盐酸博安霉素注射用原位凝胶的体内分布研究

考察盐酸博安霉素(BAM)注射用原位凝胶在裸鼠体内的扩散情况以评价原位凝胶对盐酸博安霉素的阻滞作用。以荧光染料异硫氰酸荧光素(FITC)标记盐酸博安霉素,制备异硫氰酸荧光素-盐酸博安霉素偶联物(FITC-BAM),采用透析袋透析和Sephadex G25葡聚糖凝胶柱对偶联物进行分离纯化,利用基质辅助激光解析电离/飞行时间(MALDI-TOF)质谱检测偶联效果。建立裸鼠皮下肝癌移植瘤模型,应用动物体内活体光学成像系统定时检测盐酸博安霉素在裸鼠体内的特异性分布情况。MALDI-TOF质谱检测结果显示,FITC成功与BAM发生偶联,且二者偶联的分子比主要为1∶1或2∶1。活体动物成像系统观察显示,盐酸博安霉素注射用原位凝胶组FITC-BAM的扩散较普通注射液组明显延迟。研究表明,将盐酸博安霉素制备成注射用原位凝胶制剂,能够阻滞药物在体内的释放,延长作用时间。     

小分子靶向肽（RGD）2C与力达霉素的偶联物抗肿瘤作用研究

目的：考察小分子靶向肽（RGD）：C与力达霉素的偶联物的抗肿瘤作用。方法：MTr法观察偶联物和力达霉素在体外对人口腔鳞癌KB细胞、人乳腺癌MCF-7细胞以及人肝癌Bel7402细胞的细胞毒性。克隆形成法观察其对人肝癌Bel7402细胞克隆形成的抑制作用。采用C57BIM6小鼠Lewis肺癌移植瘤模型观察其实验治疗作用。结果：MrlT法结果表明,偶联物对人口腔鳞癌KB细胞、人肝癌Bel7402细胞和人乳腺癌MCF-7细胞的细胞毒性比力达霉素分别下降13倍、46倍和186倍。克隆形成法表明．偶联物对人肝癌Bel7402细胞的克隆形成抑制率比力达霉素下降10倍。0．2,0．1,O．05mg·kg-1偶联物对C57BL／6小鼠Lewis癌皮下原发瘤的生长抑制率分别为35．8％,25．6％和10．3％;0．05mg·kg-1素对肿瘤生长的抑制率为32．4％。0．2,0．1,0．05mg·kg-1偶联物对小鼠Lewis癌肺转移的抑制率分别为69．6％,50．5％和34．2％,0．05mg·kg-1达霉素、0．05mg·kg-1力达霉素＋1mg·kg-1（RGD）：C肽的抑制率分别为53．3％和54．9％。结论：按等细胞毒性剂量来计算,偶联物体内抗肿瘤活性和抗肿瘤转移活性比力达霉素强,提示小分子靶向肽RGD与力达霉素偶联后发挥了一定的导向作用。     

微生物来源抗肿瘤先导化合物的靶向筛选研究

目的 寻找具有抗肿瘤活性的雷帕霉素结构类似物。 方法 以雷帕霉素细胞内结合蛋白RBP为靶标，运用营养依赖重组微生物工程菌筛选方法，从微生物的的次级代谢产物中筛选活性化合物；通过16S rDNA序列分析鉴定活性化合物产生菌；深层发酵制备活性物培养液并分离、纯化目标物；根据波谱检测及数据解析进行化合物结构鉴定并进行活性评价。结果 筛选获得1株活性菌株CY-365，经鉴定为放线菌链霉菌属，其代谢活性产物CY-365结构解析为15(S)-O-乙基雷帕霉素，与雷帕霉素具有相似的体外抗肿瘤活性。 结论 链霉菌CY-365经深层培养，主要代谢产物为具有抗肿瘤活性的15(S)-O-乙基雷帕霉素。     

博安霉素抗人结肠癌作用的研究

对新抗肿瘤抗生素博安霉素 (BAM)在体外的抗人结肠癌作用、对癌细胞生物大分子合成、对相关癌基因和抗癌基因的表达及 DNA损伤和修复的影响进行了研究 ,采用细胞克隆形成法测定表明 ,博安霉素对人结肠癌 HT- 2 9细胞作用的 IC50 为 3.8× 10 - 8m ol/ L;NAG酶反应测定表明 ,博安霉素作用的 IC50 比其它博来霉素类药物低一个数量级。利用 [3 H]标记物掺入法测定表明 ,BAM对人结肠癌细胞的 DNA、RNA和蛋白质合成均有强抑制作用 ,其中对蛋白质合成的抑制作用最强。 Dot blot分析表明 ,博安霉素在 10 5mol/ L明显抑制人结肠癌细胞的 c- myc和 p5 3基因的表达 ,但增强 N- ras基因的表达。中性凝胶电泳分析表明博安霉素造成的人结肠癌细胞 DNA的损伤可被修复。研究证明了博安霉素是对人结肠癌有效的抗肿瘤抗生素     

盐酸博安霉素介入治疗肝癌103例

目的：对盐酸博安霉素(BAM)介入治疗原发性肝癌的临床疗效和毒性进行评价。方法：入选103例，单药组65例，单用BAM，肝动脉介入治疗，50mg，共2～3次；联合用药组38例，每次除用BAM外加用5一氟尿嘧啶(1000mg)及丝裂霉素(10～20mg)，共2～3次。结果：单药组有效率30．8％，联合用药组有效率42．1％。BAM的主要不良反应有发热、寒战、恶心、呕吐。未见骨髓抑制及心、肝、肾功能损害，无明显肺毒性反应。结论：BAM是原发性肝癌介入治疗的有效药物。     

抗IV型胶原酶单抗与平阳霉素新型免疫偶联物的抗肿瘤作用

目的考察抗IV型胶原酶单抗3G11与平阳霉素(PYM)偶联物的抗肿瘤作用。方法采用多聚谷氨酸(PLG)为中间载体制备3G11-PLG-PYM偶联物,MTT法测定其对肿瘤细胞增殖的抑制作用,以小鼠移植性肝癌H22为模型观察体内抗肿瘤作用。结果偶联物保留了单抗3G11对IV型胶原酶的免疫活性,对体外培养H22和KB细胞的杀伤作用弱于PYM。动物实验中PYM10mg·kg-1对H22肝癌的抑制率为60·6%,而等细胞毒性剂量的3G11-PLG-PYM偶联物抑瘤率达到90·8%,与PYM相比可显著延长小鼠的中位生存时间。结论3G11-PLG-PYM偶联物对小鼠肝癌H22的抑瘤作用比PYM强,可能成为新型的抗肿瘤靶向药物。     

平阳霉素与单克隆抗体3D6及其Fab＇片段偶联物对肝癌的实验治疗

单抗3D6是抗IV型胶原酶单克隆抗体。以葡聚糖T－40（DxtranT-40)为中介体制备3D6和平阳霉素（PYM）的偶联物3D6－PYM。采用间接ELISA法、TTC抑菌效价检测法，克隆形成法，MTT法及动物移植性肿瘤模型测定偶联物的生物学活性与体内外抗肿瘤作用。实验表明，3D6－PYM偶联物与PG、KB、C26及H22细胞等于免疫学阳性反应；3D6－PYM偶联物对BEL－7402、B和H22细胞的细胞毒作用强于游离PYM。应用小鼠移植性肝癌模型观察偶联物对肝癌生长的抑制作用及长期疗效。结果显示，3DY－PYM偶联物对肿瘤生长有较强的抑制作用且在体内作用时间延长，发挥较好的疗效。第21天时，按100mg/kg剂量比较，3D6－PYM偶联物、3D6与PYM混合物及PYM的抑瘤率分别达到99．7％、95．6％、81．5％，停止治疗后，继续观察各组动物的生存时间，对照组的中位生存期脒38d，按10mg/kg剂量比较，3D6－PYM偶联物组、3D6与PYM混合物组以及PYM组的中位生存期分别为＞120、88和54d。120d时，3D6－PYM偶联物10mg/kg组未见动物死亡。以β－环糊精为中介体制备单抗3D6 Fab′片段与PYM偶联物Fab′－PYM，小鼠移植性肝癌模型皮下接种后第7天给药，Fab′－PYM偶联物组与PYM抑瘤率分别为81．0％和29．1％，以上研究结果表明，3D6－PYM及Fab′－PYM偶联物具有比游离PYM更强的抑制肝癌生长作用，特别是长期观察，其差别更为显著。     

抗IV型胶原酶单抗与平阳霉素新型免疫偶联物的抗肿瘤作用

目的考察抗IV型胶原酶单抗3G11与平阳霉素(PYM)偶联物的抗肿瘤作用。方法采用多聚谷氨酸(PLG)为中间载体制备3G11-PLG-PYM偶联物,MTT法测定其对肿瘤细胞增殖的抑制作用,以小鼠移植性肝癌H22为模型观察体内抗肿瘤作用。结果偶联物保留了单抗3G11对IV型胶原酶的免疫活性,对体外培养H22和KB细胞的杀伤作用弱于PYM。动物实验中PYM 10 mg·kg^-1对H22肝癌的抑制率为60.6%,而等细胞毒性剂量的3G11-PLG-PYM偶联物抑瘤率达到90.8%,与PYM相比可显著延长小鼠的中位生存时间。结论3G11-PLG-PYM偶联物对小鼠肝癌H22的抑瘤作用比PYM强,可能成为新型的抗肿瘤靶向药物。     

注射用盐酸博安霉素的稳定性考察

目的：考察不同温度下注射用盐酸博安霉素与0．9％氯化钠注射液配伍的稳定性,为临床合理用药提供依据。方法：采用HPLC法考察配伍液中盐酸博安霉素的含量变化,并观察配伍溶液的外观、pH和紫外吸收图谱的变化。结果：盐酸博安霉素的线性范围为30～100μg·ml。（r=0．9999）,平均回收率为99．69％,RSD=0．34％（n--9）。在实验的各个温度下,配伍液放置48h,外观、pH及紫外吸收图谱均无明显变化,而博安霉素含量有所降低。结论：博安霉素-0．9％氯化钠配伍液在冰箱内储存24h稳定;夏季、常温使用该药物时,宜现用现配,并于4h内输注完毕。     

力达霉素-单抗Fab'片段不同连接偶联物的抗肿瘤作用比较

研究力达霉素与抗Ⅳ型胶原酶单抗的不同连接键的小型化免疫偶联物的抗肿瘤作用。制备长短链的二硫键以及硫醚键抗肿瘤抗生素力达霉素(LDM)与抗Ⅳ型胶原酶单抗3G11的Fab'片段连接的免疫偶联物,ELISA法测定偶联物的免疫活性,体外细胞克隆形成测定和体内荷瘤裸小鼠模型观察两种偶联物的抗肿瘤作用。体外研究显示,Fab'-LDM偶联物部分保留了抗Ⅳ型胶原酶单抗3G11对抗原的亲和力,硫醚键偶联物对体外培养的人纤维肉瘤HT-1080细胞的杀伤作用强于LDM和二硫键偶联物。实验结果显示,LDM等剂量条件下硫醚键偶联物Fab'-SSMPB-LDM和Fab'-SMBS-LDM组的抑瘤率分别为87.7%和78.0%,二硫键偶联物Fab'-SPDP-LDM和Fab'-LCSPDP-LDM组分别为74.8%和72.3%,游离LDM组为70.9%,相比较长连接键的硫醚键偶联物抑瘤作用显著增强。Fab'-SSMPB-LDM和Fab'-SMBS-LDM组动物的平均生存时间延长为165.8%和145.2%,Fab'-SPDP-LDM和Fab'-LCSPDP-LDM组分别为82.2%和107.5%,LDM组为71.9%。与二硫键偶联物组比较...     

小分子靶向肽与力达霉素辅基蛋白偶联方法研究

建立小分子靶向肽（RGD）2C肽同时与力达霉素辅基蛋白氨基和羧基联接的方法。方法：先用交联剂SPDP与力达霉素辅基蛋白氨基端氨基反应,再用交联剂HPDP与力达霉素羧基在交联剂EDC存在时反应,最后将联接有SPDP与HPDP的力达霉素辅基蛋白与（RGD）2C肽反应。上述三个过程分别用Sephadex G15葡聚糖凝胶柱纯化以除去小分子物质。以基质辅助激光解吸电离／飞行时间质谱检测联接结果。结果：在本实验条件下,小分子靶向肽（RGD）2C肽同时与力达霉素辅基蛋白氨基与羧基成功联接,总的联接比为1：1和2：1。结论：采用交联剂SPDP和HPDP,可使小分子靶向肽同时与力达霉素氨基和羧基成功联接。     

Immunotoxins Composed of Monoclonal Antibody to α-Fetoprotein and Gelonin as a Potent Hepatoma-Targeted Drug Delivery System

Abstract

This study was carried out to evaluate our monoclonal antibody (MoAb) to α-fetoprotein (AFP), 80G, as a carrier for targeting AFP-producing hepatoma. Pharmacokinetic analysis showed that the MoAb 80G was actively incorporated into AFP-producing HuH-7N cells (xenograft of human hepatoma cell line, HuH-7) in nude mice. Four conjugates composed of MoAb 80G, and a type 1 ribosome-inactivating protein, gelonin, were prepared. They involve two disulfide-linked and two thioether-linked conjugates. The binding activity of conjugates against AFP remained as high as that of intact 80G according to enzyme-linked immunosorbent assay. The in vitro cytotoxic effects of all the conjugates were specific against AFP-producing HuH-7 cells. Of these conjugates, two containing gelonin modified with 2-iminothiolane were more potent than the others. They showed significant antitumor activity upon AFP-producing HuH-7N cells in nude mice. However, the disulfide conjugate was more toxic to mice than the thioether conjugate judging from the loss in body weight and the liver damage. These results suggest that our MoAb 80G is a suitable carrier for targeting AFP-producing hepatoma cells, and that the noncleavable thioether conjugate is promising as an AFP-producing hepatoma-targeted drug delivery system.     

Synthesis of polyrotaxane-camptothecin conjugates and evaluation of its anti-tumor effect

To prepare polyrotaxane-camptothecin conjugates and evaluate its anti-tumor effect, polyrotaxane-camptothecin conjugates were successfully synthesized, and the release behavior was performed; MTT assay and cell morphology were used to examine the inhibition of cells' proliferation effect in vitro. The experimental study of the antitumor effect on S180 mice in vivo was also performed to further evaluate the anti-tumor effect of conjugate. The result showed polyrotaxane-camptothecin conjugates can effectively inhibit the proliferation in a dose dependent effect. In vivo study and cell morphology observation of S180 mice showed significant decrease in growth of tumor, degree of tumor infiltration and blood vessel number. The result indicated anti-tumor mechanism may be through affect the angiogenesis and reduced blood supply to tumor cells and then leading to necrosis.     

Poly[N-(2-hydroxypropyl)methacrylamide] conjugates of bovine pancreatic ribonuclease (RNase A) inhibit growth of human melanoma in nude mice

Recently hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for BS-RNase modification to prevent its degradation in bloodstream or fast elimination. Polymer-conjugated BS-RNase preparations proved to be cytotoxic after intravenous or intraperitoneal application, whereas native BS-RNase was ineffective. Here RNase A unimer was conjugated with two HPMA polymers (classic and star) and their antitumor effects both in vitro and in vivo were compared with those of BS-RNase polymers. Surprisingly, the antitumor effect of RNase A conjugates was also pronounced. The RNase A conjugates (classic and star) injected intravenously to mice bearing melanoma tumor caused a significant reduction in tumor volume following ten doses of 5 and 1 mg/kg, respectively. Despite the antitumor activity observed in vivo, the in vitro tested cytotoxic activity of RNase A did not differ from that caused by native RNase A while native BS-RNase (50 microg/ml) totally inhibited DNA synthesis in treated cells. The experiments with 125I-labeled preparations demonstrated concentration-dependent internalization of native BS-RNase by tumor cells within an hour, whereas the polymer conjugate (S-BS) was not internalized. On the contrary, the in vivo experiments showed that whereas 40% of S-BS conjugate persisted in bloodstream for 24h after administration, 98% of the native BS-RNase was already eliminated. Improved antitumor activities of PHPMA-modified RNases in vivo might be ascribed to their prolonged retention in bloodstream, better proteolytic stability and resistance to the action of the ribonuclease inhibitor.     

Poly [N -(2-hydroxypropyl)methacrylamide] Conjugates of Bovine Pancreatic Ribonuclease (RNase A) Inhibit Growth of Human Melanoma in Nude Mice

Recently hydrophilic poly [N -(2-hydroxypropyl)methacrylamide] (PHPMA) was used for BS-RNase modification to prevent its degradation in bloodstream or fast elimination. Polymer-conjugated BS-RNase preparations proved to be cytotoxic after intravenous or intraperitoneal application, whereas native BS-RNase was ineffective. Here RNase A unimer was conjugated with two HPMA polymers (classic and star) and their antitumor effects both in vitro and in vivo were compared with those of BS-RNase polymers. Surprisingly, the antitumor effect of RNase A conjugates was also pronounced. The RNase A conjugates (classic and star) injected intravenously to mice bearing melanoma tumor caused a significant reduction in tumor volume following ten doses of 5 and 1 mg/kg, respectively. Despite the antitumor activity observed in vivo, the in vitro tested cytotoxic activity of RNase A did not differ from that caused by native RNase A while native BS-RNase (50 μ g/ml) totally inhibited DNA synthesis in treated cells. The experiments with 125 I-labeled preparations demonstrated concentration-dependent internalization of native BS-RNase by tumor cells within an hour, whereas the polymer conjugate (S-BS) was not internalized. On the contrary, the in vivo experiments showed that whereas 40% of S-BS conjugate persisted in bloodstream for 24 h after administration, 98% of the native BS-RNase was already eliminated. Improved antitumor activities of PHPMA-modified RNases in vivo might be ascribed to their prolonged retention in bloodstream, better proteolytic stability and resistance to the action of the ribonuclease inhibitor.     

Antitumor activity of beta-cyclodextrin polymer-camptothecin conjugates

Antitumor activity of linear, beta-cyclodextrin polymer (CDP)-camptothecin (CPT) conjugates (HGGG6, LGGG10, HG6, and HGGG10) is investigated in nude mice bearing human LS174T colon carcinoma tumors. These conjugates differ in polymer molecular mass [97 kDa (H) or 35 kDa (L)], CDP-CPT linker structure [glycine (G) or triglycine (GGG)], and CPT loading [ca. 6 wt % (6) or 10 wt % (10)]. Maximum tolerable doses (MTDs) of the three conjugates, LGGG10, HG6, and HGGG10, are determined to be 36, 9, and 9 mg of CPT/kg, respectively, while the MTD of the CDP alone exceeds 240 mg/kg (highest value investigated). The three CDP-CPT conjugates with high polymer molecular masses (HGGG6, HG6, and HGGG10) demonstrate antitumor activity at their MTDs superior to that of CPT at the same amount and to that of irinotecan at its optimal dose. They also show tumor growth inhibition that is superior to that of the conjugate containing the low-molecular mass polymer (LGGG10) at the same dose of CPT. No significant effects of CPT weight loading or linker structure on tumor growth delay are observed. However, conjugates containing G appear to be less toxic than these with GGG. These antitumor studies demonstrate that the CDP-based conjugates of CPT exhibit tumor growth inhibition superior to that of CPT or irinotecan at the conditions employed in this study. The striking observation is that a short course of treatment with the polymer conjugates gives long-term control of tumor growth that does not occur with either CPT or irinotecan. Intracellular CDPs are demonstrated by analyzing cells that were cultured in the presence of rhodamine-labeled CDP (HRhod) containing medium using both confocal microscopy and flow cytometry. The long-term therapeutic efficacy of CDP-CPT conjugates observed in mice may in part be due to the sustained release of CPT from these conjugates in the acidic, intracellular compartments since these conjugates are shown to have significantly slower release rates at acidic pH than at physiological pH.     

Synthesis and biological properties of highly sequence-specific-alkylating N-methylpyrrole-N-methylimidazole polyamide conjugates

Four new alkylating N-methylpyrrole-N-methylimidazole (PI) polyamide conjugates (1-4) with seven-base-pair (bp) recognition ability were synthesized. Evaluation of their DNA-alkylating activity clearly showed accurate alkylation at match site(s). The cytotoxicities of conjugates 1-4 were determined against six human cancer cell lines, and the effect of these conjugates on the expression levels of the whole human genome in A549 cells were also investigated. A few genes among the top 20 genes were commonly downregulated by each conjugate, which reflects their sequence specificity. Conversely, many of the top 10 genes were commonly upregulated, which may have been caused by alkylation damage to DNA. Moreover, the antitumor activities of the PI polyamide conjugates 2 and 3 were investigated using nude mice transplanted with DU145 or A549. The intravenous administration of each liposomal conjugate in water yielded tumor-suppressing effects specifically toward DU145 cells and not A549 cells, which was pertinent to cytotoxicity.     

Enhancing effects of galactosylated dendrimer/alpha-cyclodextrin conjugates on gene transfer efficiency

To improve in vitro gene transfer efficiency and/or achieve cell-specific gene delivery of polyamidoamine (PAMAM) starburst dendrimer (generation 2, G2) conjugate with alpha-cyclodextrin (alpha-CDE conjugate (G2)), we prepared alpha-CDE conjugate bearing galactose (Gal-alpha-CDE conjugates) with the various degrees of substitution of the galactose moiety (DSG) as a novel non-viral vector. The agarose gel electrophoretic studies revealed that Gal-alpha-CDE conjugates formed complexes with plasmid DNA (pDNA) and protected the degradation of pDNA by DNase I, but these effects impaired as the DSG value increased. Dendrimer and alpha-CDE conjugate exerted pDNA condensation through the complexation, but Gal-alpha-CDE conjugates did not. Gal-alpha-CDE conjugate (DSG 4) was found to have much higher gene transfer activity than dendrimer, alpha-CDE conjugate and Gal-alpha-CDE conjugates (DSG 8, 15) in HepG2, NIH3T3 and A549 cells, which are independent of the expression of the asialoglycoprotein receptor. Transfection activity of Gal-alpha-CDE conjugate (DSG 4) was insensitive to the existence of competitors (asialofetuin and galactose) and serum. In addition, no cytotoxicity after transfection of the complex of pDNA with Gal-alpha-CDE conjugate (DSG 4) was observed. These results suggest the potential use of Gal-alpha-CDE conjugate (DSG 4) as a non-viral vector in various cells.     

灵芝多糖对肿瘤细胞与内皮细胞相互作用的影响

目的:研究灵芝多糖对肿瘤细胞与内皮细胞相互作用的影响。方法:应用免疫荧光法配合使用显微镜将内皮细胞区分出来后,通过MTT法观察灵芝多糖在内皮细胞增殖分化过程中的作用,并使用相同的方法观察肿瘤细胞的增殖分化过程与灵芝多糖的关系,同时观察灵芝多糖是否干扰2种细胞间产生的黏附作用及迁移反应。结果:内皮细胞的增殖分化过程与灵芝多糖不存在明显联系,肿瘤细胞的增殖分化过程与灵芝多糖也不存在明显联系,即无明显的细胞毒性反应。但灵芝多糖可阻碍2种细胞间产生的黏附作用和迁移反应,减少肿瘤细胞的迁移百分比。结论:灵芝多糖具有抗肿瘤功效,其作用机制为阻碍肿瘤细胞的黏附作用和迁移反应。