CN100387706C - Epothilone -a microtubule stabilizer for treating tumor and blood vessel restenosis - Google Patents
Epothilone -a microtubule stabilizer for treating tumor and blood vessel restenosis Download PDFInfo
- Publication number
- CN100387706C CN100387706C CNB2004100566542A CN200410056654A CN100387706C CN 100387706 C CN100387706 C CN 100387706C CN B2004100566542 A CNB2004100566542 A CN B2004100566542A CN 200410056654 A CN200410056654 A CN 200410056654A CN 100387706 C CN100387706 C CN 100387706C
- Authority
- CN
- China
- Prior art keywords
- epothilone
- cancer
- esperamicin
- trans
- pharmaceutical composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a new bacterial strain S. Cellulosum BGS4 obtained through a directional filtration, which takes Epothilones D as the main metabolites, instead of producing Epothilones A and B; and it is stored in 'Ordinary microbial center of the Chinese microbial strain conservation management committee' on August 11,2004, whose preservation number is CGMCCNO.1206. The invention further relates to a compound trans- 9 ,10 -dehydrogenation Epothilones D, which is a new type of Epothilones derivatives produced by taking Epothilones D as a starting material. The invention also relates to a drug combination of a Epothilones compound in the preparation of anti-proliferative diseases, and the application in a coated stents that can be implanted into the body.
Description
Invention field
The present invention relates to a kind ofly obtain by directed screening, is main metabolites and do not produce the new bacterial strain S.cellulosum BGS4 of Epothilones A and B with the epothilone d.And adopt epothilone d as starting raw material, and produce and make novel esperamicin derivatives, trans-9,10-dehydrogenation epothilone d; The present invention also further relates to the application of esperamicin in the pharmaceutical composition of the anti-proliferative disease of preparation, and the application of esperamicin compounds in coating implantable intravital support.
Background technology
Esperamicin is 16 membered macrolide compounds.They at first by G.Hofle and colleague from slime bacteria Sorangium cellulosum separation and Extraction come out, main component is Epothilones A (EpothiloneA) and B (Epothilone B) (structural formula is as follows), their ratios with 2: 1 in this bacterium produce, and has antimycotic activity (referring to K.Gerth etc., 1996, J.Antibiotics 49:560-563 and German patent DE 4138042).Epothilones A and B were considered to the novel naturally occurring microtubule depolymerization inhibitor of a class afterwards.Although structurally and taxol (Taxol) and inequality, but they are having the similar mechanism of action (Hofle aspect microtubule polymerization and microtubule stabilizer, G, Deng, Angew.Chem.Int.Ed.Engl.35:1567-1569,1996 and Bollag, D.M., Deng, 1995.Cancer Res 55,2325-2333).In the medicine with cytotoxic activity of existing treatment tumour, taxol is not only very important clinically, and commercial also very successful.Therefore esperamicin is thought to continue widely can become the another carcinostatic agent of cell toxicant efficiently after the taxol.
Epothilone C (Epothilone C) and D (Epothilone D) (structural formula is as follows), promptly be respectively 12 of Epothilones A and B, 13-deoxidation-counterpart, in the esperamicin of the natural generation of research at present, epothilone d demonstrates minimum toxicity (Chou etc., Proc Natl Acad Sci USA95,15798 (1998)), and to the greatest treatment efficacy of multiple cancer and other undesired cellular proliferative disorders (referring to Harris etc., 1999, Soc.Chim.Ther.25:187).
R＝H，epothilone A R＝H，epothilone C
R＝CH
3，epothilone B R＝CH
3，epothilone D
Yet in the fermented product of natural bacterial strain S.cellulosum (So ce 90), epothilone d only exists as the trace ingredients in the esperamicin compounding mixture.And, must have the epothilone d of the fermentation process production capacity of less expensive, just has actual application and exploitation value in fact in order to satisfy the needs of clinical treatment.On the other hand, if can obtain a large amount of epothilone ds, then can utilize it to produce active better esperamicin derivatives.
Gene in the esperamicin biosynthesis gene family is carried out clonal analysis, and the heterogenous expression of esperamicin studied, derived following biosynthetic pathway: at first, by the synthetic Epothilone C of esperamicin polyketide synthase (PKSs) and D (producing) with 2: 1 ratios as intermediate product; Then, epoK gene product-Cytochrome P450 oxydase acts on Epothilone C and D, and forms epoxidation product Epothilones A and B respectively.Although in S.cellulosum, Epothilone C and D are synthesized out earlier, and its output is well below Epothilones A and B.Because far below the epoxidation counterpart of esperamicin, so the output of Epothilone C and D becomes the important factor of effective exploitation on toxicity for the Epothilone C of acyclic oxidised form and D.At present, the zymotechnique that lacks a High-efficient Production epothilone d has become and this compounds is developed to one of better anticarcinogen or other therapeutical agent hinders greatly.
Summary of the invention
The object of the present invention is to provide a kind of can mass production be the new bacterial strain Sorangium cellulosum BGS4 of main metabolites with the epothilone d, and be preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number CGMCC NO.1206 on August 11st, 2004.。
Another object of the present invention is to provide a kind of new esperamicin derivatives, trans-9,10-dehydrogenation epothilone d, it is a kind of esperamicin derivatives with better inhibition of cell proliferation performance.
Another purpose of the present invention is to provide the pharmaceutical composition of a kind of esperamicin at the anti-proliferative disease of preparation, the freeze-dried powder preparation that relates in particular to epothilone compounds is used for the treatment of the proliferative disease as cancer and other non-cancer class at the oral or parenteral form of administration of preparation; And the application of epothilone compounds in the pharmaceutical composition that coats implantable intravital support.
The present invention adopts the orthomutation screening method, and having obtained a kind of is the new bacterial strain S.cellulosum BGS4 of main metabolites with the epothilone d.The new bacterial strain that obtains is owing to lack the EpoK oxidase function, and under the identical situation of other conditions, such bacterial strain only produces Epothilone C and D as primary product, and does not produce Epothilones A and B.This dissociant can obtain by the gene mutagenesis and the directed screening (product evaluation) of one or many.Equally also can obtain the mutagenic strain of the output of epothilone d much larger than Epothilone C.The acquisition of bacterial strain S.cellulosum BGS4 and the fermenting process of producing epothilone d with this bacterial strain have been described among the embodiment.
New esperamicin derivatives provided by the invention, trans-9,10-dehydrogenation epothilone d, it is that starting raw material makes by chemical conversion with the epothilone d, shows the performance of better inhibition of cell proliferation.
Another purpose of the present invention is to provide the pharmaceutical composition of a kind of esperamicin at the anti-proliferative disease of preparation, and the application in the pharmaceutical composition that coats implantable intravital support.Esperamicin and the esperamicin mentioned afterwards are meant any esperamicin or esperamicin derivatives as mentioned herein, especially refer to epothilone d and trans-9,10-dehydrogenation epothilone d and the compound that is associated thereof.
The microtubule stabilizer esperamicin can be used for treating different types of proliferative disease, and the proliferative disease as cancer and other non-cancer class comprises colon tumor, genito-urinary system tumour, epidermis tumour, lung tumors, breast tumor and liver and gall tumour.Described proliferative disease includes but not limited to following various diseases: the cancer of head and neck and liver and courage portion; Mammary cancer, ovarian cancer, lung cancer, the cancer of the brain, intestinal cancer, leukemia, malignant tumour, and lymphoma.Can repeat this methods of treatment, to effect a radical cure cancer or to prevent the metastasis of cancer.
Esperamicin also can suppress vasculogenesis, thereby influences the growth of cancer.Utilize this character to treat to cancer with Cancer-Related disease.This character also can be used for other treatment of diseases to the angiogenesis inhibitor sensitivity, and such disease includes but not limited to the blind disease relevant with retinal vessel, sacroiliitis, especially inflammatory sacroiliitis, multiple sclerosis, vascular restenosis and psoriasis etc.On the other hand, to can be used for treating with the cell hyperplasia be the non-cancer class disease of feature to esperamicin.
Apoptosis can be induced or suppress to esperamicin, this be one to normal development and the very important normal cell death process of physiological equilibrium.The change of apoptosis signal transduction approach can cause the generation of many different human diseasess.
The typical method of modern treatment noumenal tumour is to take multiple means: surgical excision, radiotherapy and chemotherapy etc.These all are the most general therapeutic modalities, and they also unite use usually.
Chemotherapy need be to patient's dosed cells cytotoxic drug.Use this class medicament to be because they have optionally cytotoxic activity, they exceed many times to the effect of the effect comparison healthy tissues of tumor tissues.If on purpose the medicine of capacity is delivered directly to predetermined position, in the case, most medicine all has therapeutic action and has only the medicine of small portion to bring side effect.
Pharmaceutical composition of the present invention comprises at least a esperamicin or esperamicin precursor medicine (prodrug) and a kind of pharmaceutically acceptable carrier or vehicle, and it can be by the parenteral administration or/and cancer be treated in the mode administration of oral dosage form.
Multiple drug resistance makes the conventional chemotherapy medicine inoperative to patient, and this drug-fast generation is because medicine is discharged the extracellular by P-glycoprotein.In treatment cancer and other proliferative disease, esperamicin can use separately, also can unite use with other cancer therapy drug and treatment means; The esperamicin composition is in the drug effect that is used to strengthen other chemotherapeutics, and control multiple drug resistance aspect is very useful.
Esperamicin can with other chemotherapeutic together, be used for combination therapy cancer and other proliferative disease, in particular for the assisting therapy and/or the Synergistic treatment of cancer, give the esperamicin of effective dose and the combination medicament that at least a other cancer therapy drug forms for the Mammals (as the mankind) of this kind of needs treatment.For example, can can be selected from metabolic antagonist with other chemotherapeutic that esperamicin one is used from combination therapy, as Fluracil and strong pool (Gemcitabine), perhaps be selected from alkylating agent such as carboplatin (Carboplatin) and cis-platinum (Cisplatin), perhaps be selected from signal conduction depressant drug such as Iressa, Herceptin and geldanamycins and their derivative.Combination therapy can be adopted administering mode successively, just earlier uses second kind of medicament then with first kind of pharmaceutical treatment, perhaps two kinds of modes that medicament uses in treatment simultaneously.
The invention provides the methods of treatment that the anticarcinogen of Taxan (taxane) class is had the patient of resistance.Those of ordinary skill in the art can know the effective dose of epothilone compounds by inference.Typical doses to the mankind is about 0.05-200mg/kg/ days.This dosage uses with the single dose form usually, but also can develop oral preparation and administered parenterally, uses with multiple doses.
Epothilone compounds is difficult to form preparation, and reason is that their solubleness in water medium is lower, and in the aqueous solution less stable.The invention provides the application of esperamicin in the pharmaceutical composition of the anti-proliferative disease of preparation, further relate to the freeze-dried powder preparation of this pharmaceutical composition.Esperamicin is made freeze-dried, and adds carrier again before using.Carrier can be solvent or dispersion medium, water for example, ethanol, glycerol, polyethylene glycol (PEG), or its suitable mixture, and plant wet goods.More satisfactory solvent is an alcohol, alcohol/water mixtures, polyethylene glycol 300.
The invention discloses the preparation method of the esperamicin preparation of administered parenterally, wherein epothilone d begins to be dissolved in volume and accounts in the mixture of 50% the trimethyl carbinol aqueous solution freeze-drying under optimized conditions then at least.During use, the freeze-dried Cremophor that at first uses, propyleneglycoles and alcoholic acid mixture dissolve again, use newborn acidifying woods Ge Shi (Ringer ' s) reagent to be diluted to suitable administration concentration then.In addition, this freeze-dried powder preparation also can further be prepared into other injecting medicine-feeding form, as powder injection.
This freeze-dried powder preparation can further be prepared into oral administered dosage form.The oral medicine that contains this active compound can be made any normally used oral dosage form, comprises tablet, capsule, buccal tablets, oral suspension or solution, and combines with pharmaceutically acceptable neutralization buffer agent.In present method the effect of buffer reagent be temporary transient in and the hydrochloric acid in gastric juice in the gastric juice and therefore reduce the degraded of esperamicin in patient's stomach.
Antacid, for example the oxyhydroxide of aluminium and magnesium, carbonate, silicate, phosphoric acid salt are used for and hydrochloric acid in gastric juice before and after can both and taking when taking esperamicin.The combination of esperamicin and neutralization buffer agent both can be used as solid oral dosage form or can be used as oral liquid.Before taking medicine, with suitable solvent or cosolvent dissolving esperamicin and buffer composition, to form solution or suspension.The biologically active substance utilization ratio that is had thus should be at least 20%, preferably is not less than 50%.
Esperamicin also can adopt the formulation method of other preparation drugs of low aqueous solubility such as taxol to make.For example, esperamicin can form emulsion (referring to document WO 00/71163 and US6458373B1) with vitamin-E (PEG1000-tocopherol acid succinate) derivative of vitamin-E and/or PEGization.Usually, earlier esperamicin is dissolved in ethanol, the vitamin e derivative that adds vitamin-E and/or PEGization then forms treatment agent solution.Ethanol is removed, formed the aqueous solution that precursor emulsion or adding contain tensio-active agent (stablizer) then and form precursor emulsion.When being used for intravenous injection, precursor emulsion can be disperseed to form uniform emulsion.Be used to do medicinal preparation for oral administration, precursor emulsion generally places gel capsule.Formulation method than strong water-soluble esperamicin medicine also can be by the esperamicin (PEGylated esperamicin) of direct PEGization; Or by the direct water-soluble amino acid polymer of covalent attachment of the hydroxyl on the esperamicin (as 7-OH), as polyglutamic acid (poly-l-glutamic acid).The molecular weight of polyglutamic acid is 25KD to 50KD preferably, the molecule of each polyglutamic acid can be in conjunction with 5 to 25 esperamicin molecules, to increase the solubleness of compound in water medium, and reach the purpose that medicine continues to discharge and strengthen the transformation period in vivo in vivo, also can improve the characteristic of aspects such as drug toxicity, curative effect and pharmacokinetics simultaneously.
The application of esperamicin pharmaceutical composition of the present invention also relates to this pharmaceutical composition on the other hand and is used for bag by implantable medical device, particularly wraps by artery or vein blood vessel support, as airbag inflation support (balloon-expandable stents).Esperamicin is used for covered stent (being similar to the device of line-webmaster) grows to stop the blood vessel cell, thereby prevents the obstruction again of vascular restenosis or artery.
Because the SMC phenotype hyperplasia after artery is impaired is similar to the growth of tumour cell, so cancer therapy drug can be used for preventing neointima (neointimal) smooth muscle cell (smooth muscle cell, growth SMC).The support that the anti-cell proliferant agent coats can discharge this inhibitor of enough concentration in long-time, with the growth that stops inner membrance and matrix and enter into inner chamber, thus the minimizing vascular restenosis.When being used for the treatment of restenosis, comparatively ideal situation is that this compound is used for the treatment of the restenosis that postangioplasty takes place.When being used for the treatment of the restenosis that postangioplasty takes place, esperamicin before art, in the art or postoperative use, or all can in conjunction with administration on above-mentioned different opportunitys.
On the one hand, the invention provides a kind of medicament delivery method, this method adopts minimum invasion technology, for example conduit inserts or through trochar, the specific position that the implantable device of a pharmaceutical pack quilt is placed into health is tumour or site of injury for example, makes medicine continue to be discharged into these specific positions.This device can comprise a carrier matrix and the coating on this matrix.Coating on it comprises that one deck contains one or more medicinal polymer materials at least.The effect of coating is to control the release of therapeutical agent.Medicinal polymer layer can comprise hydrophilic/hydrophobic polymer composition.The optional auto-polymerization carboxylic acid of polymerization coating, cellulosic polymer, gelatinum, urethane, polyacrylic ester, polyamine class, polyvinyl alcohol, polyethylene oxide, polyester, polyethylene glycerol and saccharan etc.Especially the coating that disperses polymerizations dispersion things such as thing (BAYHYDROL etc.) and ACRYLIC EMULSION dispersion agent to prepare from for example urethane and get can be used with compound of the present invention.
Medicine described herein can be used for being aggregated the support that thing coats.Include in the solution of medicine by support is immersed, or with the solution spraying adequate time that contains medicine (for example 5 minutes) to reach the amount of effective inhibition smooth muscle cell proliferation, with the inhibition vascular restenosis; With the support drying that coats, preferably reach dry then by air-dry for some time (for example 30 minutes).The support that medicine covers can be delivered to the coronary vasodilator place by foley's tube then.
The multilayer that the invention still further relates to pharmaceutical device coats, and it is both as biocompatible surface, also as the drug delivery instrument.Because these coatings can be used on the device to sensitive high processing temperatures, so they have superiority especially, and they also can be used as the transmission medium that therapeutical agent is transported to particular target in the health.At this on the one hand, the invention discloses the application of the liquid biocompatible coating of two compositions.Further, because bioactivity surface is connected with the polymer of covalent linkage with the first layer coating, so this coating layer for good and all is connected on the matrix.So bioactivity surface has persistent biocompatibility, and first coating layer allows therapeutical agent, and for example anti-SMC proliferant agent is released, its role is to by minimizing thrombosis or SMC hyperplasia, thus the restenosis of minimizing or prevention built-in pipe postoperative.
Adopt the multilayer coating composition in a kind of mode, the bioactive composition of tool can form a second layer bag quilt.The coating composition that provides can be given the matrix biocompatibility, and in vivo single or multiple lift is coated medicine and discharge and transfer out.This coating combination comprises dispersion liquid or the emulsion that contains the organic acid functional group polymkeric substance, and can with the excessive polymeric cross-linker of the organic acid functional group reaction of polymkeric substance.Medicine itself basically with polymkeric substance or with the linking agent Fails To Respond, only be dispersed in dispersion liquid or the emulsion.With the first layer coating drying, it is combined with stromal surface, wherein unreacted a polyfunctional crosslinking agent still is present in the crosslinked coating of the first layer.Applying second layer coating on the exsiccant the first layer coating afterwards, make and form successive bioactivity surface coating layer on the matrix, the second layer coats solution or the dispersion agent that can be liquid biologically active agent, or the function equivalent that contains organic acid functional group or metal-salt of chemically modified.
In the present invention, the character that is coated matrix is depended in the selection that is used for the polymkeric substance that contains organic acid functional group of the first layer coating in dispersion liquid or the emulsion.Polymkeric substance in the first layer coating composition can be homopolymer or multipolymer, ethylene monomer unit for example, polyurethane-epoxy resin and the arbitrary combination between them etc.But, the polymkeric substance in the first layer coating composition preferably includes the urethane that contains organic acid functional group, and polyacrylic ester, and polymethacrylate etc. particularly contain inner emulsifying agent, for example emulsification such as carboxylic acid or phosphoric acid Hdyrophilic polyurethane.The concentration of polymkeric substance is about 1～40wt% in the first layer coating.The first layer coating also comprises one or more multifunctional linking agents, and it can react with organic acid functional group.The preferential multifunctional linking agent of selecting comprises polyfunctional aziridine, and multifunctional charing diimine.In the present invention, other spendable linking agents comprise the preparation of the commodity NeoCryl CX100 by name of for example Zeneca resins sale.The concentration range of linking agent accounts for 0.5～20wt% of solids component in the first layer coating composition, and ideal drying temperature scope is about 15 ℃～35 ℃.In many cases, drying at room temperature 12 hours is just passable.Therapeutical agent of the present invention should be epothilone d or its function equivalent, comprises thing of the same clan, analogue and derivative thereof etc.In this embodiment, epothilone d or its function equivalent are dispersed in first coating layer.In case enter in the body, epothilone d controllably is discharged into the environment on every side gradually from first coating layer, grows in stromal surface as prevention of SMC inhibitor or minimizing neointima.This medicine covered stent can be transferred in the coronary vasodilator by foley's tube.
The biologically active substance of the second layer coating that the present invention uses can be the saccharan that contains organic acid functional group.The top-priority saccharan bioactive agents of the present invention is glycosaminoglycan (GAGs).GAGs has a large amount of negative charges, is fit to very much by excessive, unreacted functional group on the linking agent, is connected with the first layer coating with the form of covalent linkage.The preferably preferred heparin of GAGs bioactive agents or its function equivalent.After the first and second coating layer dryings, the unreacted multifunctional linking agent by surplus makes and forms covalent linkage between the organic acid functional group of biologically active agent and the polymkeric substance and connect.See the narration of embodiment for details.
Description of drawings
Fig. 1 is the generation of esperamicin among the S.cellulosum BGS4
Fig. 2 A is the HPLC color atlas of the product that obtains from BGS4 culture XAD elutriant among the embodiment 2.
Fig. 2 B is the mass spectrum of epothilone d.
Fig. 3 is an epothilone d
1H-NMR figure
Fig. 4 epothilone d and taxol are gone into the curative effect of nonsmall-cell lung cancer model to nude mice
Embodiment
By the method for gene mutagenesis and directed screening, obtain containing the mutant strain BGS4 of the S.cellulosum of an inactivation epoK gene.
Used S.cellulosum bacterial strain So ce 90 is that (GermanCollection of Microorganisms) obtains from German microbial preservation center, and preserving number is DMS6773.
The ampoule that will contain the DMS6773 bacterial strain is transferred in the erlenmeyer flask of the 50-ml that 10ml G52 substratum is housed, and at 30 ℃, the 180rpm agitation condition was cultivated 6 days down.The culture of 5ml is transferred in the 250-ml erlenmeyer flask of 50ml G52 substratum, at 30 ℃, the 180rpm agitation condition was cultivated 3 days down.The G52 substratum comprises 0.2% yeast extract, and less salt (BioSpringer, Maison Alfort, France); 0.1%MgSO
4.7H
2O; 0.1%CaCl
2.2H
2O; 0.2% defatted soyflour Soyamine50T (Lucas Meyer, Hamburg, Germany); 0.8% potato starch Noredux A-150 (Blattmann, Waedenswil, Switzerland); 0.2% anhydroglucose; The EDTA-Fe of 1ml/L (III)-Na salt (8g/L); Regulate pH value to 7.4 (percentage ratio wherein is the quality percentage composition, down together) with KOH.
The above-mentioned culture of 0.1ml is taped against on the several petri dishs that contain nutrient agar S42.The S42 substratum comprises 0.05% tryptone, 0.15%MgSO
4.7H
2O, 1.2%HEPES and 1.2% agar are regulated pH value to 7.4 with KOH.Behind the autoclaving, in every liter of S42 substratum, add following material: 10ml filtration sterilization 10%CaCl
2.2H
2O, the 6%K of 1ml
2HPO
4, the EDTA-Fe/ sodium sulfite solution of 1ml, 40% filtration sterilization of 8ml glucose, the Spent substratum that the 5% sulfuric acid amine of 10ml and the autoclaving of 35ml are crossed.Then this plate is exposed to the open air under UV light (the maximal rays scope is 250-300nm), at 500 μ watts/cm
2Under keep 90-120 second.Cultivated 6-9 days at 30 ℃ then, up to obtaining the big independent bacterium colony of 1-2mm.By fan-shaped plastic hoop, 100-150 bacterium colony moved on to (each plate 4 part) on the petri dish that contains S42 agar respectively, cultivated 7 days at 30 ℃.About 1cm that will on agar, grow up to plastic hoop
2Colony lift is in the 50-ml erlenmeyer flask that contains 10ml G52 substratum, and at 30 ℃, the 180rpm agitation condition was cultivated 7 days down.The culture of 5ml is transferred in the 250-ml erlenmeyer flask that contains 50ml G52 substratum, and at 30 ℃, the 180rpm agitation condition was cultivated 3 days down.The culture of 10ml is transferred in the 50ml 23B3 substratum, and at 30 ℃, the 180rpm agitation condition was cultivated 7 days down.The 23B3 substratum comprises: 0.2% glucose; 2% potato starch Noredux A-150 (Blattmann, Waedenswil, Switzerland) yeast extract; 1.6% defatted soyflour Soyamine 50T (Lucas Meyer, Hamburg, Germany); The EDTA-Fe of 1ml/L (III)-Na salt (8g/L); 0.5%HEPES (Fluka, Buchs, Switzerland); 2% polystyrene XAD-16 (Rohm ﹠amp; Haas); Regulate pH value to 7.8 with NaOH.
Epothilone C that uses following method to measure to form in the culture and the culture of D:50ml filter with nylon mesh (150 μ m aperture), are retained in XAD-16 on the sieve with the less water flushing, put in the lump in the 50ml centrifuge tube with filter membrane then.The 10ml Virahol is added in the test tube.The test tube that shakes good seal under 180rpm reaches 1 hour, with the esperamicin of dissolving with resin-bonded.Next, the liquid of centrifugation 1.5ml is transferred to the supernatant liquor of about 0.8ml in the HPLC test tube with transfer pipet.The HPLC analytical results of sample is as described below.HPLC analyzes to have determined to contain the most high-load Epothilone C and D in which culture.On the part plate of above-mentioned corresponding bacterium colony (plate is deposited at 4 ℃ simultaneously), will about 1cm with plastic hoop
2The cell transfer in agar zone is in 10ml G52 substratum, and at 30 ℃, the 180rpm agitation condition was cultivated 7 days down.The culture of 5ml is transferred in the 50ml G52 substratum in the 250-ml erlenmeyer flask, and at 30 ℃, the 180rpm agitation condition was cultivated 3 days down.Transgenation has for the first time obtained to contain the bacterial strain of epoK sudden change, and has only produced Epothilone C and D.Further screening and sudden change can obtain the bacterial strain that epothilone d output improves, and just compare with Epothilone C, and the output of epothilone d is higher.The second, in the mutagenesis for the third time, method is identical with the aforesaid mutagenesis first time, selects best bacterium colony in the mutagenesis of back gene for use in one step of back.
Bacterial strain called after BGS4 with a screen mutation, and be preserved on August 11st, 2004 that " it is primary product and do not produce Epothilones A and B that China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; its preserving number is CGMCCNO.1206, this bacterial strain only produce Epothilone C and D.Fig. 1 has disclosed the generation of Epothilone C and D among the BGS4.
The cultivation of bacterial strain BGS4 and morphology are described: this bacterial strain can be at Mierocrystalline cellulose as sole carbon source, and KNO
3As the culture condition growth down of only nitrogen source, for example, growth (0.1%KNO on the filter paper on the ST2 mineral salts agar
30.1%MgSO
4.7H
2O; 0.1%CaCl
2.2H2O; 0.1%K
2HPO
40.01%MnSO
4.7H2O; 0.02%FeCl
30.002% yeast extract; The standard trace element solution; 1% agar).On this substratum, formed dark little red-brown is to black-brown sporophore (fruitingbodies).This growth bacillus (vegetative bacilli) has the typical shape of Sorangium bacterial classification (suitable tightness under phase microscope, can be observed secretly, has the terminal columned bacillus of wide circle, and average out to 3-6um is long and 1um is thick).
Main medium 1B12: yam starch Noredux A-150 (Blattmann, Waedenswil, Swizerland) 2%, defatted soyflour Soyamine 50T (Lucas Meyer, Hamburg, Germany) 1.1%, EDTA-Fe (III)-Na salt (8g/l) 1ml regulates PH to 7.8 with KOH.
Keep inoculum: the frozen cell storehouse of taking out 1ml from liquid nitrogen is inoculated among the G52 of 10ml, and at 30 ℃, 180rpm cultivated 3 days under the condition of 25mm displacement.The 5ml inoculum is added among the G52 of 45ml growth 3 days, then the culture of this 50ml was added among the G52 of 450ml growth 3 days.Later every 3-4 days, the 50ml inoculum is inoculated among the G52 of 450ml with 10% inoculum size, to carry out the successive seed culture.
Inoculum in the middle of the 20L: will contain 18L G52 culture medium and inoculate with seed culture fluid before the 2L in the fermentor tank of 30L, and continue to cultivate 3-4 days, condition is: 30 ℃, and 250rpm, 0.5L air/L/ minute, overvoltage was 0.5bars, does not control pH value.(Tegosipon, Goldschidt AG Essen), form to prevent foam to add 10ml silicone resin defoamer.
The main fermenting culture of 100L: sterilize inoculum in the middle of the inoculation 15L behind the 1B12 substratum of 100L fermentor tank adding 60L and the 2%XAD-16.Fermentation culture 6-7 days, condition was: 30 ℃, and 200rpm, 0.5L air/L/ minute, overvoltage was 0.5bars, uses H
2SO
4The control pH value is 7.6+/-0.5.
Product analysis: from fermentor tank, take out 50ml and contain polystyrene resin AmberliteXAD-16 (Rohm+Haas, Frankfurt, sample Germany).Nylon mesh with 150 μ m is filtered resin, after the less water washing, filtrate is merged in the Nunc pipe of 15ml.Adding 10ml methyl alcohol also at room temperature shook 30 minutes.Getting the supernatant liquor of 2ml after centrifugal is used for HPLC and analyzes.The sample injection is by (Inertsil in the guard column (guard column) of 4.6 * 10mm; ODS-3; 5 μ m); then with the gradient of from 35% to 100% acetonitrile; with the flow velocity of 1ml/min, under UV250nm, pass through long separator column (4.6 * 150mm, an Inertsil in 10 minutes; ODS-3,5 μ m).Epothilone d wash-out in the time of 9.9 minutes comes out, Epothilone C wash-out in the time of 9.1 minutes come out (and Epothilones A and B should be respectively when 6.5 minutes and 7.3 minutes wash-out come out).
From the 100L fermenting culture, separate meta-bolites: collect XAD with strainer.After washing with water, add 30L methyl alcohol and stir 30 minutes wash-outs.By the vaporizer concentrate eluant and remove methyl alcohol, with the water of EtOAc extracting remainder 3 times.Extract is concentrated into dried in rotatory evaporator, is dissolved in then in the 500ml methyl alcohol, with folding strainer elimination insolubles.Add solution in the Sephadex LH20 post and use methanol-eluted fractions.To contain carrying of metabolite heats up in a steamer partial concentration and is dissolved in once more to dry doubling in 50% the methyl alcohol, and be loaded in (55 * 4.8cm on the C18 reverse-phase chromatographic column, Bakerbond, 40 μ m), this chromatographic column is crossed (loading the flow velocity average out to 100ml/ minute) in advance by the methanol solution balance of 3 volumes 50%.And use 0.5L 50% successively, and 1.1L 55%, and 1.3L 60%, and the methyl alcohol of 0.5L 65% (flow velocity is 100mL/ minute) wash-out loads post.Cut is monitored at 250nm with the UV monitor.Product library is diluted to 40%, squeeze into pump on the C18 reverse-phase chromatographic column of 70ml (9 * 10cm), flow velocity average out to 360ml/ minute.With the methanol wash of 0.1L 40%, and load post with the ethanol elution of 350ml 100%.Elutriant is evaporated to dried with rotatory evaporator.(4) resuspension solid in 10ml acetone is removed insolubles with No. 2 filter paper filterings of Whatman.In acetone filtrate, add the 0.2g decolorizing carbon.Stir this mixture and reach 1 hour, use No. 50 filter paper filterings of Whatman then.Merging filtrate and rotary evaporation are to dry.The crystallization condition of epothilone d cut is: under good stirring, with the dry thing of the methyl alcohol resuspension of 15ml 100%, and slowly add 7.7ml water (reaching capacity at 66% o'clock) in beaker.Adding a small amount of (1mg) crystal seed in case of necessity forms to promote crystalline.
Fig. 2 A is the HPLC color atlas from the product of BGS4 culture XAD elutriant acquisition. Fig. 2 B is the mass spectrum of epothilone d. epothilone d
1The data of H NMR (400MHz) are seen Fig. 3, with Bruker DRX400 spectrometer CDCl under 300K of QNP zaxis gradient detection head equipment
3Record under the solution environmental.CDCl
3The chemical transformation of solution is meant
1δ 7.26 in the H spectrum.Epothilone d: HRESITOFMS m/z; C
27H
42NO
5S calculated value: 492.2778; [M+H]
+492.2775.
I is known by epothilone d degraded production methyl ketone.The Baeyer-Villiger oxidation has produced acetic ester II, can produce aldehyde III again after its process hydrolysis and the oxidation.Form undersaturated aldehyde IV by known method, it can be converted into allyl acetic acid ester V subsequently.Alkylated reaction by propenyl acetic ester V and vinyl copper VI, then by lactone cyclisation (macrolactonization) reaction, prepare novel compound 9,10-dehydrogenation epothilone d (VIII), wherein, this vinyl copper VI makes from corresponding ethene iodine (reaction scheme 3).
9,10-dehydrogenation epothilone d: HRESITOFMS m/z; C
27H
40NO
5S calculated value: 490.2622; [M+H]
+490.2631.
1H-NMR select signal: delta=5.59 (ddd, H-10), 5.54 (dd, H-9), 5.18 (ddd, H-13), 2.98 (dd, H-11a), 2.61 (dd, H-11b), 2.53 (m, H-8), 1.23 (d, 3H-25).
The preparation of freeze-dried powder preparation: the 9.86g epothilone d is wetted and be partially dissolved in and be cooled to 5 ℃ in advance, as 9: 1 (V: in trimethyl carbinol V) and the mixed solvent of water of 600ml of USP injection liquid.After drug powder was moistening fully, adding was chilled to 5 ℃ 1: 9 the trimethyl carbinol of the 600ml that is used for injection and the mixed solvent of water in advance again, and ratio is 1: 1 a mixture solution to the end, is dissolved under the lucifuge condition and carries out.The solution of above-mentioned formation under-16 ℃ through rapid freeze-drying in 48 hours (preferred about 200 millitorrs), then under 15 ℃ of high vacuum through 48 hours further dry (preferred 150 millitorrs), freeze-dried being packaged under aseptic condition in the 30ml lucifuge bottle contained the medicine of 50mg in every bottle.
The administered parenterally formulation, preparation as powder injection: during use, freeze-dried usefulness is encapsulated in the Cremophor EL of the 5.5ml in the different bottles, and the mixed solution of propyleneglycoles and dehydrated alcohol USP (20: 30: 50 (volume ratio)) dissolves again, so that last drug concentrations reaches 10mg/ml.Dissolve by rotating bottle gently, before intravenous injection, add 19ml breast acidifying Ringer ' s reagent (the every 100ml that is used to inject by every milliliter of medicine, LR comprises Sodium Chloride USP 0.6g, Sodium.alpha.-hydroxypropionate 0.31g, Repone K USP 0.03g, and Calcium dichloride dihydrate USP 0.02g), it is 0.5mg/ml that the drug solns dilution is reached ultimate density.Drug solns after this dilution can be by the dosage of transfusion input setting in to six hour.
This medicine can by oral, through intravenous injection, or two kinds of approach merge administrations.Prepare oral liquid with solvent and represented another kind oral dosage form easily.The available PEG of a suitable compound oral liquid (about 2mg/ml, intensity): ethanol: (1M, mixed solvent PH8.0) (55: 15: 30) (volume ratio) prepares and gets phosphate-buffered salt.
Preparation than highly water-soluble esperamicin prodrug: by the water-soluble polyamino acid polymer of the direct covalent attachment of the hydroxyl on the esperamicin, as polyglutamic acid (poly-l-glutamic acid).0.35g polyglutamic acid (molecular weight is 34K) is soluble in water.With 0.2M HCl pH value of solution is transferred to 2.Collecting precipitation, and use distill water dialysis, freeze-drying then obtains the polyglutamic acid of 0.29g.(75mg, repeating unit FW 170 add the epothilone d of 20mg, the dicyclohexyl carbodiimide of 15mg and the dimethyl aminopyridine of trace in DMF 0.44mmol) (1.5ml) solution at polyglutamic acid.Reaction was at room temperature carried out 4 hours or was judged finishing of reaction by thin-layer chromatography.Reaction mixture is poured in the chloroform, collecting precipitation, and vacuum-drying obtains the polyglutamic acid link coupled epothilone d of about 65mg.Change the weight ratio of epothilone d and polyglutamic acid in the reaction, can obtain containing the polymerization conjugate of different concns epothilone d.The molecule of each polyglutamic acid can be in conjunction with 5 to 25 esperamicin molecules.The esperamicin prodrug can add the aqueous solution that contains tensio-active agent (stablizer) and form precursor solution and be used for intravenous injection.Be used to do medicinal preparation for oral administration, the esperamicin prodrug can generally place gel capsule.
Other epothilone compounds or derivatives thereof all can adopt with above-mentioned similar step and make various oral or injection types.
The composition of embodiment 5 multilayer covered stents
Under agitation condition, following composition is added in the glass beaker successively,, make the first layer coating: NeoRez R981,280ml up to mixing fully; 0.5%Fluorad FC-129 storage solutions (in the water of 100ml dilute make 1ml Fluora FC-129), 10ml; 34%NH
4OH, 4ml; NeoCryl CX100,20ml; The storage liquid of 20% epothilone d in N-methyl-pyrrolidone (NMP), 20ml.NeoRez R981 (from Zeneca Resins) is a polyester based, the hydrophilic urethane of ester matter that contains carboxylic acid functional, it is stable by triethylamine, has 32% solids content under 25 ℃, and the pH value is 7.5-9.0, wherein also comprises the 5.3%NMP as cosolvent.The NeoCryl CX100 (from Zeneca Resins) that adds is as linking agent.Adding Fluorad FC-129 (from 3M) as levelling agent. ammonium hydroxide is used for the pH value of regulator solution.Second layer coating is to get by following method preparation: the Vitrum AB that adds appropriate amount continues to stir several hrs in the water of 300ml, obtains 1.2% heparin solution (Abbott).
Evenly spray stainless steel stent with the first layer coating composition, coat, then air drying 30 minutes.Repeat above-mentioned steps up to reaching necessary coating thickness.Spray second coating composition in dried first cover surface, spend the night at air drying.Support after will coating was then placed 3 hours in 50 ℃ vacuum.The coating layer that obtains has the epothilone d of controllable release, and the heparin that has covalent linkage to connect from the teeth outwards.
External antitumour activity: external antitumour activity is tested by tool multiple drug resistance (MDR) and medicaments insensitive cancer cells are used epothilone d.The ability of this compound anticancer growth is measured by sulphur rhodamine B (SRB) test (referring to Skehan etc., Natl.Cancer Inst.82:1107-1112 (1990)), and calculates half-inhibition concentration (IC50).Being determined in the different tumor cell line of kind more than 30 of external antitumour activity carried out.The source of these cells comprises the neck tumour, liver and gall tumour, mammary cancer, ovarian cancer, lung cancer, the cancer of the brain, skin carcinoma, prostate cancer, leukemia and lymphoma etc.Wherein 12 tumor cell lines have MDR characteristic (seeing the following form).All less than 100nM, and the overwhelming majority shows it not only to general non-resistant tumors cell, and the tumour cell of multi-drug resistant is had powerful antibiosis long-term job less than 10nM epothilone d to the IC50 of all these cells.This and taxol have formed distinct contrast.Taxol is to the tumor cell line of tool multi-drug resistant show activity not, and this has shown that epothilone d is better than taxol.Experimental result also shows 9, and 10-dehydrogenation epothilone d is the epothilone d derivative with better inhibition of cell proliferation performance.
External, it is that A549 and H460 produce synergistic cytotoxicity to non-small cell lung cancer cell that use is united in epothilone d and strong pool, make this unite to use to have produced than both add and stronger cell killing effect.In order to observe this synergy, these two kinds of medicaments must be with specific order administration.Earlier handle cell, and then add strong pool, promptly can be observed this synergy with epothilone d.Epothilone d can also produce synergistic effect with some other anticarcinogen.
The vivo antitumor activity:
The anti-tumor in vivo activity test of epothilone d growth have human non--carry out in the nude mouse model of minicell (non-smallcell) lung cancer A549.The subcutaneous implantation of nude mice A549 cancer cells (0.2ml, 1 * 107/ mouse) 10 days, up to the tumour size near 20mm
3After, by the tail vein infuse every other day the injection epothilone d 5 days (Q2Dx5).Measure its curative effect by measuring tumor size.
As shown in Figure 4, when finish the course of treatment, show that epothilone d is effective especially to treating this tumour.And in contrast, taxol only demonstrates partial inhibition.This marked difference in the test adds the result that above-mentioned in-vitro cell growth inhibition test obtains, and has illustrated that epothilone d has better therapeutic than other traditional cancer therapy drugs.
The anti-tumor in vivo activity of epothilone d is also tested in growth has the nude mouse model of human rectum cancer HCT-15.Inject epothilone d every day by the tail vein, injected continuously 10 days.This model is to taxol tool resistance.The result shows that taxol to this tumour inefficacy, have only 3% inhibiting rate, and the inhibiting rate of epothilone d illustrates that up to 79% epothilone d not only has better curative effect really, and can overcome the resistance of tumour.
As can be seen from the above table, trans 9 for same four kinds of cancerous cell lines, 10-dehydrogenation epothilone d shows stronger tumor-suppression activity than epothilone d.
The mechanism of action of epothilone d
Its mechanism of action of tubulin polymerization test determination with cell levels (cell-based).In the test, under 37 ℃, the VSMC or NCI/ADR-RES (the being called for short ADR) cell that are incubated in the 35mm culture dish were handled 1 hour with each compound of 1 μ m.Do not contain the PBS washed cell twice of Ca and Mg with 2ml, at room temperature use lysate (20Mm Tris, PH6.8, the 1Mm MgCl of 300 μ L
2, 2mM EDTA, 1%Triton X-100 adds proteinase inhibitor) handle cell 5-10 minute and make lysis.Lysate is transferred in the Eppendorf pipe of 1.5ml.Under the room temperature, lysate centrifugal 12 minutes at 18000g.To contain the supernatant liquor of solubility or unpolymerized tubulin and contain the granular precipitate and separate of insoluble or polymeric tubulin, and transfer in the new pipe.Then with the lysate of the 300 μ L granular precipitation that suspends again.Carry out each sample of immunoblotting assay by SDS-PAGE with 'beta '-tubulin antibody, use the relative variation of 'beta '-tubulin amount on the NIH Image computer program analysis trace then.This variation is expressed in following table by P/S ratio.Wherein P represent polymerization tubulin, S represents unpolymerized tubulin.P/S ratio is big more, and then the ability of short tubulin polymerization is just strong more.Epothilone d is VSMC and the polymerization that has all promoted tubulin in MDR cancerous cell line NIH/ADR-RES in vascular smooth muscle cell.But taxol only works to the VSMC cell, and to almost not effect of MDR cancerous cell line NIH/ADR-RES.The result shows that epothilone d not only can be used for treating cancer (particularly MDR cancer), and can be used to prevent vascular restenosis by utilizing this mechanism of action.
The short tubulin polymerization effect in cell of epothilone d and taxol
| Cell | No medicine contrast | Epothilone d | Taxol |
| VSMC | 0.3 | 5 | 4 |
| NIH/ADR-RES | 0.3 | 6 | 0.5 |
Numeral in the table is the P/S value.
The external activity that suppresses vascular smooth muscle cell growth (VSMC):
VSMC cell human and muroid is handled with epothilone d, to measure the retarding effect of this compound to these cell growths.The size of its ability to function is expressed by the IC50 value.The IC50 value of the anti-human VSMC of epothilone d is 10nM, the IC50 value of anti-muroid VSMC is 15nM, and this has illustrated that epothilone d has very strong restraining effect to the growth of smooth muscle cell in the blood vessel.This result and we use epothilone d and derivative covered stent thereof in case the conception of hemostatic tube restenosis matches.
Claims (12)
1. the new bacterial strain Sorangium cellulosumBGS4 that production is main metabolites with the epothilone d is preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number CGMCC NO.1206.
2. novel epothilone compounds, this compound are trans-9,10-dehydrogenation epothilone d.
3. trans-9, the application of 10-dehydrogenation epothilone d in the pharmaceutical composition of the anti-proliferative disease of preparation.
4. trans-9, the application of 10-dehydrogenation epothilone d in the pharmaceutical composition of preparation treatment multi-drug resistant tumour.
5. trans-9, the application in the pharmaceutical composition of preparation treatment vascular restenosis of 10-dehydrogenation epothilone d or epothilone d.
6. trans-9,10-dehydrogenation epothilone d or the mould rope D in dust slope coat application in the pharmaceutical composition of implantable intravital support in preparation.
7. trans-9, the application in the agent of preparation treatment anticancer composite medicine of 10-dehydrogenation epothilone d and at least a other cancer therapy drug.
8. application according to claim 7 is characterized in that described other cancer therapy drug is selected from metabolic antagonist Fluracil and strong pool, alkylating agent carboplatin and cis-platinum, and signal conduction depressant drug Iressa, Herceptin and geldanamycins.
9. application according to claim 7 is characterized in that described proliferative disease comprises colon tumor, genito-urinary system tumour, epidermis tumour, lung tumors, breast tumor and liver and gall tumour.
10. application according to claim 3 is characterized in that described proliferative disease is an intestinal cancer, prostate cancer, lung cancer, mammary cancer, ovarian cancer, leukemia, lymphoma, the cancer of the neck tumour of epidermis and liver and courage portion.
11. application according to claim 3 is characterized in that described pharmaceutical composition is an oral administered dosage form.
12. application according to claim 3 is characterized in that described pharmaceutical composition is an injecting medicine-feeding form.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100566542A CN100387706C (en) | 2003-08-15 | 2004-08-13 | Epothilone -a microtubule stabilizer for treating tumor and blood vessel restenosis |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN03154314 | 2003-08-15 | ||
| CN03154314.6 | 2003-08-15 | ||
| CNB2004100566542A CN100387706C (en) | 2003-08-15 | 2004-08-13 | Epothilone -a microtubule stabilizer for treating tumor and blood vessel restenosis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1629283A CN1629283A (en) | 2005-06-22 |
| CN100387706C true CN100387706C (en) | 2008-05-14 |
Family
ID=34862429
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100566542A Active CN100387706C (en) | 2003-08-15 | 2004-08-13 | Epothilone -a microtubule stabilizer for treating tumor and blood vessel restenosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100387706C (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100384419C (en) * | 2005-12-02 | 2008-04-30 | 菏泽睿鹰制药集团有限公司 | Epothilone slow-release implanting composition and use |
| CN101519404B (en) | 2008-02-29 | 2016-01-20 | 唐莉 | 15 ring thiophene ketone derivatives and preparation method thereof and application |
| CN102101863B (en) | 2009-12-17 | 2014-10-15 | 唐莉 | Novel epoxy thiazone compounds and preparation method and application thereof |
| CN107041886A (en) | 2016-02-06 | 2017-08-15 | 北京华昊中天生物技术有限公司 | Decylization oxygen epothilone derivate preparation, the application for preparing and its treating tumour |
| CN107362439B (en) * | 2017-08-14 | 2020-09-01 | 乐普（北京）医疗器械股份有限公司 | Preparation method of drug-coated balloon catheter, drug-coated balloon catheter prepared by preparation method and application of drug-coated balloon catheter |
| CN109837225B (en) * | 2018-11-15 | 2022-04-19 | 南京晶汇瀚生物科技有限公司 | Epothilone D high-producing strain and application thereof in fermentation production of epothilone D |
| CN113969255A (en) * | 2020-07-24 | 2022-01-25 | 北京华昊中天生物医药股份有限公司 | Recombinant bacterium for producing de-epoxidized epothilone B and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1254715A (en) * | 1999-11-12 | 2000-05-31 | 中国科学院上海有机化学研究所 | Isoesperamicin and its synthesizing process and usage |
-
2004
- 2004-08-13 CN CNB2004100566542A patent/CN100387706C/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1254715A (en) * | 1999-11-12 | 2000-05-31 | 中国科学院上海有机化学研究所 | Isoesperamicin and its synthesizing process and usage |
Non-Patent Citations (4)
| Title |
|---|
| Total Synthesis of Epothilone B, Epothilone D, and CisandTrans 9,10-Dehydroepothilone D.. White, J. D.等.J. Am. Chem. Soc.,Vol.123 . 2001 |
| Total Synthesis of Epothilone B, Epothilone D, and CisandTrans 9,10-Dehydroepothilone D.. White, J. D.等.J. Am. Chem. Soc.,Vol.123 . 2001 * |
| Total Synthesis of Epothilone B, Epothilone D, andCisandTrans 9,10-Dehydroepothilone D.. J.Am.Chem.Soc.123. 2001 |
| Total Synthesis of Epothilone B, Epothilone D, andCisandTrans 9,10-Dehydroepothilone D.. J.Am.Chem.Soc.123. 2001 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1629283A (en) | 2005-06-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Altmann et al. | Anticancer drugs from nature—natural products as a unique source of new microtubule-stabilizing agents | |
| RU2268306C2 (en) | Enzymatic method for preparing epotilones, crystalline form of epotilone b and its using | |
| Cavallaro et al. | Synthesis, physico-chemical and biological characterization of a paclitaxel macromolecular prodrug | |
| CN100387706C (en) | Epothilone -a microtubule stabilizer for treating tumor and blood vessel restenosis | |
| CN104163823B (en) | A kind of camptothecine and Artesunate conjugate and preparation method thereof and application | |
| CN101747326B (en) | 18-macrolide epothilone compound and application thereof | |
| CN101160294A (en) | Anti-cancer and anti-microbial oxazolidinones and analogues | |
| CN103739616A (en) | Thiazolyl-containing rapamycin type derivative and application thereof | |
| Zafar et al. | New insights into the anticancer therapeutic potential of maytansine and its derivatives | |
| JP2005507880A (en) | Wortmannin analogs and methods of using them | |
| CN104478890A (en) | All-trans retinoic acid-camptothecin anticancer drug conjugate as well as preparation method and application thereof | |
| CN1686115A (en) | Indole-3-methanol and its dimer application in preparation of medicnie for preventing and treating bred blood vessel disease | |
| CN102336904B (en) | Multivalent polyglycol (PEG) modifier for camptothecin and derivatives thereof and application of multivalent PEG modifier | |
| CN104629036A (en) | Water-soluble triptolide prodrug with polyethylene glycol as carrier as well as preparation method and application thereof | |
| CN102558178A (en) | Tabersonine derivative, pharmaceutical compositions, preparing method and medical application thereof | |
| CN104327097A (en) | Triazole derivatives of rapamycin and application | |
| CN104530081B (en) | The azacyclo-derivant of rapamycin and purposes | |
| CN103183722B (en) | Glyoxalase I inhibitor, preparation method and medical application thereof | |
| CN101011579B (en) | Application of cytoplasm-targeting chitosan oligosaccharide-aliphatic acid grafting medicinal-vector colloidal cluster | |
| CN101463029B (en) | Taxane derivative, and preparation and use thereof | |
| CN111773377B (en) | Application of anidulafungin in preparation of antitumor drugs and antitumor drugs | |
| CN102210668B (en) | Application of cantharidin and derivants thereof in preparation of tumor chemotherapy sensitivity enhancing medicine | |
| CN101177425B (en) | Novel epothilones compound as well as preparation method and use thereof | |
| AU2010306279A1 (en) | Inclusion complex of deoxypodophyllotoxin of cyclodextrin, preparation method, use for treament of cancer thereof | |
| CN101198318A (en) | Antibiotique composition |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20090925 Address after: Beijing city Daxing District Demao Road 8, zip code: 100076 Co-patentee after: NORTH CHINA PHARMACEUTICAL Co.,Ltd. Patentee after: BEIJING BIOSTAR TECHNOLOGIES, Ltd. Address before: Beijing city Daxing District Demao Road 8, zip code: 100076 Patentee before: BEIJING BIOSTAR TECHNOLOGIES, Ltd. |
|
| CP03 | Change of name, title or address |
Address after: Room 310, floor 3, building 3, No. 88, Kechuang 6th Street, Beijing Economic and Technological Development Zone, Daxing District, Beijing 100176 Patentee after: Beijing Huahao Zhongtian biomedical Co.,Ltd. Patentee after: NORTH CHINA PHARMACEUTICAL Co.,Ltd. Address before: 100076 Beijing city Daxing District Demao Road No. 8 Patentee before: BEIJING BIOSTAR TECHNOLOGIES, Ltd. Patentee before: NORTH CHINA PHARMACEUTICAL Co.,Ltd. |
|
| CP03 | Change of name, title or address | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211125 Address after: Room 310, floor 3, building 3, No. 88, Kechuang 6th Street, Beijing Economic and Technological Development Zone, Daxing District, Beijing 100176 Patentee after: Beijing Huahao Zhongtian biomedical Co.,Ltd. Address before: Room 310, floor 3, building 3, No. 88, Kechuang 6th Street, Beijing Economic and Technological Development Zone, Daxing District, Beijing 100176 Patentee before: Beijing Huahao Zhongtian biomedical Co.,Ltd. Patentee before: NORTH CHINA PHARMACEUTICAL Co.,Ltd. |
|
| TR01 | Transfer of patent right |