Method for preparing epothilones B and use thereof

A technology of epothilone and adsorbent, which is applied in the field of chronic myeloid leukemia drugs, can solve problems such as complex drug resistance mechanisms, and achieve the effects of easy large-scale production, strong applicability, and low cost

Active Publication Date: 2008-01-16
HUNAN DINUO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

② Epothilone has no cytotoxic activity of paclitaxel
The mechanism of drug resistance of CML cells to Gleevec is complex...

Method used

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  • Method for preparing epothilones B and use thereof
  • Method for preparing epothilones B and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment one seed liquid culture

[0038] Take the frozen strains and transfer them to 50ml medium, the medium components are potato starch 8g / L, glucose 2g / L, soybean powder 2g / L, yeast extract 2g / L, etc. After culturing for 24 hours, transfer to a plurality of 500ml No. I culture medium, cultivate the seed solution for 72 hours, and transfer to a fermenter for fermentation.

Embodiment 2

[0039] Embodiment two fermentation, extract crude extract

[0040] 1) Add 65L medium to a 100L fermenter, and the medium components are potato starch 1g / L, glucose 10g / L, soybean powder 30g / L, yeast extract 15g / L, etc. Sterilize at 121°C for 20 minutes. Transplant under aseptic conditions. During the fermentation process, set the ventilation rate to 25L / min, the pressure to 0.4Mpa, the rotational speed to 100r / min, the temperature to 32°C, and the pH to 7.4. After culturing for 12 hours, 1% of the total volume of AB-8 macroporous adsorption resin was added. Harvest after culturing for 100 h.

[0041] Filter the fermented liquid with a thick cloth to obtain the adsorbent AB-8, and extract it twice with 2 times the volume of isopropanol for 1 hour each time. The isopropanol solutions were combined, concentrated to 1 / 10 by distillation under reduced pressure, and the concentrated isopropanol was extracted three times with ethyl acetate to obtain a crude epothilone B extract. ...

Embodiment 3

[0048] Embodiment three: Separation and purification of epothilone B

[0049] The crude extract containing epothilone B is separated and purified by two steps to obtain epothilone B with a purity higher than 95%.

[0050] The first step: Sephadex LH-20 column chromatography

[0051] After the extract was dissolved in methanol, the supernatant was collected by centrifugation and added to the top of Sephadex LH-20 column. Mobile phase: methanol, the mobile phase flow rate is 10cm / h. Analysis by HPLC high performance liquid chromatography (filler: Kromasil C 18 5μm, size: 4.6mm×150mm. Mobile phase: methanol: water = 7:3. Detection wavelength: UV230nm). Fractions containing epothilone B were combined for further purification.

[0052] The second step: C18 reversed-phase chromatographic separation

[0053] The fraction separated by Sephadex LH-20 was further purified by C18 reverse phase chromatography column. Mobile phase: methanol: water = 70:30, detection wavelength: UV...

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Abstract

The invention relates to a preparation method of epothilones B. In particularly, the invention provides a technique method to prepare epothilones B through adopting 100 to 500 liters fermentor to ferment and to separate epothilones B in large scale. The invention also relates to the preparation and application of epothilones B in preparation of gleevec resistance chronic myeloid leukemia treatment drug.

Description

technical field [0001] The invention belongs to the technical field of pharmacy, and specifically relates to a preparation method of epothilone B, a preparation process and an application thereof for preparing a drug for treating Gleevec-resistant chronic myelogenous leukemia. Background technique [0002] Epothilone is a secondary metabolite produced by the myxobacteria Sorangium Cellulosum discovered by GBF microbiologists in the late 1980s. In 1987, two new macrolides were isolated and named epothilone A and B, and it was found that they had inhibitory activity against cultured fungal pathogens. In 1994, the National Cancer Society of the United States found that epothilone B had inhibitory activity on tumor cells, and Merck laboratory found that it had at least the same effect as paclitaxel in the tubulin polymerization experiment, which proved that epothilone B had an inhibitory effect on tumor cells fact. [0003] Further research found that epothilone has superior a...

Claims

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Application Information

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IPC IPC(8): C12P17/18C12R1/01
Inventor 张庆林王赫肖凤君王石齐李学林易志恒
Owner HUNAN DINUO PHARMA
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