素(norcantharidin,NCTD)具有抑瘤作用,本文研究它对血管生成的作用,以及对人乳腺癌MCF-7细胞鸡胚绒毛尿囊膜(chorioallantoci membrane,CAM)移植肿瘤血管生成的影响。方法:①建立鸡胚绒毛尿囊膜模型,将明胶海绵载体置于两条前卵黄静脉间血管的CAM上,将不同剂量的NCTD加在载体上,以生理盐水对照,观察记录载体周围血管分支点数,计算血管生成抑制率。②建立人乳腺癌MCF-7细胞鸡胚移植模型。给予不同剂量的NCTD20μl[72、36、18μg/(胚.d)],以生理盐水对照,计算血管生成抑制率。结果:NCTD对以明胶海绵为载体的鸡胚绒毛尿囊膜血管生成具抑制作用,72、36、18、9μg NCTD组血管生成抑制率分别为77.7%、62.9%、50.6%及33.0%,并且呈剂量依赖性。移植肿瘤可诱发血管生成,NCTD能明显抑制MCF-7鸡胚移植肿瘤血管生成,72、36、18μg NCTD对移植瘤诱导的血管抑制率分别为66.2%、39.3%和22.8%。结论:NCTD能明显抑制鸡胚绒毛尿囊膜新生血管的生成,同时能抑制人乳腺癌MCF-7细胞鸡胚移植肿瘤的血管生成。其抗肿瘤作用与抑制肿瘤血管生成有关。     

3H-去甲斑蝥素小鼠体内药代动力学与组织分布

健康小鼠灌服³H-去甲斑蝥素溶液，收集不同时间血、组织、尿和粪，采用氚标记示踪法测定放射活性，计算³H-去甲斑蝥素血浓度、单位组织放射活性及尿和粪累积排泄率，DAS软件拟合小鼠去甲斑蝥素药代动力学模型，计算其药代动力学参数，评价³H-去甲斑蝥素小鼠体内的吸收、分布及排泄过程。结果表明，小鼠灌服³H-去甲斑蝥素0.5 h后血中放射活性达峰值；15 min后小肠、胆囊、胃、肾上腺、肾脏、心脏、子宫等放射活性较高，此后逐渐降低，但3 h后胆囊、肾上腺、子宫放射活性仍较高，肾脏、肝脏等组织则较低；24 h后粪便和尿液累积排泄率分别为65.40%和1.33%。因此，小鼠口服³H-去甲斑蝥素吸收迅速，血中分布明显高于其他组织；胆囊、肾上腺、子宫分布多且持久，肝脏分布少且消除快，肾脏则分布多，但消除也快。³H-去甲斑蝥素主要经肾脏排泄，极少量经粪便排泄。由于所测放射活性为去甲斑蝥素原形及代谢物之和，应对其体内转化过程及可能代谢产物做进一步研究。     

去甲斑蝥素聚乳酸-聚乙二醇纳米粒的制备及细胞毒性实验

目的:制备去甲斑蝥素纳米粒(norcantharidin nanoparticles,NCTD-NP),并考察其缓释特性和细胞毒性。方法:以聚乳酸-聚乙二醇为载体,采用相分离法制备NCTD-NP。用激光粒度仪测定纳米粒的粒径,以透射电镜观察其形态,以HPLC法测定其包封率,以紫外分光光度法考察其体外释放特性。应用四甲基偶氮唑盐微量酶反应比色法(MTT法)比较NCTD-NP和NCTD对胆囊癌GBC-SD细胞的细胞毒性。结果:纳米粒的粒径为(97.4±14.5)nm,表面光滑,包封率为(51.7±1.32)%,具有良好缓释性能。相同浓度的NCTD-NP和NCTD的细胞毒性在24 h之前基本相同,但NCTD-NP对GBC-SD细胞的生长有更持久的抑制效果,在IC50浓度48 h时仍抑制(56.42±9.45)%,而NCTD组仅抑制(23.14±3.77)%,两组有显著差异(P<0.01)。结论:NCTD-NP具有良好的缓释性能,并且可较长时间抑制胆囊癌GBC-SD细胞的生长。     

去甲斑蝥素诱导小鼠肺纤维瘤L929细胞凋亡

目的：研究去甲斑蝥素(norcantharidin，NCTD)抑制小鼠肺纤维瘤(L929)细胞的作用机制。方法：用MTT法测定细胞生长抑制率。采用Hoechst 33258染色、DNA凝胶电泳和乳酸脱氢酶(LDH)活力检测细胞凋亡。用免疫印迹法(Western blot)分析了细胞外信号调节激酶(extracellular signal-regulated kinase，ERK)和磷酸化ERK、c-Jun N-末端激酶(C-Jun N-terminal kinase，JNK)和磷酸化JNK蛋白表达的变化。结果：NCTD诱导L929细胞凋亡。JNK抑制剂(SP600125)与ERK抑制剂(PD98059)，可明显抑制NCTD对细胞的杀伤作用。同时磷酸化ERK和磷酸化JNK表达增加。结论：NCTD对L929细胞的细胞毒作用是通过诱导其凋亡而发生的，且呈时间剂量依赖性。这种作用可能与JNK，ERK通路激活有关。     

氮取代去甲斑蝥酰亚胺衍生物的合成及抗惊厥活性研究

目的合成氮取代去甲斑蝥酰亚胺衍生物，发现新的抗惊厥先导化合物。方法呋喃和马来酐经过Diels—Alder反应生成去甲去氢斑蝥素，在钯炭催化下还原成去甲斑蝥素，与取代苯胺缩合成氮一苯基去甲斑蝥酰亚胺的衍生物。结果合成的大多数化合物都具有抗惊厥活性。结论化合物7、9和10可能成为新的抗惊厥先导化合物。     

去甲基斑蝥素-壳聚糖纳米粒的表征和体外释放研究

目的研制粒径小于200 nm的去甲基斑蝥素-壳聚糖纳米粒,并对其质量进行评价,对其结构、体外释放性能进行研究。方法以低相对分子质量的壳聚糖为载体,通过离子诱导法,制备去甲基斑蝥素-壳聚糖纳米粒,并考察纳米粒的形态、粒径、药物包封率、载药量、回收率、表面氨基以及体外释放特征。同时,采用红外光谱(IR)、X-射线衍射技术(XRD)和差示扫描量热法(DSC)对其结构进行了表征。结果制备的去甲基斑蝥素-壳聚糖纳米粒粒径小(131±11)nm、大小分布均匀(多分散指数PDI 0.183),外观呈球形,药物包封率45.12%,载药量7.3%,回收率99.62%,且70 min后体外释药完全。IR、XRD与DSC分析表明,壳聚糖已发生交联并形成了新的物相。结论低相对分子质量的壳聚糖有望成为制备载药纳米粒的优良材料。     

The effect of cantharidins on leukemic stem cells

To identify an agent with specific activity against leukemic stem cells (LSCs), we evaluated compounds that targeted hepatic leukemia factor (HLF), a gene implicated in hematopoietic stem cell (HSCs) regulation, that we found overexpressed in LSCs. Cantharidin, a natural toxin from blister beetles, used as medicinal agent since antiquity, has been described to modulate the HLF competitor NFIL3 and is under clinical evaluation as an antitumor and antimetastatic agent. The molecule is not a substrate for multidrug resistant pumps and does not cause myelosuppression, and therefore it represents a promising compound for selective ablation of LSCs. Cantharidin and norcantharidin, a derivative with reduced toxicity, decreased HLF protein levels and induced apoptosis in the AML cell line MV4‐11 by modulating the expression of several molecules that govern survival pathway, including HLF, SLUG, NFIL3 and c‐myc, thereby inducing p53 and the mitochondrial caspase cascade. In vitro, cantharidin readily targeted primary AML stem and progenitor cells in contrast to conventional chemotherapeutic agents, such as Ara‐C and daunorubicin, that mainly targeted more differentiated leukemic cells. In vitro the compound did not exhibit a therapeutic window, being equally toxic to normal HSCs and LSCs. In vivo cantharidin did not produce myelosuppression. Because of dose‐limiting toxicity in vivo, neither cantharidin nor norcantharidin proved therapeutical benefit in AML xenograft models as a single agent. However, its potent in vitro LSC activity and pathway targeting may still be exploited clinically with a new generation of cantharidin derivatives or formulations and with appropriate drug combinations. © 2008 Wiley‐Liss, Inc.     

去甲斑蝥素用于单克隆抗体为载体的肿瘤导向治疗实验研究

用单抗３Ａ５制备与去甲斑蝥素（ＮＣＴＤ）的偶联物。ＥＬＩＳＡ检测结果表明：３Ａ５ ＮＣＴＤ偶联物保持对人肝癌ＢＥＬ ７４０２细胞的免疫反应性。克隆生成法测定结果显示：３Ａ５ ＮＣＴＤ具有比ＮＣＴＤ更强的细胞毒性,两者的ＩＣ５０分别为２．３ｍｇ／Ｌ和４．２ｍｇ／Ｌ。小鼠腹腔内移植Ｈ２２肝癌,腹膜内给药（ｉｐ）,３Ａ５ ＮＣＴＤ（３ｍｇ／ｋｇ）和ＮＣＴＤ（３ｍｇ／ｋｇ）的延长寿命值（ＩＬＳ）分别为１６８％和４２％。裸鼠移植人肝癌ＢＥＬ ７４０２细胞,静脉给药（ｉｖ）,ＮＣＴＤ的肿瘤抑制率为１５％（Ｐ＞０．０５）,３Ａ５ ＮＣＴＤ为６２％（Ｐ＜０．０１）;瘤结周围给药（ｐｔ）,ＮＣＴＤ的肿瘤抑制率为４７％（Ｐ＜０．０５）,３Ａ５ ＮＣＴＤ为７８％（Ｐ＜０．０１）。结果表明,与ＮＣＴＤ相比,３Ａ５ ＮＣＴＤ对裸鼠移植人肝癌和腹腔肿瘤具有更强的治疗作用。以上结果提示单抗与ＮＣＴＤ偶联物在肿瘤的导向治疗中有较好的疗效。