利用茉莉酸类结合mRNA差示技术研究紫杉醇生物合成机制

差示从红豆杉悬浮细胞中克隆出茉莉酸类诱导表达的基因，并用异源表达系统对有关基因进行功能鉴定，从而阐明紫杉醇生物合成机制，为今后觖红豆杉悬浮法生产细胞紫杉醇产量低且不稳定的难题理论和技术手段。     

红豆杉的细胞工程学研究进展

利用红豆杉Taxus的培养细胞作为提取抗癌新药紫杉醇(Taxol)的原料来源的研究已取得了极大进展。到目前已知对红豆杉属的10个种或变种进行过细胞培养的研究,有些种培养细胞的紫杉醇含量高于原植物。通过加入生物合成前体和/或真菌诱导子等措施可以显著提高红豆杉培养细胞中紫杉醇的含量。紫杉醇酶标免疫含量测定的研究成功标志着培养细胞中紫杉醇的微量含量测定可以快速进行,为快速筛选高产紫杉醇细胞系提供了有力的检测手段。红豆杉的毛状根培养、固定化培养及胚培养等均已研究成功,为生产紫杉醇提供了多条途径。     

生物合成紫杉醇的研究进展

紫杉醇是红豆杉及其内生真菌产生的萜类次级代谢产物,作为有效的抗癌药物,目前生产主要依赖于红豆杉,供求矛盾十分突出。为解决紫杉醇来源匮乏的问题,开展了多项代替红豆杉的研究。本文总结近年红豆杉细胞培养和微生物组合生物合成方面的研究进展,供生物合成紫杉醇的进一步研究参考。     

紫杉醇与三尖杉宁碱分离方法的改进

紫杉醇对卵巢癌和乳腺癌的治疗效果使之在临床和化学上都受到了极大重视。然而由于它在天然资源中存在量甚少,且与同类物三尖杉宁碱 cephalomannine 密不可分,使得从天然资源中分离紫杉醇的工艺十分复杂。在几种红豆杉属植物中,如短叶红豆杉、东北红豆杉和云南红豆杉,紫杉醇含量为0.001％～0.08％,三尖杉宁碱含量为     

从红豆杉树皮浸膏中提取紫杉醇初分离工艺的研究

从红豆杉树皮浸膏中高效提取紫杉醇。方法;采用液－液萃取、固相萃取、硅胶柱层析、氧化铝柱层析、薄层层析五种分离方法对紫杉醇进行了初分离。结果：优化的紫杉醇初分离方案为红豆杉树皮提取物先经氧化铝柱,再上固相萃取柱进行分离提取。结论：该工艺分离所得紫杉醇浓度可达４０％以上,紫杉醇的回收率超过１０％。     

红豆杉资源的生物工程研究新进展

紫杉醇 (taxol)是 2 0世纪 70年代由 Wani等 [1 ]从短叶红豆杉 Taxus brevifolia树皮中提取出来的具有独特抗癌作用的天然产物 ,被认为是治疗卵巢癌的首选药物 ,近年来又不断发现它对其它癌症的治疗作用 ,是一种非常有发展前途的抗癌新药。到目前为止 ,发现紫杉醇只存在于裸子植物红豆杉科的红豆杉属 Taxus L .和澳洲红豆杉属种中。由于红豆杉类植物多属珍稀物种 ,数量稀少 ,生长缓慢 ,紫杉醇的含量又非常低 ,而全球每年需要紫杉醇 2 0 0～ 30 0 kg,所以靠砍伐树木提取的方法远不能满足人们对紫杉醇日益增长的需要。迄今为止 ,人们已从各…     

从西藏红豆杉的内寄生真菌中分离紫杉醇

紫杉醇对人体的某些肿瘤有活性作用。树皮中紫杉醇含量低且提取颇难。紫杉醇虽可化学全合成与半合成获得,但步骤复杂、价格昂贵。降低成本获取紫杉醇的理想的方法是通过微生物发酵的途径。1994年Stierle等首次从安德列亚菌(Taxomycesandreanae)中分离出紫杉醇,但含量仅为纳克级水平。现报道从另一种红豆杉内寄生真菌中分离出紫杉醇,并且在培养物中的聚积量每升可达微克级水平。　　该内寄生真菌是从喜马拉雅山脉海拔1500～3000m丘陵地带的西藏红豆杉小枝的内皮中分离得到的,Sutton博士鉴定该菌为小孢盘多毛孢(Pestalotiopsismicrospora)。…     

紫杉醇放疗增敏在头颈部肿瘤治疗中的临床运用

紫杉醇(paclitaxel,商品名taxol泰素)是一种由红豆杉属植物的树皮和针叶中提取的化合物,是近年来研究开发出的化学结构新颖、作用机理独特的新型抗肿瘤药.     

紫杉醇的研究进展

长期以来，科学家不断寻求抗癌生物资源及天然药物，并先后从动植物及海洋生物中提取了不少抗癌天然产物，紫杉醇的发现，就是一重大突破。1紫杉醇的研究、开发史^[1.2]红豆杉俗称：紫杉，属植物界裸子植物亚门、松杉纲、红豆杉目、红豆杉科中红豆杉属。早在1856年Lucas就从浆     

紫杉醇的提取，分离与检测研究进展

介绍从红豆杉属植物或培养组织中获得紫杉醇的有关提取，分离和检测方法的研究进展，同时探讨了影响紫杉醇含量的因素 和提高产量的途径。     

Stability, compatibility, and plasticizer extraction of taxol (NSC-125973) injection diluted in infusion solutions and stored in various containers.

The stability of taxol (NSC-125973) in various diluents and containers was determined, and the extent of leaching of di(2-ethylhexyl) phthalate (DEHP) from polyvinyl chloride (PVC) bags caused by the taxol formulation was measured. A taxol formulation consisting of a 6-mg/mL solution of taxol in 50% polyoxyethylated castor oil and 50% dehydrated ethanol was added to 50- and 100-mL glass bottles, PVC infusion bags, and polyolefin containers containing 5% dextrose injection or 0.9% sodium chloride injection to give initial nominal taxol concentrations of 0.3, 0.6, 0.9, and 1.2 mg/mL. The containers were maintained at 20-23 degrees C for 12-24 hours. Samples were assayed by stability-indicating high-performance liquid chromatography, and clarity was determined visually. An experiment was run to ascertain whether DEHP would leach from a PVC administration set during a simulated infusion. There was no substantial loss of taxol over 24 hours. Filtration through a membrane resulted in no loss of taxol. All the solutions initially appeared hazy. Solutions stored in PVC bags became more hazy with time than solutions stored in glass or polyolefin containers. The haze seen in PVC bags was traced to leaching of DEHP. Agitation had no effect on the extent of leaching. Leaching was also seen during simulated delivery through PVC administration sets. No DEHP was detected when solutions were stored in glass or polyolefin containers and infused through polyethylene-lined sets. At the dilutions studied, taxol was visually and chemically stable for up to 24 hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aurora A激酶抑制剂VX-680对人子宫内膜癌细胞化疗敏感性的影响

目的 探讨抑制子宫内膜癌细胞中Aurora A表达与细胞对紫杉醇敏感性的相关性,明确Aurora A低表达影响紫杉醇对子宫内膜癌的治疗效果.方法 以Aurora激酶抑制剂VX-680抑制子宫内膜癌细胞株Ishikawa中的Aurora A表达,再将细胞用不同浓度紫杉醇处理,Western blot检测Aurora A蛋白受抑制情况,用流式细胞仪分析Aurora A表达下调后细胞生长抑制率和细胞凋亡率的变化.结果 Aurora A表达下调使紫杉醇时细胞的生长抑制率和细胞凋亡率均升高(P＜0.01).结论 人子宫内膜癌细胞中Aurora A表达与细胞对紫杉醇敏感性具有相关性,抑制Aurora A可望提高紫杉醇对子宫内膜癌的治疗效果.     

Taxol and related diterpenoids of Taxus sp.: important acquisitions in the treatment of cancer]

Phytochemical and pharmacological studies on Taxus sp extracts have resulted in the isolation and the identification of several diterpenoids, and the discovery of the potent antitumor activity of taxol. This natural compound displays a unique mechanism of action as it stabilizes microtubules and inhibits their depolymerization into free tubulin. The emergence of taxol from the screening of natural products shows the benefits and the potent interest of these constituents in the search of drugs exhibiting novel mechanisms of action. The most pressing problem in taxol or taxol derivates commercialization is the drug supply. Alternative sources of taxol are now under investigation, mainly the total synthesis of taxol, the hemisynthesis of taxol or of taxotere from 10-deacetylbaccatine III, the in vitro production of taxol by cell or tissue cultures, and the prospection of new natural sources of taxol.     

The protective effect of beta-1,3-D-glucan on taxol-induced hepatotoxicity: a histopathological and stereological study

The present study was undertaken to determine the histopathological and quantitative effects of the antineoplastic agent, taxol, on the liver. The protective effects of the strong antioxidant, β-1,3-D-glucan, against liver damage induced by taxol were also investigated. Mice were divided into four main treatment groups: control, taxol, β-1,3-D-glucan, and taxol+β-1,3-D-glucan. Each group was further subdivided into six subgroups, according to time of sacrifice (6, 12, 24, and 48 hours and 7 and 14 days). After the experiments, quantitative and histopathological changes in liver were examined by light microscopy and modern stereological systems. Stereological results indicated that the portal triad area of the taxol group was significantly reduced, compared to the controls at 12 hours, whereas in the taxol plus β-glucan and β-glucan groups, the means were similar to those of the controls. There was no statistically significant difference in the numerical density of hepatocytes with time between the control and other groups. The histopathological results indicated an increased, time-dependent degeneration and necrosis of the liver tissues in mice in the taxol group. Regenerative changes in livers of mice in the taxol plus β-glucan group were observed, when compared with those of the taxol group. Stereological and histopathological results suggest that β-glucan may reduce taxol-induced hepatic damage by blocking the change in the portal area and suppressing processes leading to necrosis.     

Development of a Polymeric Surgical Paste Formulation for Taxol

Purpose. To develop and characterize a biodegradable polymeric sustained release surgical paste formulation for taxol. Methods. Taxol was incorporated into poly(-caprolactone) (PCL) or blends of PCL with methoxypolyethylene glycol, MW 350 (MePEG). The surgical pastes were characterized using gel permeation chromatography, thermal analysis, scanning electron microscopy, and a tensile strength tester. In vitro release data for taxol from the surgical paste formulations was carried out at 37°C in phosphate buffered saline, pH 7.4, using an HPLC assay for taxol. Antiangiogenic activity of the formulations were assessed using a chick chorioallantoic membrane assay (CAM). Results. The addition of up to 30% MePEG in PCL decreased the melting point of PCL by 5°C and the tensile strength by 152.7 N/cm² to 26.7 N/cm² but increased the degree of PCL crystallinity from 42% to 51%. Taxol showed a biphasic in vitro release profile composed of a burst phase lasting 1 or 2 days followed by a period of slow sustained drug release. There was no significant difference in the release profiles of taxol from two different sources of PCL. The addition of MePEG increased the amount of water taken up by the polymer blends but decreased the rate of taxol release. The formulations were shown to have antiangiogenic activity by the CAM assay at levels as low as 0.1% taxol using 3 mg surgical paste pellets. Conclusions. Our surgical paste formulations for taxol give sustained release while having physical properties which can be adjusted using additives.     

红豆杉浸膏在氧化铝催化下紫杉醇的生成研究

目的 :研究在氧化铝层析初分离红豆杉浸膏中紫杉醇增量的来源。方法 :采用C18 硅胶反相层析、氧化铝层析及硅胶层析等方法对物料进行处理 ,HPLC分析紫杉醇的含量。结果 :紫杉醇增量主要来源于红豆杉浸膏中 7 表 紫杉醇的碱性氧化铝催化下的异构化。结论 :优化 7 表 紫杉醇的异构化条件 ,可以增加 7 表 紫杉醇向紫杉醇的转化。     

Synthesis and evaluation of some water-soluble prodrugs and derivatives of taxol with antitumor activity.

The synthesis and evaluation of some 2'- and 7-amino acid derivatives of taxol (1) are reported. Reaction of taxol with N-protected amino acids gave 2'-N-protected amino acid esters of taxol. However, deprotection of the amino group and subsequent isolation of products were complex and only successful when formic acid was used to deprotect a t-BOC protecting group. Esterification of taxol using N,N-dialkylated amino acids gave 2'-amino acid esters of taxol, 2'-(N,N-dimethylglycyl)taxol (4) and 2'-[3-(N,N-diethylamino)propionyl]taxol as its methanesulfonic acid salt (5b), in good yield. The 7-derivatives, 7-(N,N-dimethylglycyl)taxol (9) and 7-L-alanyltaxol (12), were prepared by two alternate methods. In the first approach, the 2'-hydroxyl group was protected using the [(2,2,2-trichloro-ethyl)oxy]carbonyl, or troc, protecting group followed by the esterification of the 7-hydroxyl and subsequent deprotection of the amino and troc groups. In the second approach, taxol was allowed to react with more than 2 molar equiv of the N-protected amino acids or N,N-dialkylated amino acids to give 2',7-diamino acid esters of taxol. For the protected amino acids, the deprotection of the amino group followed by removal of the 2'-substituent gave the 7-amino acid esters of taxol. The methanesulfonic acid salts of both 2'- and 7-amino acid esters showed improved solubility ranging from 2 to greater than 10 mg/mL. The 7-derivatives were effective in promoting microtubule assembly in vitro while 2'-derivatives showed little in vitro activity. The derivatives 2'-(N,N-dimethylglycyl)taxol (4) and 2'-[3-(N,N-diethylamino)propionyl]taxol (5) inhibited proliferation of B16 melanoma cells to an extent similar to that of taxol, while the other derivatives were about 50% as cytotoxic. In a mammary tumor screen, 2'-[3-(N,N-diethylamino)propionyl]taxol showed the greatest antitumor activity compared to the other analogues. The lower activities of the 7-derivatives in inhibiting tumor growth and melanoma cell proliferation (although they were almost as active as taxol in inducing microtubule assembly in vitro) may be due to differences in drug uptake by the cells. The similar cytotoxic and antitumor activities of the 2'-analogues and taxol can be explained by their conversion to taxol or an active taxol metabolite. Therefore, the 2'-analogues appear to behave as prodrugs and have the potential to be developed as chemotherapeutic agents.     

新型14β-侧链紫杉醇衍生物的合成及构效关系研究

以生物合成得到的紫杉烷 sinenxan A为起始原料,合成一系列新的紫杉醇衍生物,以寻找高效低毒、抗瘤谱广、综合性能好的新一代紫杉醇类抗癌药,并进行构效关系研究。从半合成的紫杉烷中间体7出发,分别经5步和6步反应成功地合成了4位羟基和4位乙酸酯两类共8个新的14β 侧链紫杉醇衍生物,2位基团为苯甲酸酯、间氯苯甲酸酯、正戊酸酯和苯乙酸酯。将目标化合物连同已合成的2个14β-侧链紫杉醇衍生物进行了微管聚合试验和体外肿瘤细胞抑制试验。所有化合物在浓度为10μmol·L^-1时对微管无作用。在体外肿瘤细胞抑制试验中,大部分化合物显示边缘细胞毒活性。14β-侧链紫杉醇衍生物的构效关系与13α-侧链紫杉醇衍生物有所不同,2位脂肪酸酯与2位芳香酸酯活性相当,表明2位基团的改变对活性无明显影响。4位羟基衍生物的活性好于4位乙酸酯。     

紫杉醇生产新工艺研究

从红豆杉茎皮提取紫杉醇，采用１％柠檬酸渗漉、柱色谱分离、溴加成后柱色谱分离的新工艺，可以获得比较满意的结果。     

High sensitivity ELISA determination of taxol in various human biological fluids

Taxol (paclitaxel)--the natural product isolated from Pacific yew (Taxus brevifolia)--is a novel agent with high activity in the treatment of patients with several malignant tumors including those resistant to other cytotoxic drugs. The therapeutic index of this promising anticancer drug could be further increased by the exploration of its pharmacokinetic pharmacodynamic relationship in cancer patients. Since taxol is highly protein bound, a very specific and highly sensitive analytical method is required in order to determine free, protein unbound and biologically active taxol species in human physiological fluids: plasma; plasma ultrafiltrate; and salivary fluids. In order to accomplish this, a new indirect competitive enzyme-linked immunosorbent assay (ELISA), for quantitating such a low bioactive taxol concentration level, has been developed in our laboratories. This method uses taxol competitive inhibition of mouse anti-taxol antibodies binding to the solid phase coated antigen 7-succinyltaxol-bovine serum albumin. This indicates recognition of the active taxol in the solution phase, where a diluted horseradish peroxidase labeled goat anti-mouse enzyme conjugate is used. While employing this technique, after systematic optimization of the experimental conditions, we are able to detect the anticipated taxol in plasma ultrafiltrate and salivary fluids at the concentration level of subpicogram per milliliter. The working range of the assay is approximately five orders in magnitude, i.e. from pg ml(-1) to 100 ng ml(-1). The clinical part of this study verified the working range of the ELISA method using samples of physiological fluids from a cancer patient treated with 3 h intravenous (i.v.) infusion of this drug. Our results of taxol determination in plasma, plasma ultrafiltrate and saliva demonstrate the applicability of the newly developed ELISA method for further pharmacokinetic studies of free, biologically active taxol species in cancer patients.