西罗莫司高产菌株与野生菌株中FKBP25的初步比较

目的　对比西罗莫司高产突变菌株———吸水链霉菌R2 7、R388、R4 4 1和野生菌株———吸水链霉菌ATCC 36 7817的蛋白质电泳图谱及FKBP2 5含量的差异 ,探讨FKBP2 5与西罗莫司产量的可能关系。方法　制备、分离高产突变株与野生菌株的菌体蛋白质并进行PPIase活性测定 ,比较 3株西罗莫司高产突变株与野生菌株的蛋白质电泳图谱及PPI ase酶活性。结果　西罗莫司低产的野生菌株的FKBP2 5含量比西罗莫司高产突变菌株高。结论　初步认为在吸水链霉菌中西罗莫司的产量与FKBP2 5量的多寡呈现一定的相关性     

强效免疫抑制剂西罗莫司类似物FIM-Rap-02的分离与鉴定

从西罗莫司产生菌吸水链霉菌突变株FC904-30的发酵产物中,分离纯化获得西罗莫司类似物FIM-Rap-02的晶体。对FIM-Rap-02进行理化性质、光谱等分析,并通过单晶X-Ray衍射确定构型,结果证实它的结构与prolylrapamycin同质。     

吸水链霉菌FC-904发酵代谢产物29-O-去甲基雷帕霉素的分离和结构鉴定

目的 从西罗莫司产生菌吸水链霉菌FC904的发酵液中分离纯化代谢产物FIM-R85，鉴定其化学结构。方法 采用硅胶柱层析和制备液相分离纯化获得FIM-R85，进行理化性质以及NMR、UV、IR和HRMS等波谱分析，鉴定其化学结构。 结果 纯化获得HPLC纯度为98.7%的FIM-R85，其为西罗莫司类似物，与29-O-去甲基雷帕霉素同质。结论 29-O-去甲基雷帕霉素为西罗莫司产生菌吸水链霉菌FC904的发酵代谢产物之一。     

西罗莫司同分异构体Rap-D1的分离与鉴定

从西罗莫司产生菌的突变株FC904-25的发酵产物中分离获得化合物Rap-D1的晶体。对其理化性质包括UV、IR、LC-MS、NMR谱等进行分析，结果表明Rap-D-与西罗莫司为同分异构体，具有相同的平面结构。通过单晶X-衍射确定了构型，它与文献报道经化学半合成获得的28-epirapamyein同质。     

用庆大霉素抗性筛选法选育西罗莫司高产菌株

用紫外线对吸水链霉菌（Streptomyces hygroscopicus）NP03进行诱变，再采用庆大霉索抗性筛选法进行处理，剂量分别为0、0．01、0．03、0．05和0．08μg／ml，共获得113株抗性突变株，其中西罗莫司产量高于出发菌株的有51株，突变株NP03-74#的生产能力达到出发菌株的134％，且遗传特性稳定。     

新型强效免疫抑制剂西罗莫司F904的研究

从福建省福州市的土壤中分离到的链霉菌 FC90 4 ,经分类鉴定、菌株培养确定为吸水链霉菌。其发酵代谢产物 F90 4具有抗真菌、抗增殖和比环孢素强的免疫抑制活性。分离、纯化、结晶 ,得 F90 4 ,经理化性质、光谱分析和 X-射线单晶衍射研究证明 F90 4为三烯大环内酯新型强效免疫抑制剂西罗莫司。     

链霉菌702抗药性致死突变标志微波诱变筛选研究

目的 筛选出产抗真菌活性物质的高产链霉菌702突变株.方法 分别以链霉菌702菌株为试验材料和以庆大霉素为敏感抗生素建立链霉菌702孢子致死突变标志的微波诱变筛选模型,通过微波对链霉菌702菌株孢子进行不同时间的诱变处理,将诱变处理后的孢予悬液涂布于含致死浓度的庆大霉素的PDA平板培养基上,获得抗庆大霉素突变株,分别挑取单个抗药性突变菌株进行摇瓶初筛和复筛.生物效价测定采用一剂量法.结果 微波处理30s对菌株的致死率可达70.53%,抗药性突变率高达23.13%,获得的抗药性突变株经过摇瓶初筛和复筛,获得高产突变株20-29-47菌株,产抗真菌活件物质的摇瓶发酵单位达到1478μg/ml,比出发菌株发酵单位986μg/ml提高T49.9%.结论 采用抗药性致死突变标志的微波诱变筛选模型可以获得产抗真菌活性物质的链霉菌702高产菌株.     

雷帕霉素产生菌突变株FC904-107发酵产生7-O-ethyl-rapamycin

在雷帕霉素产生菌吸水链霉菌FC904突变株FC904-107的发酵液中发现一个雷帕霉素同系物FIM-4025,用反相硅胶柱层析等提取纯化并获得晶体。经理化性质、UV、IR、LC-MS、NMR等波谱分析,单晶X-Ray衍射确定构型,结果表明雷帕霉素同系物FIM-4025与雷帕霉素化学半合成的衍生物7-O-乙基雷帕霉素同质。     

游动放线菌ATCC 12065生物转化强效免疫抑制剂西罗莫司

游动放线菌ATCC 12065生物转化强效免疫抑制剂西罗莫司后产生与西罗莫司不同的4个未知组分(未知组分1,2,3,4),HPLC保留时间分别为25.41min,34.13min,48.46min和52.24 min(西罗莫司的保留时间为26.62min)。经UV、HPLC及LC-MS分析表明,转化产物为西罗莫司衍生物。未知组分1和未知组分2的分子量为916,比西罗莫司的分子量多2;未知组分3和未知组分4的分子量900,比西罗莫司的分子量少14。     

他克莫司高产菌株的理性化选育

目的 理性化选育他克莫司高产菌株.方法应用化学和物理诱变剂对他克莫司产生菌进行菌种诱变,并利用特定的选择剂即莽草酸和哌可酸及其相应的结构类似物作为选择压力筛选突变株.然后对特定的正突变株进行他克莫司生物合成的营养学研究.结果与结论 获得一株高产突变株6H-23-7,它的他克莫司相对发酵效价比出发菌株弗氏链霉菌类群Str...     

新型强效免疫抑制剂F904的结构测定

分析吸水链霉菌 (Streptomyces hygroscopicus) FC90 4产生的具有免疫抑制活性的化合物 F90 4的理化性质及光谱学性质 (UV、IR、NMR、X- Ray衍射 ) ,证实化合物 F90 4的分子结构及空间构型与西罗莫司(sirolimus,又称 rapamycin)同质。     

Mutagenesis of the rapamycin producer Streptomyces hygroscopicus FC904

Rapamycin (RPM) is produced by Streptomyces hygroscopicus FC904 isolated from soil in Fuzhou, China. It is a triene macrolide antibiotic with potential application as an immunosuppressant and drug for human gene therapy. In an attempt to improve rapamycin production, mutation and screening of the parent culture have been carried out. Thousands of survivors were obtained after mutagenesis by NTG (3 mg/ml) and UV (30 W, 15 cm, 30 seconds) of spore suspensions. None showed improved production of RPM. We determined the susceptibility to antibiotics of S. hygroscopicus FC904 by two fold dilutions of antibiotics in oatmeal agar plates. It was found that the strain was resistant to penicillin, erythromycin, RPM, tetracycline and chloramphenicol, but susceptible to mitomycin C (MIC, 10 microg/ml) and aminoglycosides such as gentamicin (MIC, 0.1 microg/ml), kanamycin (MIC, 0.1 microg/ml) and streptomycin (MIC, 0.3 microg/ml). Protoplasts of strain FC904 were prepared after finding the best conditions for their formation. They were treated with gentamicin, erythromycin, mitomycin C and NTG. Surprisingly, gentamicin was especially effective for obtaining higher RPM-producing mutants. Mutant C14 was selected by exposing the protoplasts of the parent strain FC904 to 1 microg/ml of gentamicin at 28 degrees C for 2 hours. A higher RPM-producing mutant (C14-1) was obtained from the protoplasts of mutant C14 treated with gentamicin, and its titer was 60% higher than that of the parent strain FC904 by HPLC analysis. Another improved mutant (C14-2) was obtained from the spores of mutant C 14 treated with 1 microg/ml of gentamicin plus 2 mg/ml of NTG at 28 degrees C for 2 hours. Mutant C14-2 had a titer 124% higher than FC904. The possible mechanism for the effect of gentamicin by using protoplasts or spore suspensions will be discussed, i.e. the possibility of gentamicin being a mutagen or a selective agent.     

3L自动发酵罐中西罗莫司的发酵研究

采用美国NBS公司生产的BIOFLO3000型3L发酵罐进行西罗莫司发酵代谢过程的考察和代谢参数的累积,建立了较为适应的西罗莫司发酵条件,罐上发酵水平与摇瓶发酵水平相当.     

西罗莫司产生菌Streptomyces hygroscopicus WY—93的诱变育种与代谢研究

研究了不同诱变方法对西罗莫司产生菌正变率的影响,发现紫外线单一因子处理、光复活较为有效;用这一条件处理得到--正变株UV-8-61,其效价比出发菌株Z27高2～3倍,产生抗生素水平经连续传代证明非常稳定。本文还研究了菌落形态与发酵效价以及诱变后正变率与死亡率的关系,表明孢子丰富的梅花型或面包型菌株发酵效价较高。以紫外线单一因子、光复活处理死亡率在99.90%～99.95%时正变率较高。另外,从代谢角度对高产株与原株对碳、氮源的利用及葡萄糖-6-磷酸脱氢酶的活性进行了深入研究,发现两者在生长速度,碳、氮源的利用方面存在显著差别,尤其是高产株葡萄糖-6-磷酸脱氢酶的活性进行了深入研究,发现两者在生长速度,碳、氮源的利用方面存在显著差别,尤其是高产株葡萄糖-6-磷酸脱氢酶的活性明显高于原株。由此推断,高产株UV-8-61西罗莫司产量的提高有可能是由于菌体生理代谢旺盛,参与戊糖一级代谢的G-6-P脱氢酶活性提高,由此增加了作为西罗莫司生物合成环已烷前体的莽草酸的供给。     

Sirolimus-itraconazole interaction in a hematopoietic stem cell transplant recipient

Interactions between azole antifungal agents and immunosuppressants that are metabolized by cytochrome P450 3A4 (chiefly calcineurin inhibitors) are well documented. Interactions between itraconazole and sirolimus are known to occur in patients after solid organ transplantation, but interactions in hematopoietic stem cell transplant (HSCT) recipients have yet to be reported in the literature. We describe an allogeneic HSCT recipient who experienced supratherapeutic trough levels of sirolimus as a result of its coadministration with itraconazole. This patient was a 20-year-old African-American man who underwent HSCT for treatment of myelodysplastic syndrome with severe aplastic anemia. After several regimen changes, the patient received oral itraconazole 200 mg every 12 hours and sirolimus at a dosage of 7 mg/day on days 76-80 and 5 mg/day on days 81 and 82. His sirolimus whole blood trough levels were 17.5 and 35.6 ng/ml on days 80 and 82, respectively (therapeutic range 5-15 ng/ml). An interaction between itraconazole and sirolimus was suspected, and sirolimus was withheld on days 83-90. On day 90, the patient's sirolimus trough level had normalized to 4.4 ng/ml. Sirolimus was resumed at 1-2 mg/day, with adjustments as needed to maintain trough levels of 10-15 ng/ml. Both the itraconazole and sirolimus were eventually were discontinued. The patient died, however, from a disseminated adenovirus infection leading to end-organ failure. Sirolimus is extremely sensitive to the inhibitory potential of azole antifungals. We propose that itraconazole also has a potent effect on sirolimus metabolism. Preemptive sirolimus dosage reduction and close monitoring of its whole blood trough levels are required whenever this combination is considered to avoid immunosuppressant toxicity in already critically ill patients.     

Combination treatment with sirolimus and ciclosporin in clinical renal transplantation: A comprehensive review

Sirolimus is a potent new immunosuppressive agent that has been shown to reduce the incidence of acute rejection episodes among renal transplant recipients as well as provide a unique approach to optimize treatment outcomes in difficult transplant situations. Owing to its properties as a critical-dose drug, therapeutic concentration monitoring of sirolimus readily compensates for intra- and interpatient variability and drug interactions with a variety of other agents such as ciclosporin. This review summarizes the results that demonstrate the efficacy of sirolimus in combination treatment with ciclosporin in human renal transplantation, as well as its potential in alternative therapeutic modalities in a broad range of transplant recipients. The clinical trials for SDZ-RAD, a macrocyclic lactone immunosuppressant structurally similar to sirolimus, also are reviewed herein. SDZ-RAD was developed in an attempt to improve the pharmacokinetic characteristics of sirolimus, particularly to increase the extent and reproducibility of its oral bioavailability and to reduce the extensive tissue distribution by virtue of its greater polarity. (c) 2001 Prous Science. All rights reserved.     

Comparison of pharmacokinetics of the sirolimus-eluting stent in Japanese patients with those in American patients

The aim of this study was to evaluate the pharmacokinetics of the sirolimus-eluting stent (SES) implanted in 20 Japanese patients with angina pectoris and compare it with that in US study. Bx VELOCITY stent loaded with a total sirolimus dose of 150 mug was used in this study. Ten patients were treated by single-SES (group 1) and 10 patients were treated by double-SES (group 2). Sirolimus levels in whole blood were serially measured in the 2 groups after the SES implantation and compared with the pharmacokinetics in US study. We also evaluated the side effect of sirolimus, major adverse clinical events, and binary angiographic restenosis at 8 months after the SES implantation. Peak concentrations were observed approximately 4 hours after the SES implantation, and sirolimus half-lives were approximately 120 hours in each group. Mean peak sirolimus levels were 0.86 and 2.00 ng/mL for the group 1 and group 2, respectively. The peak concentrations of sirolimus in this study were twice higher in Japanese than in Americans, but they were much lower than effective concentration of sirolimus when orally administrated as an immunosuppressive agent. There were no side effects of sirolimus and no binary angiographic restenosis in any patients. One patient had target vessel revascularization at 8 months after the SES implantation. Although blood concentrations of sirolimus in Japanese patients after SES implantation are somewhat higher than those in American patients, its level is extremely low compared with the systemic administration, indicating the same clinical benefits by the SES could be safely expected in Japanese patients.