86 results about "Epothilone B" patented technology

The invention relates to a new process for concentrating epothilones in culture media, a new process for the production of epothilones, a new process for separating epothilones A and B and a new strain obtained by mutagenesis for the production of epothilones, as well as aspects related thereto. New crystal forms of epothilone B are also described.

The present invention relates to improved methods for the production, isolation and purification of epothilone B. These methods include, for example, a fermentation process for the production of epothilone B, isolation via adsorption onto a resin, and subsequent purification.

Disclosed is an anti-cancer drugs slow release agent containing Epothilone which comprises slow release microspheres and dissolvent, wherein the slow release microballoons comprise anti-cancer active constituents and slow release auxiliary materials, the dissolvent being specific dissolvent containing suspension adjuvant. The anticancer active constituents include Epothilone, Epothilone derivatives, Epothilone B, Epothilone D and combination of anti-cancer drugs selected from phosphoinositide-3-kinase inhibitor, of pyrimidine analogues and/or DNA restoring enzyme inhibitor, the slow release auxiliary materials include polylactic acid and its copolymer, polyethylene glycol, PLA-COOH copolymer, di-aliphatic acid and sebacylic acid copolymer, poly(erucic aciddipolymer-sebacylic acid), poly(fumaric acid-sebacylic acid), Polifeprosan, polylactic acid and other biocompatible high polymers, the viscosity of the suspension adjuvant is 100-3000cp (at 20-30 deg C), and is selected from sodium carboxymethylcellulose. The anticancer active constituents and the slow release microspheres can also be prepared into slow release implanting agent for intra-tumor or around-tumor injection or placement for the effective suppression of tumor growth and for the appreciable enhancement for curative effects of non-operative treatments such as chemotherapy.

The invention relates to an anti-cancer sustained release injection containing epothilone derivative, consisting of sustained microspheres and menstruum. The sustained microspheres comprise anti-cancer drugs selected from taxane, alkylating agent and/or plant alkaloid and the like, the epothilone derivative and sustained release auxiliary material. The menstruum is a special menstruum containing suspending agent. The epothilone derivative is selected from epothilone B, epothilone D, iso-epothilone D, BMS-247550, azaepothilone B, furan epothilone D or BMS-310705. The sustained release auxiliary material is selected from poly-dl-lactide, the glycolic acid copolymer of the poly-dl-lactide, polyethyleneglycol, the polylactide copolymer of the polyethyleneglycol, carboxyl terminated polylactide copolymer, fatty acid and decanedioic acid copolymer, etc. The suspending agent is selected from carboxymethyl cellulose and the like with the viscosity of 100cp to 3000cp (under the temperature of 25 DEG C to 30 DEG C). The sustained release microsphere can also be made into a sustained release implant. The sustained release injection is injected or arranged in or around the tumour and can release drug at partial position for 40 days approximately, therefore, the sustained release injection improves the local drug concentration selectively and enhances the treatment effect of non-operative treatments, such as radiotherapy, chemotherapy and the like at the same time.

The invention relates to an anti-cancer sustained release injection containing epothilone derivative, consisting of sustained microspheres and menstruum. The sustained microspheres comprise anti-cancer drugs selected from taxane, alkylating agent and/or plant alkaloid and the like, the epothilone derivative and sustained release auxiliary material. The menstruum is a special menstruum containing suspending agent. The epothilone derivative is selected from epothilone B, epothilone D, iso-epothilone D, BMS-247550, azaepothilone B, furan epothilone D or BMS-310705. The sustained release auxiliary material is selected from poly-dl-lactide, the glycolic acid copolymer of the poly-dl-lactide, polyethyleneglycol, the polylactide copolymer of the polyethyleneglycol, carboxyl terminated polylactide copolymer, fatty acid and decanedioic acid copolymer, etc. The suspending agent is selected from carboxymethyl cellulose and the like with the viscosity of 100cp to 3000cp (under the temperature of 25 DEG C to 30 DEG C). The sustained release microsphere can also be made into a sustained release implant. The sustained release injection is injected or arranged in or around the tumour and can release drug at partial position for 40 days approximately, therefore, the sustained release injection improves the local drug concentration selectively and enhances the treatment effect of non-operative treatments, such as radiotherapy, chemotherapy and the like at the same time.

The invention discloses a fermentation production method based on epothilone B metabolic pathways and belongs to the technical field of fermentation engineering. The method comprises the following steps of: 1) inoculating Sorangium cellulosum into an M26 culture medium, carrying out shake cultivation to obtain a seed solution; 2) inoculating the seed solution into fermentation liquor containing resin, carrying out shake culturing for 72-120 hours at 25-35 DEG C, adding a precursor and small molecular substances into the fermentation liquor containing resin; and 3) filtering the fermentation liquor after the culture is finished, collecting the resin, washing the resin and oscillating and extracting with methyl alcohol to obtain extract liquor containing the epothilone B. According to the invention, the precursor and small molecular substances related to the biosynthetic pathways obtained by screening are added into the fermentation liquor, and the constructive metabolism pathways of the epothilone B are disturbed, so that the excellent bacterial strain gives full play to the ability of anabolism and the fermentation level of the epothilone B is improved greatly, thus the fermentation cost is reduced and the commercial process of the anti-cancer drug is promoted.

The present invention is directed to novel acyloins, their derivatives, methods for their production and their use for the production of novel epothilones and their derivatives. In addition, the invention is directed to the building blocks for epothilone synthesis, methods for their production and the use of synthetic building blocks for the production of epothilones and their derivatives.

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.

The invention discloses a fermentation production process of Epothilone B, i.e. a process for increasing the fermentation output of Epothilone B of Epothilone B production bacterial strain sprangium cellulosum. The process comprises the following steps: 1) adding a molecular imprinting polymer adsorbent of the Epothilone B in the fermentation process; 2) selecting soybean meal, corn starch and the like suitable for sprangium cellulosum production; and 3) adding glucose pulses in the fermentation process and the like. So the fermentation production output of the sprangium cellulosum of the epothilone B achieves 50mg / L.

The invention discloses a method for preparing an epothilone molecularly imprinted polymer film by adopting a mixed template and belongs to the technical field of molecular imprinting. The method comprises the following steps: 1, after mixing template molecule epothilone B and epothilone D, adding a functional monomer and a cross-linking agent, sufficiently and uniformly mixing to obtain a mixture, adding the mixture into a pore-foaming agent, uniformly mixing under the ultrasonic condition, standing at room temperature, then adding an initiator and uniformly mixing to obtain mixed liquid; 2, after soaking a matrix film in the mixed liquid, taking out the matrix film, placing the matrix film between two glass sheets, removing air bubbles, placing the glass sheets in a sealed bag, filling nitrogen, sealing up the bag, irradiating the bag by an ultraviolet lamp, separating the glass sheets to obtain the epothilone molecularly imprinted polymer film. The method is easy to operate; the reaction conditions are easy to control; the preparation cost is low; the film is easy to carry out readsorption and elution and has a high repeated utilization rate; not only are the defects of long polymerization time and requirement for screening of a rodlike molecularly imprinted polymer overcome, but also elution time is shortened.

A method for making epothilones and epothilone analogs is described, as are novel compounds made by the method. One embodiment of the method was used to synthesize epothilone B by a convergent approach that entailed Wittig coupling of an ylide derived from phosphonium bromide with an aldehyde. The former was prepared from propargyl alcohol by a nine-step pathway which installed both trisubstituted double bonds with clean Z configuration. Macrolactonization of a resulting seco acid provided the following intermediate diene epothilone analog. Selective saturation of the 9,10-olefin and subsequent epoxidation provided epothilone B.

The invention discloses a preparation method of an epothilone B molecularly imprinted polymer and belongs to the technical field of chemical engineering. The preparation method comprises the following specific steps of: dissolving a template molecule (epothilone B), a functional monomer, a cross-linking agent and an initiator into a pore-foaming agent according to a certain mol ratio, wherein the pore-foaming agent can be a mixed solution of methanol and acetonitrile; after reaction, grinding and sieving the obtained product to obtain particles with the particle size being about 40-60mum; fully washing the obtained particles in a mixed solution of acetic acid and methanol according to the ratio being (1: 9)-(3:7) (v:v) so as to remove the template molecule; and freezing and drying to obtain the epothilone B molecularly imprinted polymer. The epothilone B molecularly imprinted polymer prepared by the preparation method disclosed by the invention can be used for specifically and selectively adsorbing the epothilone B and has greater application value in the production process of the epothilone B by adopting a liquid fermenting method.

The invention discloses a method for preparing an epothilone molecularly imprinted polymer by adopting a mixed template and belongs to the technical field of molecular imprinting. The method comprises the following steps: 1, uniformly mixing epothilone B and epothilone D according to a certain proportion to obtain template molecules, adding a functional monomer and a cross-linking agent into the template molecules, uniformly mixing to form a mixture, adding the mixture into a pore-foaming agent, uniformly mixing under the ultrasonic condition, standing at room temperature, then adding an initiator and uniformly mixing to obtain mixed liquid; 2, performing the polymerization reaction on the mixed liquid at a temperature of 40 to 60 DEG C to obtain a blocky polymer, then crushing, grinding, sieving, repeatedly carrying out sedimentation by acetone, drying to obtain polymer particles, repeatedly carrying out Soxhlet extraction on the polymer particles and drying to obtain the epothilone molecularly imprinted polymer. The epothilone molecularly imprinted polymer prepared by the method has higher affinity on epothilone, has a better adsorption effect and has wide application value and market prospect.

The invention discloses a sorangium cellulosum fermentation medium, which comprises a carbon source, a nitrogen source, inorganic salts, a macroporous absorption resin, and water. The fermentation medium also comprises amino acid. When the fermentation medium is used for preparing the epothilone B by fermentation culture of sorangium cellulosum, the performance of strains is stable, the growth is rapid, and the yield of the product epothilone B is obviously improved. The invention also provides a fermentation method for producing the epothilone B by the fermentation culture of the sorangium cellulosum by using the sorangium cellulosum fermentation medium.

The invention discloses a method for producing epothilone B based on coupling of microbial fermentation and membrane separation techniques. The invention proposes a separation and fermentation coupling method mainly aiming at the problems that toxicity and feedback inhibition on cells are generated due to increase of a product epothilone B in the fermentation process, and bacteria growth in the fermentation process is affected. The method comprises the following steps: fermenting and cultivating, filtering by a micro-filtration membrane, carrying out ultrafiltration and nanofiltration, feeding materials, concentrating and extracting. The concentration of the fermentation liquor product epothilone B is reduced to a lower level, the growth inhibition action on the strain is relieved, the yield of the epothilone B is improved, the production cost is reduced, post-treatment of a fermentation liquor and refining of the product are facilitated, and large-scale production becomes possible, so that the method has a good industrial production prospect.

The invention relates to an epothilone B separating and extracting method based on a membrane filtration technology. The method comprises the following steps: adsorbing a fermentation liquid by using a macroporous resin; then leaching the absorbed macroporous resin by using ethyl acetate and collecting a leaching liquid; filtering the leaching liquid by using an ultrafiltration membrane; washing and filtering the leaching liquid by using the ethyl acetate; combining filtrates; concentrating; performing detection through preparative HPLC (High Performance Liquid Chromatography); and crystallizing the epothilone B component obtained in a separating manner, thereby obtaining the epothilone B. The method is moderate in condition, simple and convenient to operate, few in separating steps and good in selectivity, and can be used for obviously increasing the yield of the epothilone B, thereby providing conditions more favorable for industrial production. The method with combination of membrane filtration and crystallization is adopted, so that a part of macromolecular impurities can be effectively removed; meanwhile, the application of an organic reagent is avoided, so that the industrial production pollution is reduced. Thus, the loss of the epothilone B in other complicated preparation processes is avoided. The epothilone B prepared in a crystallization manner has relatively high purity. Thus, the epothilone B is broad in application prospect and economic benefit.

The invention concretely discloses a fermentation method for producing epothilone B, and belongs to the field of biological fermentation. According to the method, during culturing of a seeding tank, ammonia hydroxide is added in a flowing mode to adjust the pH value, so that a lot of seed thalli are obtained; during fermentation, ammonia hydroxide or propanoic acid is added in a flowing mode to regulate the pH value, in this way, the yield of the epothilone B is increased, the regulation procedures are simplified, production cost is lowered, industrialized production is easy, and application prospects are good.

The invention discloses a method for improving sorangium cellulosum's epothilone B yield by genetic engineering. The method includes: introducing a Vitreoscilla hemoglobin gene vgb into an expression vector PBEP43 to obtain a recombinant plasmid PBEP43-vgb, and then subjecting the recombinant plasmid PBEP43-vgb to electrotransformation into sorangium cellulosum So ce M4, thus obtaining a sorangium cellulosum So ce M4 recombinant strain, and realizing improving the sorangium cellulosum So ce M4's epothilone B yield. According to the invention, the electrotransformation method is employed to transform the gene of sorangium cellulosum So ce M4, thereby greatly improving the yield of epothilone B. The method provided by the invention has broad application prospects, and research in the aspect is not reported to date.

The invention relates to an improved method for epothilone B extraction and purification, and belongs to the technical field of biological separation. According to the method, most of impurities are removed by a low-temperature refrigeration method, solid-liquid separation is achieved by changing density, the purity of finished products is further improved by a two-time crystal method, rate-limiting steps such as silica-gel column chromatography are decreased, an epothilone B extraction process is simplified, operation cost is reduced, and the method has a wider application prospect.

The invention relates to a preparation method of epothilone D and B, which comprises the following steps: reacting carboxylic acid (6) and hydroxyl compound (7) to obtain an ester (8), reacting with a key intermediate (9) in an Aldol mode to obtain a compound (10), carrying out hydroxy protection with proper protective group to obtain a compound (11), carrying out RCM reaction to close the cycle to obtain a compound (12), removing the hydroxy protective group to obtain the compound epothilone D, and epoxidating to obtain the epothilone B.

The invention discloses an epothilone B separating and extracting method based on the molecularly imprinted membrane filtering technology. The method comprises the following steps: fermentation liquid is adsorbed by macroporous resin and resolved by ethyl acetate to obtain a crude product; the crude product is filtered by a micro-filtration membrane and an ultrafiltration membrane to remove macromolecules and other impurities; filtrate is filtered by the epothilone B molecularly imprinted membrane to obtain pure epothilone B liquid; and finally, the pure epothilone B liquid is obtained through crystallization. The method is convenient for continuous operation, is easy to enlarge, simple to operate, low in cost and high in selectivity, can rapidly separate the epothilone B molecules from a mixture of structural analogues, and can be put into industrial production easily.