CN101732310B - Application of epothilone B - Google Patents
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- CN101732310B CN101732310B CN2009102593725A CN200910259372A CN101732310B CN 101732310 B CN101732310 B CN 101732310B CN 2009102593725 A CN2009102593725 A CN 2009102593725A CN 200910259372 A CN200910259372 A CN 200910259372A CN 101732310 B CN101732310 B CN 101732310B
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Abstract
The invention relates to application of epothilone B in preparing a medicament for treating Gleevec medicament resistant chronic myelogenous leukemia.
Description
The application is that application number is 200610091035.6, the applying date is on July 12nd, 2006, denomination of invention is divided an application for the Chinese patent application of " preparation method of epothilone B and purposes ".
Technical field
The invention belongs to the pharmaceutical technology field, specifically, relate to the purposes of preparation method, preparation process and the drug-fast chronic granulocytic leukemia medicine of preparation treatment imatinib mesylate thereof of epothilone B.
Background technology
Epothilones is the secondary metabolite that the slime bacteria sorangium cellulosum SorangiumCellulosum of microbiologist's discovery of eighties of last century late nineteen eighties GBF produces.Isolated two kinds of new macrolide called after ebomycin A and B in 1987, experiment finds that the fungal pathogens of cultivating is had the activity of inhibition.American National cancer association in 1994 finds that epothilone B has the activity of inhibition to tumor cell, the Merck laboratory finds that it and paclitaxel have same function at least in the tubulin polymerization experiment, proved that Epothilones has the inhibiting fact to tumor cell.
Discover that further Epothilones has the antitumor characteristic more superior than paclitaxel.1. Epothilones is better than taxol soluble, and is simple in structure, helps chemosynthesis Epothilones and structure derivatization.2. Epothilones does not have paclitaxel intracellular toxin activity.3. different with paclitaxel, Epothilones is also kept very big cytotoxicity in the multidrug resistance sexual cell of P P-glycoprotein expression type.4. in the human cell system of the paclitaxel sensitivity of research such as Richard, epothilone B has bigger antiproliferative activity than ebomycin A and paclitaxel, and ebomycin A often the activity than paclitaxel is lower.In addition, Epothilones still has sensitivity to strain multidrug resistance rectal cancer system and paclitaxel resistance ovarian cancer system.Epothilones has several allied substances to enter the clinical research stage at present.Mainly comprise Epothilone B (EPO B), Epothilone D and BMS-247550.In the preliminary clinical experiment to 300 patients, this chemical compound has definite curative effect to colon cancer, breast carcinoma, kidney and ovarian cancer.
R
1＝Me R
2＝H epothilone?A
R
1＝Me R
2＝Me epothilone?B
That publishes at present passes through fermenting process of preparing epothilone B method production cost height, the fermenting process of preparing technology and the isolation and purification method of the extensive and cheap and simple of the research of still needing.
(Chronic myeloid leukemia is one of modal malignant disease of blood system CML) to chronic granulocytic leukemia, and annual morbidity is about 0.36/10 ten thousand at home, accounts for leukemic the 3rd.The molecular biological characteristics of CML is to have the bcr/abl fusion gene, and it is the fusion rotein P of 210KDa that this gene produces molecular weight by transcription and translation
210 Bcr/ Abl, this fusion rotein has the tyrosine kinase activity that increases unusually, makes hematopoietic stem cell take place to transform unusually and cause CML to take place.K562 cell line derives from acute transformation phase patient CML, P
210 Bcr/ablBeing positive, is the cell model of research CML classics.
The front chemicals of treatment CML is the imatinib mesylate (Glivec, or title STI571) of calendar year 2001 new listing at present
[1]Belong to α-aniline pyrimidine derivative, the mechanism of imatinib mesylate effect is effect ATP and tyrosine-kinase enzyme binding site, and specificity suppresses the ABL tyrosine kinase.Though use imatinib mesylate acute transformation phase CML is alleviated fully, most patients is shorter to the phase of replying of imatinib mesylate, and final cell makes clinical recurrence to its drug resistance
(2)The CML cell is to the drug resistance mechanism more complicated of imatinib mesylate.Because imatinib mesylate listing new drug does not soon also have good method to overcome CML to its drug resistance at present.People are to attempt delaying or stoping the CML cell that imatinib mesylate is produced drug resistance to CML treatment research at present, wherein by the reactive compound of screening discovery with imatinib mesylate different structure, different mechanism of action, overcome the drug resistance of cell to imatinib mesylate, or merge to use the drug resistance that overcomes cell with imatinib mesylate, become an important directions of CML treatment research.
Summary of the invention
The invention provides a kind of large scale fermentation and produce the method for epothilone B.
Specifically, the inventive method comprises the steps:
(a). seed liquor was cultivated 12～96 hours at ferment tank, added 1%～10% adsorbent, the absorption metabolite
(b). fermenting stops fermentation after 100-150 hour, thick cloth filtering fermentating liquid results adsorbent
(c). adsorbent with isopropanol extraction after concentrating under reduced pressure or use water infiltration, reuse ethyl acetate extraction rotary evaporation must contain the epothilone B crude extract to doing
(d). after extract uses the lower alcohol dissolving, earlier through mixed liquor or single the use lower alcohol eluting of normal pressure column chromatography with lower alcohol and alkyl chloride
(e) .HPLC detects and merges the fraction that contains epothilone B, and concentrating under reduced pressure is further purified by liquid chromatograph behind certain volume, obtains the epothilone B of purification.
Seed liquor among the step a can be changed over to cultivate in the culture medium after the amplification culture step by step again and obtain in 1～5 day by frozen strain, strain can be No. 15384, commercial strain such as sorangium cellulosum ATCC through ultraviolet or mutagenic agent mutation strain, or for separating other sorangium cellulosum kinds that obtain in the soil.
The preferred low pole macroporous adsorbent resin of adsorbent is as AB-8, DA201 etc.
The used culture medium of fermenting comprises starch 1～35g/L, sugar 1～10g/L, analysis for soybean powder 2～30g/L, yeast extract 2～30g/L; Also can add acid, the alkali of inorganic salt or adjusting pH value, for example add CaCl
2.2H
2O 1g/L, MgSO
4.7H
2O 1g/L, Fe-EDTA 8mg/L.Described sugar can be selected monosaccharide or disaccharide for use, as glucose, fructose, sucrose etc.
Among the step b, can add glucose or compound material (starch, analysis for soybean powder etc.) in the sweat, improve the output of epothilone B.
Among the step c, be 2～3: 1 with the volume/weight ratio of adsorbent preferably during with isopropanol extraction, gradation is extracted 2～3 times, is concentrated into 1/10～1/5 of original volume
In the steps d, the column chromatography filler can be selected silica gel or sephadex lh-20 for use, and lower alcohol can be selected methanol or ethanol for use, and alkyl chloride can be selected dichloromethane or chloroform for use.
The anti-phase C-18 preparative liquid chromatography of preferred employing carries out purification among the step e.
The epothilone B that this method prepares is through structural identification, consistent with bibliographical information (table 1)
Table 1. epothilone B
1H-NMR and
13C-NMR
Epothilone B preparation method provided by the present invention has following advantage:
1) suitability is strong, is suitable for multiple industrial strain
2) realized the production domesticization of equipment, reagent, with low cost
3) easy and simple to handle, be easy to large-scale production
On the other hand, the present invention also provides a kind of pharmaceutical composition of epothilone B, contains epothilone B and Polyethylene Glycol.Said composition can be made into injection or lyophilized powder, and for example the Polyethylene Glycol that is dissolved in the suitable molecular weight size by epothilone B prepares.Its specification can be preferably every 2.5mg～25mg according to the using dosage adjustment.Face the time spent with normal saline dilution or be dissolved into injection of debita spissitudo posterior vein or intravenous drip.
The inventor finds that also epothilone B is inhibited to the drug-fast K562 cell growth of imatinib mesylate, and this chemical compound can be used for treating the drug-fast chronic granulocytic leukemia of imatinib mesylate.
Description of drawings
The separation of Fig. 1 .C18 reversed phase chromatography chromatographic column obtains ebomycin A and B
Fig. 2. the epothilone B purity analysis chromatogram of purification
Fig. 3 .EpoA and EpoB suppress the growth activity curve chart to the drug-fast K562 cell of imatinib mesylate
The specific embodiment
In order to further specify embodiments of the present invention and purposes, describe with reference to following examples.Embodiment is in order to explain rather than to limit the scope of the invention by any way, and some change that those skilled in the art are made within the scope of the claims and adjusting also should be thought and belongs to scope of the present invention.
Embodiment one seed liquor is cultivated
Get frozen strain, change the 50ml culture medium over to, medium component is potato starch 8g/L, glucose 2g/L, analysis for soybean powder 2g/L, yeast extract 2g/L etc.Cultivate after 24 hours, change a plurality of 500ml I culture medium over to, seed liquor is cultivated 72h, changes ferment tank over to.
Crude extract is extracted in embodiment two fermentations
1) add the 65L culture medium in 100 liters of fermentation tanks, medium component is potato starch 1g/L, glucose 10g/L, analysis for soybean powder 30g/L, yeast extract 15g/L etc.121 ℃ of sterilization 20min.Transferred species under the aseptic condition is provided with ventilation 25L/min in the sweat, pressure 0.4Mpa, rotating speed 100r/min, 32 ℃ of temperature, pH7.4.After cultivating 12h, add the AB-8 macroporous adsorbent resin of cumulative volume 1%.Gather in the crops after cultivating 100h.
Thick cloth filtering fermentating liquid obtains adsorbent A B-8, with the isopropanol extraction of 2 times of volumes 2 times, and each 1 hour.Merge aqueous isopropanol, distilling under reduced pressure is concentrated into 1/10, and the isopropyl alcohol after the extraction of reuse ethyl acetate concentrates 3 times obtains the epothilone B crude extract.
2) add 160 liters of culture medium in the 250L fermentation tank, medium component is potato starch 15g/L, fructose 4g/L, analysis for soybean powder 25g/L, yeast extract 2g/L etc.121 ℃ of sterilization 30min.Change strain over to, fermentation condition is the same, behind the cultivation 48h, adds the AB-8 macroporous adsorbent resin of cumulative volume 5%.After cultivating 100h, add into glucose solution.Continue to ferment to 120h results.
Thick cloth filtering fermentating liquid obtains adsorbent A B-8.With the isopropanol extraction of 3 times of volumes 3 times, each 0.5 hour.Merge aqueous isopropanol, distilling under reduced pressure is concentrated into 1/5, and the isopropyl alcohol after the extraction of reuse ethyl acetate concentrates 3 times obtains the epothilone B crude extract.
3) add 160 liters of culture medium in the 250L fermentation tank, medium component is potato starch 20g/L, sucrose 1g/L, analysis for soybean powder 15g/L, yeast extract 30g/L etc.121 ℃ of sterilization 20min.Change strain over to, fermentation condition is the same, behind the cultivation 96h, adds the AB-8 macroporous adsorbent resin of cumulative volume 10%.After cultivating 120h, add into culture fluid.Continue to ferment to 150h results.
Thick cloth filtering fermentating liquid obtains adsorbent A B-8.Behind water infiltration, add the ethyl acetate extracting twice of 3 times of volumes again, each 1 hour, filter out ethyl acetate, rotary evaporation obtains containing the epothilone B crude extract to doing.
4) add 70 liters of culture medium in the 100L fermentation tank, medium component is potato starch 35g/L, glucose 8g/L, analysis for soybean powder 2g/L, yeast extract 20g/L etc.121 ℃ of sterilization 20min.Change strain over to, fermentation condition is the same, behind the cultivation 72h, adds the AB-8 macroporous adsorbent resin of cumulative volume 8%.Gather in the crops after cultivating 150h.
Thick cloth filtering fermentating liquid obtains adsorbent A B-8.Behind water infiltration, the ethyl acetate that adds 4 times of volumes again extracts three times, and each 0.5 hour, filter out ethyl acetate, rotary evaporation obtains containing the epothilone B crude extract to doing.
Embodiment three: the separation and purification of epothilone B
The crude extract that contains epothilone B obtains purity through two step separation and purification and is higher than 95% epothilone B.
The first step: sephadex lh-20 column chromatography
After extract used dissolve with methanol, the centrifuging and taking supernatant added to polydextran gel Sephadex LH-20 capital.Mobile phase: methanol, flow rate of mobile phase are 10cm/h.Through HPLC efficient liquid phase chromatographic analysis (filler: Kromasil C
185 μ m, size: 4.6mm * 150mm.Mobile phase: methanol: water=7: 3.Detect wavelength: UV230nm).Merge and contain the epothilone B fraction, carry out next step purification.
Second step: C18 reversed phase chromatography separation
Fraction after Sephadex LH-20 separates is further purified by C18 reversed phase chromatography chromatographic column.Mobile phase: methanol: water=70: 30, detect wavelength: UV230.As shown in Figure 1.Purified epothilone B purity analysis such as accompanying drawing 2.The 1HNMR of the epothilone B of purification and 13CNMR such as table 1, consistent with bibliographical information.
Embodiment four: epothilone B is to the effect of imatinib mesylate drug resistance K562 cell inhibiting
Imatinib mesylate drug resistance K562 cell is constantly to form with inducing under the imatinib mesylate effect from the K562 cell, and the drug resistance multiple is 75 times.
The epothilone B that in imatinib mesylate drug resistance K562 cell culture fluid (10% calf serum PRMI-1640 culture fluid), adds various dose, 37 ℃, 5%CO
2Cultivate after 72 hours in the incubator, add 20 μ l MTT (tetramethyl azo azoles salt) solution, continued to hatch 4 hours, inhale and remove cell suspension, add 120 μ l DMSO dissolution precipitation things, microplate reader 570nm measures its optical density value down.Each sample concentration is three holes, and positive control is vincristine (n=3).Ebomycin A B is to imatinib mesylate drug resistance K562 cell inhibiting effect such as accompanying drawing 3.
Embodiment five: the epothilone B preparation
Epothilone B is mixed with 5mg/ml solution after dissolving with PEG400, and injection is made in packing after the aseptic filtration.Time spent injects or intravenous drip with normal saline dilution posterior vein.
Epothilone B is mixed with 10mg/ml solution after dissolving with Macrogol 600, and the lyophilized formulations of every bottled amount 10mg is made in packing after the aseptic filtration, lyophilizing.Time spent injects or intravenous drip with water for injection or normal saline dilution posterior vein.
List of references
1.Altmann?KH.Recent?developments?in?the?chemical?biology?of?epothilones.Curr?Pharm?Des.2005；11(13)：1595-613.
2.Bollag?DM.Epothilones：novel?microtubule-stabilising?agents.Expert?Opin?Investig?Drugs.1997Jul；6(7)：867-73.
3.Gerth?K，Bedorf?N，Hofle?G?et?al.，Epothilons?A?and?B：antifungal?and?cytotoxic?compoundsfrom?Sorangium?cellulosum(Myxobacteria).Production，physico-chemical?and?biologicalproperties.J?Antibiot(Tokyo).1996Jun；49(6)：560-3.
4.Mukai?M，Che?XF，Furukawa?T，Sumizawa?Tet?al.，Reversal?of?the?resistance?to?STI571?inhuman?chronic?myelogenous?leukemia?K562?cells.Cancer?Sci.2003Jun；94(6)：557-63.
Claims (1)
1. epothilone B is used for the treatment of purposes in the medicine of the drug-fast chronic granulocytic leukemia of imatinib mesylate in preparation.
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| CN2009102593725A CN101732310B (en) | 2006-07-12 | 2006-07-12 | Application of epothilone B |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1489466A (en) * | 2001-01-25 | 2004-04-14 | ����˹�ж�-����˹˹������˾ | Parenteral dosage containing epothilones |
| CN1496256A (en) * | 2001-03-14 | 2004-05-12 | ����˹�ж�-����˹˹������˾ | Combination of EPOTHILONE analogs and chemotherapeutic agents for treatment of proliferative diseases |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1489466A (en) * | 2001-01-25 | 2004-04-14 | ����˹�ж�-����˹˹������˾ | Parenteral dosage containing epothilones |
| CN1496256A (en) * | 2001-03-14 | 2004-05-12 | ����˹�ж�-����˹˹������˾ | Combination of EPOTHILONE analogs and chemotherapeutic agents for treatment of proliferative diseases |
Non-Patent Citations (3)
| Title |
|---|
| Mukai et al.Reversal of the resistance to STI571 in human chronic myelogenous leukemia K562 cells.《cancer sci》.2003,第94卷(第6期),557-563. * |
| O.Polgar et al.ABC transporters in the balance: is there a role in multidrug resistance?.《Biochemical Society Transactions》.2005,第33卷241-245. * |
| T.O"Reilly et al.Patupilone (epothilone B, EPO906) and imatinib (STI571, Glivec) in combination display enhanced antitumour activity in vivo against experimental rat C6 glioma.《Cancer Chemother Pharmacol》.2005,第55卷307-317. * |
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Effective date of registration: 20151030 Address after: 410329 Liuyang County, Hunan biological pharmaceutical industrial park, Central Garden Road Patentee after: Hunan Dinuo Pharmaceutical Co., Ltd. Address before: 410329 Liuyang County, Hunan biological pharmaceutical industrial park, Central Garden Road Patentee before: Hunan Dinuo Pharmaceutical Co., Ltd. Patentee before: Emission and Radiation Medical Research Institute |
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Address after: 410005 Kangping Road, Liuyang economic and Technological Development Zone, Changsha, Hunan, 165 Patentee after: Hunan Dino pharmaceutical Limited by Share Ltd Address before: 410329 Huan Yuan East Road, Liuyang biological medicine industrial park, Hunan Patentee before: Hunan Dinuo Pharmaceutical Co., Ltd. |
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