Enzymatic method for preparing epotilones, crystalline form of epotilone b and its using

FIELD: biotechnology, microbiology, medicine, pharmacy.

SUBSTANCE: invention relates to a method for preparing and concentrating cytotoxic substances, in particular, epotilones and their using for preparing pharmaceutical compositions used in treatment of proliferative diseases. Method for concentrating epotilones involves addition of a complex-forming component taken among the group of cyclodextrins to an aqueous cultural medium used in growing the producer, - Sorangium cellulosum. Also, invention describes a cultural medium used for growing S. cellulosum that contains the abovementioned complex-forming component and a method for preparing epotilones by using this medium. Invention represents a method for separating prepared epotilones being primarily epotilones A and B by method of reverse column chromatography and a (lower)-alkylcyanide containing eluent. Invention describes crystalline form of epotilone B and its using in pharmaceutical composition for treatment of proliferative diseases and a method for treatment of these diseases. Invention provides preparing epotilones in the industrial scale.

EFFECT: improved preparing method.

16 cl, 6 tbl, 4 ex

 

The present invention relates to new biotechnological method of getting that can be used on an industrial scale to obtain epothilones primarily to method the concentration of these compounds in the culture medium, as well as to the new strain, intending to obtain these compounds by enzymatic. The invention also relates to new crystalline forms epothilones primarily epothilone In, which can be obtained using the proposed method, to their use for the preparation of pharmaceutical compositions to new pharmaceutical compositions comprising these novel crystalline forms, and/or to the use of these compounds for the treatment of proliferative diseases such as tumors, or for the preparation of pharmaceutical compositions that can be used for such treatment.

Background of invention

Among the known cytotoxic active substances for the treatment of tumors, plays an important role in stabilizing microtubules agent Taxol® (Paclitaxel; Bristol-Myers Squibb), which is in great demand in the market. However, Taxol has a number of disadvantages. In particular, one of the problems associated with its poor solubility in water. As a result of this Taxol® you must enter in the composition together with Cremophor® EL (p is litoxetine castor oil; BASF, Ludwigshafen, Germany). Cremophor® EL has serious side effects; for example, it causes allergic reactions, at least in one case even led to the death of the patient.

Although belonging to the class of taxane stabilizing microtubules anticancer agents described as "contributing, perhaps the most important contribution to the Arsenal of pharmaceutical agents to fight cancer in the last decade (see E.K. Rowinsky, Ann. rev. Med. 48, 353-374 (1997)), however, despite the commercial success of Taxol® these connections are really not yet considered a major achievement in cancer chemotherapy. Treatment with Taxol® associated with a variety of serious side effects, and several classes of primary solid tumors, namely, tumors of the colon and prostate carcinoma sensitive to this connection only to a small extent (Rowinsky E.K, see above). In addition, the effectiveness of Taxol can influence and even completely neutralize the mechanisms of acquired resistance based mostly on the overexpression of phosphoproteins, which act as so-called "pumps" for active substances, as well as resistance to many drugs, which is a consequence of the overexpression of transport glycoprotein for many drugs, i.e. P-glycoprotein is".

Epothilone a and b represent a new class of stabilizing microtubules cytotoxic active substances (see Gerth, K., and others, J.Antibiot. 49, 560-563 (1966)) of the formula I

where R means a hydrogen atom (epothilone A) or methyl (epothilone).

These compounds have the following advantages compared with Taxol®:

a) they have a better solubility in water, which simplifies the preparation of compositions based on them,

b) there is evidence that in experiments on cell cultures, they also show activity against cell proliferation in which the activity is associated with P-glycoprotein "suction pump" has developed resistance to many drugs, than due to resistance to treatment with other chemotherapeutic agents, including Taxol® (see Bolag D.M., and others, "Epothilones, a new class of mirotubule-stabililizing agents with Taxollike mechanism of action", Cancer Research 55, 2325-2333 (1995)), and

C) it was found that they show a very high efficiency in vitro against resistant to Taxol® line cell carcinoma of the ovary with modified β-tubulin (see Kowalski R.J., and others, J.Biol. Chem. 272(4), 2543-2541 (1997)).

Epothilone can be applied in the pharmaceutical industry, for example, for the treatment of tumors, similar to that described for Taxol (see for example, US 5641803, US 5496804, US 5565478).

However, that is s epothilone can be used for pharmaceutical purposes on a large scale, these connections need to obtain in adequate quantities.

To date, the literature describes the extraction of natural substances, primarily epothilones, from myxobacteria, mainly from cells of strain Sorangium cellulosum Soce90 (which is deposited under the number 6773 in the German collection of microorganisms and cell cultures, see WO 93/10121). To obtain a satisfactory concentration of natural substances in the culture medium, especially epothilone, and their subsequent extraction previously always added as adsorbate resin based on polystyrene, for example Amberlite XAD-1180 (firm Rohm&Haas, Frankfurt, Germany).

However, the disadvantage of this process is that if it is applied in large-scale production, many problems arise. So, for example, causes damage to the valves resin beads, there is a possibility of blockage of the tubes, and the apparatus may be subjected to high wear due to mechanical friction. The resin beads are porous and thus have a large area of the inner surface (about 825 m2/g resin). Another problem associated with sterilization as a prisoner in the resin air is not autoclaved. Thus, the addition of resins virtually eliminates large scale application of this process. On the other hand, without adding balls smo is s in the culture medium cannot be achieved satisfactorily concentration epothilones.

In the claimed invention was unexpectedly found a way to solve this dilemma, allowing you to reach a satisfactory concentration of natural substances using microorganisms, primarily mycobacteria that produce epothilone, such as epothilone a or b, in particular to achieve a satisfactory concentration epothilones a and b in the culture medium without the addition of resins, thereby allowing you to resolve the above technical problems associated with obtaining these compounds, especially epothilone, on an industrial scale.

Detailed description of the invention

One of the objects of the present invention is a method for concentrating epothilones primarily epothilone and/or, in particular epothilone In, in the culture medium in order to obtain these compounds in biotechnological scale, in this case the implementation of the method is to use microorganisms producing these compounds, primarily myxobacteria (as producers of natural substances), and medium was added complexing component, which is soluble in the culture medium.

Another object of the invention is a culture medium with an appropriate complexing component and microorganisms, primarily mixed bacteria, in particular of the genus Sorangium, which can be used for producerof is of epothilones, first of all epothilone a and/or B.

Another object of the invention is a method for epothilones primarily epothilone and/or In mainly two pure compounds, in particular epothilone, which differs in that epothilone receive through the preparation of culture medium for the biotechnological production of these compounds, which is intended for cultivation of microorganisms as producers of natural substances, primarily mycobacteria that produce these compounds, and in which the added complexing component, soluble in culture medium, followed by cleaning and, if necessary, by separation epothilones, for example epothilone and epothilone Century

The next object of the invention is a method of separation epothilones primarily epothilones a and b, characterized by the use of column chromatography with reversed phase and eluent comprising (ness.) alkylene.

Another object of the invention is obtained by mutagenesis strain Sorangium cellulosum that in otherwise identical conditions produces a greater number epothilones than strain Sorangium cellulosum Soce90.

The object of the invention are also new crystalline forms epothilone Century

General concepts that are used above and below in the description have the following meanings./p>

If the above and below in the description reference is made to literature, they are included in the description to the extent that this is necessary.

Console "(ness.)" always means that the radical contains up to 7 carbon atoms, in particular up to 4 carbon atoms and may be branched or unbranched. (Ness.)the alkyl may be, for example, unbranched, or mono - or multiply branched and is for example, methyl, ethyl, propyl, such as the isopropyl or n-propyl, butyl, such as isobutyl, sec-butyl, tert-butyl or n-butyl, or also of pencil, such as amyl or n-pentyl.

The culture medium for the biotechnological production of epothilones with producing these compounds by microorganisms as producers of natural substances, primarily with mixed bacteria, mainly represents the environment that is intended for cultivation relevant (naturally occurring or obtained by cultivation or in part, by genetic modification of microorganisms, especially strains of mycobacteria that can produce these compounds, in particular myxobacteria of the genus Sorangium, preferably (in particular for the production epothilone) microorganism type Sorangium cellulosum Soce90 (see WO 93/10121), or obtained from him or from parts of mexab is Cheri biomaterial (e.g., using mutagenesis or recombinant technology gene), primarily appropriately obtained strain, in particular strain, designated as VSE/10, or its mutants, and which, in addition to water preferably contains other conventional and suitable components of cultural media, such as biopolymers, sugar, amino acids, salts, nucleic acids, vitamins, antibiotics, synthetic chemicals, growth factors, extracts of biological materials, such as yeast or extracts of other cells, soya flour, starch such as potato starch and/or micronutrients, such as iron ions in the form of complex, or an acceptable combination of all or some of these components and/or similar additives. Appropriate culture medium known to specialists in this field or can be prepared by known methods (see, for example, the compositions of the culture media described as examples in the present description or WO 93/10121). The preferred strain of this type of bacteria is a strain obtained by UV mutagenesis and selected on the basis of higher education epothilone and/or compared with the strain of Sorangium cellulosum Soce90 that is deposited at the DSM on the number 6773, first of all mutant VSE/10, which was deposited under the number DSM 11999 9 Febr what I 1998 in the German collection of microorganisms and cell cultures (Deutsche Sammlung von Mikroorganismen und Zeilkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124, Braunschweig, Germany).

Cultivation of strain VSE/10 and morphological description of the strain

The strain can grow on cellulose as the sole source of carbon and energy with the addition of potassium nitrate as the sole nitrogen source, such as filter paper covering the agar ST21 with mineral salts (0.1% of KNO3, of 0.1% MgSO4•7H2O, 0,1% CaCl2•2H2Oh, 0,1%2HPO40.01% of MnSO4•7H2O, 0.02% of FeCl3, 0,002% yeast extract, standard trace element solution, 1% agar). This environment is formed fruit bodies from dark reddish brown to blackish brown. They consist of small sporangiola (with a diameter of about 15-30 microns) and are present in the form dense formations and groups of different size.

Vegetative bacilli have the form typical of Sorangium (relatively compact, in the study using a phase-contrast microscope are dark, cylindrical sticks with wide rounded ends, with the average length of 3-6 microns and a width of 1 μm).

Epothilone are primarily epothilone a and/or b, but also other epothilone, for example epothilone C and D, described in applications WO 97/19086 and WO 9822461, epothilone E and F, described in WO 98/22461, and other epothilone, which can be obtained by using the appropriate microorganisms.

Water-soluble complexing component is a first vodorastvorimoe oligo - or polypeptide is derived, or, in particular, oligo - or polysaccharide derivative cyclic or spiral structure, which forms an intramolecular cavity, and due to their sufficiently hydrophobic properties has the ability to contact epothilone, primarily epothilones and/or epothilone Century. Especially preferred water-soluble complexing component selected from the group including cyclodextrins or (in particular) cyclodextrine derivatives and mixtures thereof.

Cyclodextrins are cyclic (α-1,4)-linked oligosaccharides α-D-glucopyranose with a relatively hydrophobic Central cavity and hydrophilic outer surface.

It should be noted, in particular, the following compounds (numbers in parentheses indicated the number of glucose residues per molecule): α-cyclodextrin (6), β-cyclodextrin (7), γ-cyclodextrin (8), δ-cyclodextrin (9), ε-cyclodextrin (10), ζ-cyclodextrin (11), η-cyclodextrin (12) and θ-cyclodextrin (13). The most preferred ablauts the δ -cyclodextrin and especially α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin or a mixture thereof.

Cyclodextrine derivatives primarily represent derivatives of the above cyclodextrins, especially α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin, in particular cyclodextrins, in which one or more up to all of the hydroxy groups (3 radical glucose) transformed with the formation of simple or complex ester. Ethers primarily represent simple alkalemia esters, preferably (ness.)alkalemia esters, such as methyl or ethyl ester and propyl or butyl ether, arylhydroxylamine esters, such as phenylketone(ness.)alkilany, preferably phenylhydroxylamine ether, hydroxyalkyl esters, in particular hydroxy(ness.)alkalemia esters, preferably 2-hydroxyethyloxy ether, hydroxypropionic ether such as 2-hydroxypropionic ether, or hydroxybutanoic ether such as 2-hydroxybutanoic ether, carboxyaniline esters, in particular carboxy(ness.) alkalemia esters, preferably carboxymethoxy or carboxitherapy ether, derivateservlet carboxyaniline esters, in particular derivationally carboxy(ness.)alkilany ether, in which derivational carboxypropyl is the tsya esterified or amidinophenoxy carboxypropyl (primarily aminocarbonyl, mono - or di(ness.)alkylaminocarbonyl, morpholino-, piperidino-, pyrrolidino or piperazinylcarbonyl, or alkoxycarbonyl), in particular (ness.)alkoxycarbonyl-(NISS)alkalemia esters, for example methoxycarbonylpropionyl ether or ethoxycarbonylphenyl ether, sulfoalkyl esters, in particular sulfo(ness.)alkalemia esters, preferably sulfobutyl ether, cyclodextrin, in which one or more Oh-groups etherification using a radical of the formula

-O-[Alk-O]n-H

where Alk denotes alkyl, preferably (ness.)alkyl, and n denotes an integer from 2 to 12, preferably 2 to 5, in particular 2 or 3, a cyclodextrin in which one or more Oh-groups amerivision using a radical of the formula

where R' denotes hydrogen atom, hydroxy, -O-(Alk-O)z-H, -O-(Alk(-R)-O)p-N or-O-(Alk(-R)-O)q-Alk-CO-Y, with Alk in all cases refer to alkyl, preferably (ness.)alkyl, m, n, p, q, and z represent an integer from 1 to 12, preferably 1 to 5, in particular 1 to 3, a Y means OR1or NR2R3where R1, R2and R3independently from each other represent a hydrogen atom or (ness.)alkyl, or R2and R3together with the attached nitrogen atom represent morpholino, piperidino, pyrrolidino or piperazine derivatives, or branched CEC shall dextrine, which contain ether groups or acetals formed with other molecules of sugars, preferably glucosyl-, diglycosyl(G2-β-cyclodextrin), maltose or demultiplication or N-acetylglucosaminyl, glucosaminyl-, N-acetylgalactosamine or galactosaminidase.

Esters primarily represent alkanolamine esters, in particular (ness.) alkanolamine esters, such as acetylene ethers of cyclodextrins.

It is also possible to use cyclodextrins, in which two or more of these different groups of ester groups and esters are present simultaneously.

Can also apply a mixture of two or more of these cyclodextrins and/or cyclodextrine derivatives.

Preference is given, in particular, α-, βor γ-cyclodextrins or their simple (ness.)alkilany esters, such as methyl-β-cyclodextrin or particularly preferably 2,6-di-O-methyl-β-cyclodextrin, or preferably their simple hydroxy(ness.)alkilany esters, such as 2-hydroxypropyl-α-, 2-hydroxypropyl-βor 2-hydroxypropyl-γ-cyclodextrin.

Cyclodextrins or cyclodextrine derivative added to the culture medium preferably at a concentration of 0.02 to 10, more preferably 0.05 to 5, especially PR is doctitle 0,1-4, such as 0.1-2% (wt./vol.).

Cyclodextrins or cyclodextrine derivatives are known or can be obtained by known methods (see, for example, US 3459731, US 4383992, US 4535152, US 4659696, EP 0094157, EP 0149197, EP 0197571, EP 0300526, EP 0320032, EP 0499322, EP 0503710, EP 0818469, WO 90/12035, WO 91/11200, WO 93/19061, WO 95/08993, WO 96/14090, GB 2189245, DE 3118218, DE 3317064 and listed in these publications links, which also apply to the synthesis of cyclodextrins or cyclodextrine derivatives, or see also .Loftsson and ..Brewster, Pharmaceutical Applications of Cyclodextrins: Drug Solubilization and Stabilisation, Journal of Pharmaceutical Science 85 (10): 1017-1025 (1996); R.A.Rajewski and V.J.Stella, Pharmaceutical Applications of Cyclodextrins: In vivo Drug Delivery, Journal of Pharmaceutical Science 85 (II): 1142-1168 (1996)).

In the description below, the process of the cleansing of the term "epothilone" mean aptilon, which can be obtained from the corresponding microorganism, preferably epothilone C, D, E, F, or particularly preferably epothilone And, most preferably epothilone Century, If not indicated otherwise, when using the term "epothilone" imply this concept in a broad sense, including individual epothilone or mixtures thereof.

The process epothilones carried out by standard methods, primarily the division of culture on liquid phase (centrifugate) and solid phase (cells) using filtration or centrifugation (laboratory centrifuge or Sep'a is ATOR). Part of the (main) epothilones under centrifugate or in the filtrate, then directly mixed with a synthetic resin, such as resin based on polystyrene as the matrix (hereinafter in the present description also denoted simply as polystyrene), such as Amberlite XAD-16 [the company Rohm & Haas GmbH, Frankfurt, Germany) or Diaion HP-20 [Resindion S.R.L., Mitsubishi Chemical Co., Milan] (preferably in a volume ratio of centrifugal:resin from about 10:1 to 100:1, more preferably about 50:1). After contact with the resin, continuing preferably from 0.25 to 50 hours, particularly preferably from 0.8 to 22 h, the resin is separated, for example by filtration or centrifugation. If necessary, after the adsorption of the resin is washed with a highly polar solvent, preferably water. Then carry out the desorption epothilone using a suitable solvent, preferably alcohol, in particular isopropanol. Phase solvent, preferably isopropanol phase, then removed from the solvent, preferably by the prior, simultaneous or subsequent addition of water, in particular in the evaporator with circulation, thus conducting, if necessary, the concentration, and the resulting aqueous phase is extracted with a solvent suitable for the formation of a second phase, such as ester, n is the sample (ness.)alkanol(ness.)alkanoate, as a rule, ethyl acetate or isopropylacetate. In this way epothilone transferred into the organic phase. Then the organic phase is concentrated to the desired extent, preferably to dryness, for example using a rotary evaporator.

Then perform additional processing using the following stages, where stage purification using chromatography with reversed phase elution nitrile is an offer in the invention stage and as a result is required, while other stages are optional:

molecular filtration (gel chromatography), for example on a column of such material, as Sephadex LH-20, Pharmacia, Uppsala, Sweden), using alcohol, such as methanol as eluent;

Department epothilones using chromatography with reversed phase after their dissolution in a suitable solvent and elution with a mixture of nitrile/water (required), preferably characterized in that the chromatography is performed on a column of such material, as a predominantly material RP-18, which carries the hydrocarbon chain, such as hydrocarbon chains containing 18 carbon atoms, and the eluent includes nitrile, preferably (ness.)alkynylaryl, in particular acetonitrile, primarily using a mixture of nitrile/water, preferably a mixture of acetonitrile/water, p is edocfile in the ratio of nitrile and water from about 1:99 to 99:1, first of all, from 1:9 to 9:1, for example from 2:8 to 7:3, in particular 3:7 or 4:6;

- single or multiple extraction residue (preferably after evaporation) in the two-phase system comprising water and a water-immiscible solvent, preferably an ester, in particular (ness.)alkyl(ness.)alkanoate, such as ethyl acetate or isopropylacetate;

- adsorption chromatography, in particular using a column of silica gel and elution with appropriate solvent or mixture of solvents, preferably a mixture of ester/hydrocarbon, for example (ness.)alkyl(ness.)alkanoate/S4-C10alkane, preferably ethyl or isopropylacetate/n-hexane, and the ratio of ester and hydrocarbon is preferably from 99:1 to 1:99, more preferably from 10:1 to 1:10, for example 4:1;

- dissolution residue, which can be obtained after concentration in an appropriate solvent, such as alcohol, for example methanol;

the mixture of activated carbon and its removal;

- recrystallization, for example from suitable solvents or mixtures of solvents, such as esters, mixtures of ester/hydrocarbon or alcohols, preferably ethyl or isopropylacetate:toluene in a ratio of from 1:10 to 10:1, preferably 2:3 (epothilone A) or methanol or utilize is at (epothilone);

between stages the resulting solution or suspension optionally concentrating and/or liquid and solid components are separated from each other, in particular by filtration or by centrifugation of the solutions/suspensions. Below, a more precise description can preferably be applied to the above-mentioned separate stages.

Processing and purification is preferably carried out as follows.

After collecting the culture is separated into the liquid phase (centrifugate) and solid phase (cells) by centrifugation (laboratory centrifuge or separator). The main part epothilones under centrifugate, then directly mixed with polystyrene, such as Amberlite XAD-16 [the company Rohm & Haas GmbH, Frankfurt, Germany) or Diaion HP-20 [Resindion S.R.L., Mitsubishi Chemical Co., Milan] (preferably in a volume ratio of centrifugal:resin from about 10:1 to 100:1, more preferably about 50:1)and stirred in the mixer. At this stage epothilone transfer of cyclodextrin on the resin. After contact with the resin, lasting approximately 1 h, the resin is separated by centrifugation or filtration. Adsorption epothilone the resin can also be carried out on a chromatographic column, introducing resin to the column and disperse centrifugal on the resin. After adsorption of the resin washed with water. Desorption epothilones of the resins is carried out using isopropanol. Then from isopropanole the phases are separated isopropanol, preferably by addition of water, in particular in the evaporator with circulation, and the resulting aqueous phase is extracted with ethyl acetate. Epothilone transferred from the aqueous phase in an ethyl acetate phase. Then an ethyl acetate extract was concentrated to dryness using a rotary evaporator. The initial concentration epothilones then carried out using molecular filtration (using, for example, Sephadex LH-20 [Pharmacia, Uppsala, Sweden] and methanol as eluent). Obtained after molecular filtration fraction, which correspond to the highest peaks (peak fractions), contain epothilone and ive shared content epothilones exceed 10%. Then carry out the separation of these peak fractions, which contain epothilone a and B in the mixture, the individual components using chromatography with reversed phase, for example RP-18 phase (phase, modified alkyl radicals contain 18 carbon atoms in the chain), using the appropriate eluent, preferably containing a nitrile, such as acetonitrile (allowing more efficient separation than when using, for example, alcohols such as methanol). Epothilone And eluted earlier than epothilone Century Containing epothilone In peak fractions can still include a small amount of the quality epothilone And, which can be removed more separation on RP-18. At the completion of the faction, including epothilone And crystallized directly from a mixture of ethyl acetate:toluene (2:3), and the fraction comprising epothilone - from methanol or ethyl acetate.

Preferred embodiments of the inventions

The invention preferably relates to a method for concentrating epothilones, preferably epothilone and/or, in particular epothilone, in culture medium, which is designed for biotechnological production of (-) connection (s) using a microorganism suitable for such receipt, preferably strain Sorangium, particularly preferably of the type strain Sorangium cellulosum Soce90, or its mutant, in particular strain, designated as VSE/10, and which contains water and other usual ingredients of cultural media, and medium was added cyclodextrin or cyclodextrine derivative or a mixture of two or more the amounts of these compounds, preferably one or more, preferably one, two or more cyclodextrins selected from the group including α-cyclodextrin (6), β-cyclodextrin (7), γ-cyclodextrin (8), δ-cyclodextrin (9), ε-cyclodextrin (10), ζ-cyclodextrin (11), η- cyclodextrin (12) and θ-cyclodextrin (13), particularly preferably the α -cyclodextrin, β-cyclodextrin or γ-cyclodextrin, or primarily cyclodextrine derivative or a mixture cyclodextrine derivatives selected from derivatives of cyclodextrin in which one or more up to all of the hydroxy groups are converted with the formation of simple Olkiluoto ether, preferably (ness.)Olkiluoto ether, such as methyl or ethyl ester and propyl or butyl ether, arylhydroxylamine ether, such as phenylketone(ness.)alkilany, preferably phenylhydroxylamine ether, hydroxyalkyl ether, in particular hydroxy(ness.)Olkiluoto ether, preferably 2-hydroxyethylamide ether, hydroxypropylamino ether such as 2-hydroxypropionic ether, or hydroxybutyrate ether such as 2-hydroxybutanoic ether, carboxycellulose ether, in particular carboxy(ness.)Olkiluoto ether, preferably carboxymethylamino or carboxycellulose ether, derivatizing carboxycellulose ether, in particular derivatizing carboxy(ness.)Olkiluoto ether, in which derivational carboxypropyl is aminocarbonyl, mono - or di-(ness.)alkylaminocarbonyl, morpholino-, piperidino-, pyrrolidino or piperazinylcarbonyl or alkoxycarbonyl, in particular (ness.)alkoxycarbonyl, such as before occhialino (ness.) alkoxycarbonyl -(ness.)alkilany ether, for example methoxycarbonylpropionyl ether or ethoxycarbonylphenyl ether, sulfoalkyl ether, in particular sulfo(ness.)Olkiluoto ether, preferably sulfobutyl ether cyclodextrin in which one or more Oh-groups etherification using a radical of the formula

-O-[Alk-O]nMr.

where Alk denotes an alkyl, preferably (ness.)alkyl, and n denotes an integer from 2 to 12, preferably 2 to 5, in particular 2 or 3, a cyclodextrin in which one or more Oh-groups etherification using a radical of the formula

where R' denotes hydrogen atom, hydroxy, -O-(Alk-O)z-H, -O-(Alk(-R)-O)p-N or-O-(Alk(-R)-O)q-Alk-CO-Y, with Alk in all cases refer to alkyl, preferably (ness.) alkyl, m, n, p, q, and z represent an integer from 1 to 12, preferably 1 to 5, in particular 1 to 3, a Y indicates OR1or NR2R3where R1, R2and R3independently from each other represent a hydrogen atom or (ness.)alkyl, or R2and R3together with the attached nitrogen atom represent morpholino, piperidino, pyrrolidino or piperazine derivatives, or a branched cyclodextrin, which contains ether group or acetals formed with other molecules of sugars, preferably glucosyl-, diglycosyl(G2-β-cyclodextrin, maltose or demultiplication or N-acetylglucosaminyl - glucosaminyl-, N-acetylgalactosamine or galactosaminidase, or (NISS)alkanoyl, such as complex acetyloxy ether cyclodextrin.

Especially preferred is a method in which the cyclodextrin and/or cyclodextrine derivative added to the culture medium at a concentration of 0.02 to 10, preferably 0.05 to 10, more preferably 0.05 to 5, particularly preferably 0.1 to 4, such as 0.1-2% (wt./vol.).

Especially preferred is the method according to any of the two preceding paragraphs, according to which cyclodextrine derivative selected from cyclodextrin, preferably β-cyclodextrin and hydroxy(ness.)alkylcyclohexane, preferably 2-hydroxypropyl-α-, -β- or -γ-cyclodextrin, or a mixture thereof, comprising one or more of these cyclodextrins, and the most preferred is 2-hydroxypropyl-β-cyclodextrin.

The invention also relates, in particular, to the culture medium, which comprises a cyclodextrin, cyclodextrine derivative or a mixture of two or more complexing component selected from the group comprising cyclodextrins and cyclodextrine derivatives, particularly preferably a cyclodextrin or cyclodextrine derivative is, specified in the third from the previous paragraph, in particular in the second of the previous paragraph, or a mixture comprising one or more of these compounds, and which is used for cultivation of the microorganism which can produce epothilone, preferably epothilone and/or epothilone, preferably a strain of the genus Sorangium, preferably strain Sorangium cellulosum, for example strain Soce90 or its derived mutant, in particular strain VSE/10.

Another object of the invention is a method for epothilone a and/or b, preferably in the form of two pure compounds, in particular epothilone, which differs in that epothilone share, for example, by centrifugation, solid and liquid phase (centrifugate), thus prepared culture medium with the purpose of the biotechnological production of these compounds, as described above, in the environment add komleksoobrazuyuschee component that is soluble in the culture medium, in particular cyclodextrin, cyclodextrine derivative or a mixture of two or more cyclodextrins and/or cyclodextrine derivatives, centrifugal mixed with the resin, preferably polystyrene, or expanded through a column Packed with such a resin, the resin if necessary, washed with water, epothilone(s) is desorbed from the resin with a polar process is Italia, preferably alcohol, primarily (ness.)alkanol, such as isopropanol, optionally concentrated by the prior, simultaneous or subsequent addition of water, add organic solvent which is not miscible with water, for example an ester such as ethyl acetate, and epothilone(s) are transferred into the organic phase, for example by stirring or mixing, the organic phase is optionally concentrated, epothilone obtained from organic solvent concentrate using molecular sieves for compounds with low molecular weight, and then the fractions containing epothilone, preferably epothilone a and/or b, is subjected to separation using a column with reversed phase preferably the elution eluent containing a nitrile, such as acetonitrile (or in another embodiment, the eluent containing alcohol, such as methanol), while epothilone a and b extracted separately, and if necessary, they can be further concentrated using recrystallization.

Another preferred object of the invention is a method of separation epothilones primarily epothilones a and b, which differs in that use chromatography on a column of reversed phase with an eluent containing (ness.)alkylene, and chromatography is performed on this is the material of the column, primarily on the material RP-18, which carries the hydrocarbon chain, such as hydrocarbon chains containing 18 carbon atoms, and used including nitrile eluent, which preferably is a (ness.)alkynylaryl, in particular acetonitrile, primarily a mixture of nitrile/water, preferably a mixture of acetonitrile/water, preferably in the ratio of nitrile and water from about 1:99 to 99:1, especially from 1:9 to 9:1, for example from 2:8 to 7:3, in particular 3:7 or 4:6. This separation can be performed after filtration through a molecular sieve or preferably directly using residue after adsorption epothilones of culture medium containing complexing component on the resin. The advantage of separation by elution solvent containing (ness.)alkylene compared with the use of alcohols, such as methanol, is more efficient separation epothilones a and B.

The invention preferably relates to a method for epothilones, which provides:

a) concentration epothilones primarily epothilone and/or, in particular epothilone In, in the culture medium for the biotechnological production of (-) connection(-I), which produces a microorganism suitable for such a obtain, first of all strain Sorangium, particularly preferably of the type strain Sorangium celluosum Soce90, or its mutant, in particular strain, designated as VSE/10, and which contains water and other usual ingredients of cultural media, and medium was added cyclodextrin or cyclodextrine derivative or a mixture of two or more of these compounds, preferably one or more, preferably one, two or more cyclodextrins selected from the group including α-cyclodextrin (6), β-cyclodextrin (7), γ-cyclodextrin (8), δ-cyclodextrin (9), ε-cyclodextrin (10), ζ-cyclodextrin (11), η-cyclodextrin (12) and θ-cyclodextrin (13), particularly preferably α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin, or primarily cyclodextrine derivative or a mixture cyclodextrine derivatives selected from derivatives of cyclodextrin in which one or more up to all of the hydroxy groups are converted with the formation of simple Olkiluoto ether, preferably (ness.)Olkiluoto, such as methyl or ethyl ester and propyl or butyl ether, arylhydroxylamine ether, such as phenylketone(ness.)alkilany, preferably phenylhydroxylamine ether, hydroxyalkyl ether, in particular hydroxy(NISS,)Olkiluoto ether, preferably 2-hydroxyethylamide ether, hydroxypropylamino EF is RA, such as 2-hydroxypropionic ether, or hydroxybutyrate ether such as 2-hydroxybutanoic ether, carboxycellulose ether, in particular carboxy(ness.)Olkiluoto ether, preferably carboxymethylamino or carboxycellulose ether, derivatizing carboxycellulose ether, in particular derivatizing carboxy(ness.)Olkiluoto ether, in which derivational carboxypropyl is aminocarbonyl, mono - or di-(ness.)alkylaminocarbonyl, morpholino-, piperidino-, pyrrolidino or piperazinylcarbonyl or alkoxycarbonyl, in particular (ness.)alkoxycarbonyl, such as preferably (ness.)alkoxycarbonyl-(NISS)alkilany ether, for example methoxycarbonylpropionyl ether or ethoxycarbonylphenyl ether, sulfoalkyl ether, in particular sulfo(ness.)Olkiluoto ether, preferably sulfobutyl ether cyclodextrin in which one or more Oh-groups etherification using a radical of the formula

-O-[Alk-O]nMr.

where Alk denotes an alkyl, preferably (ness.)alkyl, and n denotes an integer from 2 to 12, preferably 2 to 5, in particular 2 or 3, a cyclodextrin in which one or more Oh-groups etherification using a radical of the formula

where R' denotes hydrogen atom, hydroxy, -O-(Alk-O)sub> z-H, -O-(Alk(-R)-O)p-N or-O-(Alk(-R)-O)q-Alk-CO-Y, with Alk in all cases refer to alkyl, preferably (ness.)alkyl, m, n, p, q, and z represent an integer from 1 to 12, preferably 1 to 5, in particular 1 to 3, a Y indicates OR1or NR2R3where R1, R2and R3independently from each other represent a hydrogen atom or (ness.)alkyl, or R2and R3together with the attached nitrogen atom represent morpholino, piperidino, pyrrolidino or piperazine derivatives, or a branched cyclodextrin, which contains ether group or acetals formed with other molecules of sugars, preferably glucosyl-, diglycosyl(G2-β-cyclodextrin), maltose or demultiplication or N-acetylglucosaminyl, glucosaminyl-, N-acetylgalactosamine or galactosaminidase, or (NISS)alkanoyl, such as complex acetyloxy ether cyclodextrin, and

b) separation epothilones primarily epothilones a and b, which differs in that use chromatography on a column of reversed phase with an eluent containing (ness.)alkylene, and chromatography is performed on this material column, primarily on the material RP-18, which carries the hydrocarbon chain, such as hydrocarbon chains containing 18 carbon atoms, and used including nitrile eluent, preferably, the bottom is.)alkynylaryl, in particular acetonitrile, primarily a mixture of alkynylaryl/water, preferably a mixture of acetonitrile/water, preferably in the ratio of nitrile and water from about 1:99 to 99:1, especially from 1:9 to 9:1, for example from 2:8 to 7:3, in particular 3:7 or 4:6, then if necessary use an additional stage for pererabotki and cleanup.

The invention also relates, in particular, to a mutant derived from strain Sorangium cellulosum Soce90, first of all, the strain of Sorangium cellulosum, which can be obtained by mutagenesis, preferably using one or more stages of UV-induced mutagenesis (in particular with UV irradiation in the wavelength range from 200 to 400, especially from 250 to 300 nm) with subsequent selection after each stage of mutants with enhanced ability to produce epothilone (in particular, by means of which increases the concentration epothilones in culture medium), and this strain in identical in other respects the conditions produces a greater number epothilones primarily epothilone and/or than the strain Sorangium cellulosum Soce90, is first and foremost a strain Sorangium cellulosum VSE/10.

The invention preferably applies to the individual stages of the process described in the examples, or any combination, to the culture medium described in the present description, the crystal Faure the am and described the strain.

The invention also relates to new crystalline forms epothilone In, primarily crystalline form epothilone, described as a modification, and, in particular, to the form, described as a modification of A.

Crystalline forms primarily can be distinguished on the basis of their x-rays. Characterization of solid organic compounds preferably used radiographs obtained using a diffractometer and Cu-Kα1as the source of irradiation. X-rays are particularly preferred for determining the crystalline modification substances. With the aim to characterize the obtained crystalline modification a and crystalline modification In epothilone In measurements carried out in the interval of values of the angle (29) from 2° to 35° when using samples of the substance, which is maintained at room temperature.

The data thus obtained x-rays (angle of reflection and the intensity of the most important lines for crystalline modification (modification A) epothilone In the following table 1A:

Intensity
7,7very strong
10,6weak
13,6average
14.4Vaverage
15,5average
16,4weak
16,8weak
17,1weak
the 17.3weak
17,7weak

Intensity
18,5weak
20,7strong
of 21.2strong
of 21.9weak
22,4weak
23,3strong
25,9average
31,2weak
32,0average

The invention also relates, in particular, to a new crystalline form epothilone, which differs in that its melting temperature is more than 120°primarily between 120 and 128°With, in particular 124-125°C. Unexpectedly, it was found that this value is much higher than the values previously known from the literature. The invention preferably relates to a crystalline form epothilone In, which is characterized by the presence of x-rays characteristic of the crystalline forms A, and the melting point is use 120° With primarily 120-128°With, in particular 124-125°C.

The data thus obtained x-rays (angle of reflection and the intensity of the most important lines) of crystalline modification (modification) epothilone In the following table 1B:

Intensity
6,9very strong
8,0weak
8,3average
10,8strong
11,5average
12,4weak
13,1strong
15,5weak

Intensity
16,2weak
16,7average
18,1average
18,6average
20,4weak
20,9strong
21,3weak
a 21.5very poor
22,5average
24,2weak
25,1average

New the crystalline forms are especially stable, in particular crystalline form And is more thermodynamically stable, and they result suitable as active substances for solid forms of administration, for storage in a solid form or for storage in the form of intermediate products (which are particularly high stability), designed to receive solid or liquid forms of administration.

The invention also relates to the use of new crystalline forms, preferably crystalline form, especially the crystalline form A (all identified hereinafter as active ingredient), in the preparation of pharmaceutical compositions, novel pharmaceutical compositions which contain these new crystalline forms and/or their use for the treatment of proliferative diseases such as tumors. Further, in the context of the present description, if mentioned pharmaceutical preparations or compositions that contain or include active substance, in the case of liquid compositions or compositions that do not contain crystalline form, as such, always refer to and pharmaceuticals obtained with the use of crystalline forms (for example, solutions for infusion obtained with the use of crystalline form a or b epothilone), even if they are not already contain sootvetstvuyuschengo form (for example, with regard to the fact that they are in solution).

The invention also preferably relates to the use of new crystalline forms epothilone, preferably crystalline form, especially the crystalline form And, in the preparation of pharmaceutical compositions, characterized by the mixing of the new crystalline form epothilone In one or more media.

The invention also relates to a method of treating a warm-blooded animal suffering from a proliferative disease, characterized by the introduction epothilone B in a dose effective for the treatment of this disease, in the form of one of the new crystalline forms of warm-blooded animals which need such treatment, including, in particular, treatment with such compositions, which are prepared using one of the new crystalline forms, and/or the adoption of new crystalline forms epothilone In for such treatment.

For the preparation of pharmaceutical compositions, the active substance can be used, for example, so that the pharmaceutical composition contains an effective amount of active ingredient in combination or in a mixture with a sufficient quantity of one or more organic or inorganic, solid or liquid pharmaceutically acceptable carriers.

Izaberete is s also relates to pharmaceutical compositions, suitable for introduction warm-blooded animal, especially a human, for the treatment of proliferative diseases such as tumor, this composition contains the active substance in an amount necessary to treat the disease in combination with a pharmaceutically acceptable carrier.

The pharmaceutical compositions according to the invention is a composition suitable for enteral, especially nasal, rectal or oral, preferably parenteral, especially intramuscular or intravenous injection, warm-blooded animals, especially humans, and they contain an effective dose of the active substances individually, or in combination with a sufficient amount of pharmaceutically acceptable carrier. The dose of active ingredient depends on the type of warm-blooded animal, body weight, age and individual condition, specific pharmacokinetic characteristics, underlying disease and route of administration.

The pharmaceutical compositions contain from about 0,0001% to about 95%, preferably from 0.001% to 10% or 20%, or to about 90% active ingredient. The pharmaceutical compositions according to the invention can be, for example, in the form of standard dosage forms, for example in the form of ampoules, vials, suppositories, pills, tablets or capsules.

Pharmaceutical is the cue compositions of the present invention are prepared by known methods, for example, by conventional dissolution, freeze-drying, mixing, granulating or other processes.

Solutions of active ingredient, and also suspensions, in particular aqueous solutions or suspensions, when it is possible, for example in the case of lyophilised compositions that contain the active substance as such or in combination with a pharmaceutically acceptable carrier, for example mannitol, preferably used in the form of solutions or suspensions prepared just before the introduction. The pharmaceutical compositions can be sterile and/or contain excipients, such as preservatives, stabilizers, wetting or emulsifying agents, substances that increase solubility, salts for regulating osmotic pressure and/or buffers, and they can be prepared by known methods, for example by the usual processes of dissolution or lyophilization. These solutions or suspensions may contain substances which increase the viscosity, such as carboxymethylcellulose sodium, carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatin.

Suspensions contain oil as the oil component of the vegetable, synthetic or semi-synthetic oils, which are usually used for injection. The most preferred examples are, in particular, liquid esters of fatty the acids, which contain as the acid component of the fatty acid with a long chain consisting of 8-22, preferably 12-22 carbon atoms, for example lauric acid, tridesilon acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, arachidonic acid, beenbuy acid or corresponding unsaturated acids, for example oleic acid, elaidic acid, erucic acid, brussilov acid or linoleic acid, and optionally additives as preservatives, such as vitamin E, β-carotene or 3,5-di-tert-butyl-4-hydroxytoluene. The alcohol component of these fatty acid esters preferably has up to 6 carbon atoms and is a mono - or polysiloxanes, for example mono-, di - or trihydroxide, for example methanol, ethanol, propanol, butanol or pentanol, or its isomer, but preferably the glycol and glycerin. In particular, as examples can be mentioned the following esters of fatty acids: profilerita, isopropyl, "Labrafil M 2375" (polyoxyethyleneglycol, Gattefosse, Paris), "Miglyol 812" (triglyceride of saturated fatty acids with a chain length from 8 to 12 carbon atoms, Hills AG, Germany), but especially vegetable oils, such as cottonseed oil, almond oil, olive oil, castor oil, sesame is the second oil soybean oil and especially peanut butter.

Preparations for injection or infusion is prepared by conventional methods in a sterile environment; the same applies to the filling compositions vials or bubbles and closing containers.

The preferred solution for infusion, which contains the active ingredient and pharmaceutically acceptable organic solvent.

Pharmaceutically acceptable organic solvents that can be used in the composition according to the invention can be selected from such solvents, which are known to experts in this field. The solvent is preferably selected from alcohol, for example absolute ethanol, mixtures of ethanol with water, preferably 70%ethanol, polyethylene glycol 300, polyethylene glycol 400, polypropylenglycol and N-methylpyrrolidone, preferably of polypropyleneglycol or 70%ethanol.

The active substance is present in the composition in a concentration of from 0.001 to 100 mg/ml, preferably from about 0.05 to 5 mg/l or 5 to 50 mg/ml

Compositions of this type are easy to store in vials or ampoules. Typically, bubbles or ampoules made of glass, such as borosilicate glass. The vials or ampoules can be of any amount, which is usually used in this field. They preferably have a size large enough the La-fill composition is 0.5-5 ml

Before the introduction of the active substance to the patient the composition should be diluted with an aqueous medium suitable for intravenous administration.

Preferably the solution for infusion has the same or almost the same osmotic pressure as that of the shared water body. Thus, the aqueous medium preferably contains an agent to maintain isotonicity, which gives the solution for infusion is the same or almost the same osmotic pressure as that of the total water in the body.

Agent to maintain isotonicity can be selected from any known in this area substances, for example, can represent mannitol, dextrose, glucose or sodium chloride. Preferably the agent to maintain isotonicity is a glucose or sodium chloride. Agents to maintain isotonicity can be used in amounts which give the solution for infusion is the same or almost the same osmotic pressure as that of the shared water body. The exact necessary amount can be determined using conventional experiments, and they may depend on the composition of the solution for infusion and the type of agent to maintain isotonicity.

The concentration of the agent to maintain isotonicity in the aquatic environment depends on the type of each of the agents. When using glucose, it is oncentrate is preferably from 1 to 5% wt./about., more preferably 5% wt./about. When the agent is to maintain isotonicity is a sodium chloride, it is preferably used in quantities of up to 1% wt./about, preferably about 0.9% wt./about.

Solution for infusion may be diluted with an aqueous medium. The used amount of the aqueous medium is selected depending on the desired concentration of active substance in the solution for infusion. The solution for infusion is preferably produced by mixing the contents of the vial or ampoule, representing a concentrate for infusion (see above), the water environment, bringing with water environment up to 200-1000 ml Solution for infusion may contain other additives that are commonly used in compositions for intravenous administration. These additives include antioxidants.

Antioxidants can be used to protect the active substance from oxidative decomposition. Antioxidants can be selected from antioxidants, known to specialists in this field and is suitable for intravenous compositions. The amount of antioxidant can be determined using conventional experiments. Alternatively, antioxidants or in addition to anti-oxidant activity can be achieved by preventing contact of oxygen (air) solution for infusion. Typically, it is the goal of capacity, containing solution for infusion, can be treated with an inert gas such as nitrogen, which is a simple operation.

Solutions for infusion may be obtained by mixing the contents of a vial or ampoule with an aqueous medium, such as 5%glucose solution in water for injection (VDI) in a suitable container, such as container or vessel for infusion.

Capacity for solution for infusion can be selected from any conventional containers, materials which do not react with the specified solution for infusion. This can be used, in particular, glass containers, preferably made of borosilicate glass, although it is preferable to use plastic containers such as plastic bottles for infusion.

Plastic containers primarily can be a vessel of thermoplastic polymers. Plastic can optionally include additives, such as plasticizers, fillers, antioxidants, antistatic agents and other additives known in this field.

Plastics that are suitable according to the present invention must also be resistant to the high temperatures required for sterilization. The preferred plastic bottles for infusion are cylinders made of such well-known specialists in the field of plastics like PVC.

<> Can be applied capacity, the dimensions of which vary widely. When choosing the size of the tank first of all, consider the solubility epothilones in water environment, ease of handling and, if necessary, the ability of the vessel to storage. It is preferable to use the capacity, the capacity of which may be approximately 200-1000 ml solution for infusion.

With regard to their preferred for inclusion in compositions of properties, new crystalline forms epothilone In the invention are most suitable for simple and reproducible preparation of the solution for infusion. However, the new crystalline form of especially suitable for the preparation of pharmaceutical compositions, which contain the active substance in solid form, for example, compositions for oral administration.

Pharmaceutical compositions for oral administration can be obtained by combining the active substance with solid carriers, optionally granulating the resulting mixture and further processing the mixture, if necessary after adding acceptable adjuvants, with taking tablets, dragee cores or capsules. It is also possible to apply to them a coating of plastic substrates, which enable effective substance to diffuse or be released within the specified number is two.

Suitable pharmaceutically acceptable carriers are primarily fillers such as lactose, saccharose, mannitol or sorbitol, preparations based on cellulose and/or calcium phosphates, for example tricalcium phosphate or calcium bicarbonate, and binders, such as starches, for example corn, wheat, rice or potato starch, gelatin, tragakant, methylcellulose, hypromellose, carboxymethylcellulose sodium and/or polyvinylpyrrolidone, and/or, if necessary, leavening agents, such as the abovementioned starches, cross-linked vinylpyrrolidone, agar, alginic acid or its salts, such as sodium alginate. Adjuvants primarily represent improves the fluidity agents and oiling agents, such as silicates, talc, stearic acid or its salts, such as magnesium stearate or calcium, and/or polyethylene glycol. The dragee cores if necessary, put the appropriate resistant to the action of gastric juices coating, using, inter alia, concentrated solutions of sugars, hummurabi, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or using solutions to cover in acceptable organic solvents or in order to obtain resistant to the action of gastric juices coating solutions acceptable drugs C is lulzy, such as ethyl cellulose phthalate or phthalate of hydroxypropylmethylcellulose. Capsules are dry capsules consisting of gelatin or pectin and, if necessary, softener, such as glycerol or sorbitol. Dry capsules can contain the active substance in the form of granules, for example, fillers, such as lactose, binders, such as starches, and/or oiling agents, such as talc or magnesium stearate, and optionally stabilisers. Capsules, soft coated active substance may be present in soluble or dispersible form, to which is added oily adjuvants such as fatty oils, paraffin, or liquid propylene glycols, it also can be added stabilizers and/or anti-bacterial additives. In tablets or dragee coatings can be added dyes or pigments, for example to identify or distinguish different doses of active ingredient.

Use for the treatment of proliferative diseases one of the crystalline forms In the form And preferably carried out by introducing crystalline form (preferably the same as for the above application in the form of solution for infusion) warm-blooded animal, especially a human in a dose that can be defined at the level from 20%to 13%, preferably from 25 to 100% of the maximum tolerated dose (MTD), using standard methods, for example using a modified Fibonacci series, in which successively increasing doses of 100%, 67%, 50% and 40%, and then for all subsequent doses of 33%, and if necessary, enter another and a few extra doses in the range greater than the first dose, each dose is given after such time period, which allows the patient undergoing treatment, to recover after previous treatment, in particular after 1 week or more after the first treatment, preferably after 2-10 weeks, particularly preferably 3-6 weeks after each of the previous processing. In General, this treatment regimen, according to which a high dose is administered once, twice or several times with a sufficiently long intervals between injections, allowing to occur restoration, has the advantage compared with more frequent treatments with lower doses, because it allows more sparse and short-term hospitalization, and lets hope for a higher anticancer activity. Recommended for people dose epothilone In is preferably from 0.1 to 50 mg/m2, more preferably from 0.2 to 10 mg/m2.

Below the invention Provillus is recorded on the examples not limiting its scope.

Precautions: When working with epothilone due to their high toxicity if necessary, to use protection.

Used in the description 750-liter fermenter is a fermenter of treated steel, obtained from Alpha AG, Nidau, Switzerland.

Example 1: Obtaining strain VSE/10 by mutation and selection

Used strain is a mutant strain of SESS/10 (which is deposited under the number DSM 11999 in the German collection of microorganisms and cell cultures 9 February 1998), obtained from strain Sorangium cellulosum Soce90 as a result of mutation and selection, as described below. In liquid medium the mutant VSE/10 forms typical for the genus Sorangium sticks with rounded ends, length 3 to 6 μm and a width of about 1 μm. Strain Sorangium cellulosum Soce90 was obtained from the German collection of microorganisms and cell cultures under the number DSM 6773.

Obtaining mutant VSE/10 includes 3 stages mutations using UV irradiation and selection of individual colonies. This procedure involves the following stages.

Cultivation of ampoules

Cells DSM6773 of the vials are transferred into 10 ml of medium G52 in the Erlenmeyer flask 50 ml and incubated for 6 days in a mixer at a speed of 180 rpm and at 30°C. 5 ml of this culture is transferred into 50 ml of medium G52 (in which Abu Erlenmeyer volume of 200 ml) and incubated for 3 days in a stirrer at a speed of 180 rpm and at 30° C.

The first stage of obtaining mutations using UV and selection

Aliquots of 0.1 ml of the above culture were seeded in several Petri dishes containing agar medium S42. Then each Cup is treated with UV light (maximum wavelength of irradiation is 250-300 nm) for 90 or 120 s at 500 µw/cm2. Then Cup incubated for 7-9 days at 30°to obtain isolated colonies with a diameter of 1-2 mm and Then using a plastic loop of cells from each of 100-150 colonies were seeded separately in the sectors of Petri dishes containing agar S42 (4 sectors per Cup) and incubated for 7 days at 30°C. the Cells are grown on an area of about 1 cm2agar, transferred using a plastic loop in 10 ml of medium G52 in the Erlenmeyer flask 50 ml and incubated for 7 days in a stirrer at a rotation speed of 180 rpm and at 30°C. 5 ml of this culture is transferred into 50 ml of medium G52 (in the Erlenmeyer flask 200 ml) and incubated for 3 days in a stirrer at a speed of 180 rpm and at 30°C. 10 ml of this culture is transferred into 50 ml of medium US and incubated for 7 days in the mixer when the rotation speed of 180 rpm and at 30°C.

With the purpose of definition in this culture of quantities epothilone and epothilone use In the following process. 50 ml of the culture was filtered through a nylon sieve (pore size of 150 μm), keep the, which on the sieve polystyrene Amberlite XAD16 washed with a small amount of water and then placed together with the filter in the centrifuge tube of 50 ml volume (firm Falcon Labware, Becton Dickinson AG, Immengasse 7, 4056 Basel). In the tube with the filter, add 10 ml isopropanol (>99%). Then tightly closed and the tube is shaken for 1 h at 180 rpm for dissolution epothilone a and b, which are connected with resin (polystyrene), in isopropanol. Then 1.5 ml of the centrifuged liquid and about 0.8 ml of supernatant added with a pipette into a column for GHUR. GHUR-analysis of these samples is conducted according to the procedure described below in the section relating to the analysis of the product using GHUR. Using GHUR analysis determine which crop has the highest content epothilone Century From the above sector cups corresponding to this colony (cups at this time stored at 4°C)cells from agar surface of approximately 1 cm2transferred using a plastic loop in 10 ml of medium G52 in the Erlenmeyer flask 50 ml and incubated for 7 days in a stirrer at a rotation speed of 180 rpm and at 30°C. 5 ml of this culture is transferred into 50 ml of medium G52 (in the Erlenmeyer flask 200 ml) and incubated for 3 days in a stirrer at a speed of 180 rpm and at 30°C.

The second and third stages of the mutation using UV and selection

This procedure corresponds exactly to that described above for the first stage of obtaining mutations using UV, but for the second mutagenesis using the best of the colonies, C is annoy in the first stage of UV mutagenesis. For the third stage of mutagenesis, respectively, using the best of the colonies selected at the second stage UV mutagenesis. The best of the colonies after the third cycle stages of obtaining mutations using UV after selection of the strains obtained in respect of the increased production epothilone b corresponds to the mutant VSE/10.

Storing strain

10 ml of the 3-day culture in the environment G52 (50 ml medium in an Erlenmeyer flask of 200 ml, 30°C and 180 rpm) and transferred into 50 ml of medium B (in the Erlenmeyer flask 200 ml) and incubated for 3 days in a stirrer at a speed of 180 rpm and at 30°C. an Aliquot of this culture volume of 1 ml allocate in the most homogeneous form (before each selection culture shaken manually in the Erlenmeyer flask) with polystyrene Amberlite XAD16 (adsorbing polystyrene, the company Rohm & Haas, Frankfurt, Germany), and then bring in cryoprobes volume of 1 ml type Nunc (A/S Nunc, DK 4000, Roskilde, Denmark) and stored at -70°or in liquid nitrogen.

Cultivation of strains of these vials is carried out by heating them in air at room temperature, and then transfer the entire contents of cryoprobes in 10 ml of medium G52 in the Erlenmeyer flask 50 ml and incubation for 5-7 days in a stirrer at a speed of 180 rpm and at 30°C.

Environment

Wednesday G52:

yeast extract with a low salt concentration
(firm Springer, Maisons Alfort, France)2 g/l
MgSO4(7 H2Oh)1 g/l
CaCl2(2 H2Oh)1 g/l
defatted soy flour (firm Mucedola S.r.1, Settimo Milan, Italy)2 g/l
potato starch Noredux
(firm Blattmann, Wadenswil, Switzerland)8 g/l
anhydrous glucose2 g/l
Fe-add 8 g/l (product # 03625, firm
Fluka Chemie AG, Switzerland) pH value of 7.4
adjusted with KOH1 g/l

Sterilization: 20 minutes at 120°

Agar medium S42

Corresponds to the environment described in S.Jaoua and others, Plasmid 28, 157-165 (1992).

Wednesday V

glucose2 g/l
potato starch Noredux (firm Blattmann,
Wadenswil, Switzerland)20 g/l
defatted soy flour
(firm Mucedola S.r.L., Settimo Milan, Italy)16 g/l
Fe-add (product # 03625, the company luka,
Buchs, Switzerland)8 g/l
HEPES (company Fluka, Buchs, Switzerland)5 g/l
polystyrene XAD16 (firm Rohm and Haas)2 vol.%

divisioona water

the pH value adjusted to 7.8 with NaOH

Sterilization: 20 minutes at 120°

(HEPES denotes 4-(2-hydroxyethyl)piperazine-1-econsultancy acid)

Example 2: Cultivation with the aim of obtaining epothilones

Strain: Sorangium cellulosum Soce90 VSE/10 (example 1).

Storage of strains in liquid N2as described in example 1.

Environment

Pre-culture and sub-culture: Wednesday G52

Main culture: Wednesday 1V12.

Wednesday G52

yeast extract with a low salt concentration (firm BioSpringer, Maisons Alfort, France)2 g/l
MgSO4(7 H2Oh)1 g/l
CaCl2(2H2O)1 g/l
defatted soy flour Soyamin 50T (the company Lucas Meyer, Hamburg, Germany)2 g/l
potato starch Noredux A-150 (firm Blattmann, Wadenswil, Switzerland)8 g/l
anhydrous glucose2 g/l
Fe(III)-Na-salt etc (8 g/l)1 ml/l

the pH value of 7.4 correcti owano using KON

Sterilization: 20 minutes at 120°C.

Wednesday 1V12

potato starch Noredux A-150
(firm Blattmann, Wadenswil, Switzerland)20 g/l
defatted soy flour Soyamin 50T
(the company Lucas Meyer, Hamburg, Germany)11 g/l
Fe(III)-Na-salt etc (8 g/l)8 mg/l

the pH value adjusted to 7.8 with KOH

Sterilization: 20 minutes at 120°C.

Adding cyclodextrins and cyclodextrin derivatives

Different concentrations of cyclodextrins (company Fluka, Buchs, Switzerland, or Wacker Chemie, Munich, Germany) are sterilized separately and added to the environment 1V12 before sowing culture.

Cultivation

1 ml of suspension supernatant of strain Sorangium cellulosum Soce90 VSE/10 ampoules with liquid N2transferred into 10 ml of medium G52 (in the Erlenmeyer flask 50 ml) and incubated for 3 days in a mixer with offset 25 mm at a speed of 180 rpm and at 30°C. 5 ml of this culture is transferred into 45 ml of medium G52 (in the Erlenmeyer flask 200 ml) and incubated for 3 days in a mixer with offset 25 mm at a speed of 180 rpm and at 30°C. 50 ml of this culture were then added to 450 ml of medium G52 (in flask Erlenmeyer volume of 2 l) and incubated for 3 day is in the mixer with an offset of 50 mm at a speed of 180 rpm and at 30° C.

Maintaining culture

Culture is subcultured every 3-4 days by adding 50 ml culture to 450 ml of medium G52 (in the Erlenmeyer flask 2 l). All experiments and fermentation processes carried out, using as source supported this culture.

Experiments in the flask

(I) Pre-culture in shake flask

Using as source 500 ml supported culture, 1×450 ml of medium G52 seeded with 50 ml of supported culture and incubated for 4 days in a mixer with an offset of 50 mm at a speed of 180 rpm and at 30°C.

(II) the Main culture in shake flask

40 ml of medium 1V12 plus 5 g/l powder 4-morpholinepropanesulfonic acid (pug) (in the Erlenmeyer flask 200 ml) is mixed with 5 ml of 10-fold concentrated solution of cyclodextrin, seeded 10 ml of pre-culture and incubated for 5 days in a mixer with an offset of 50 mm at a speed of 180 rpm and at 30°C.

Fermentation

The process of fermentation is carried out in a volume of 10 l, 100 l and 500 l Fermentation at 20 l and 100 l serve as an intermediate stage of cultivation. While pre-crops and catch crops sown in the form of a supported culture in a concentration of 10 vol.%, the main culture is seeded with about 20. %intermediate culture. It is important to note, is that unlike the mix of cultures ingredients environments for fermentation expect in terms of a finite amount of culture, including the inoculate. For example, if you combine 18 l medium + 2 l inoculum, the substance is weighed at the rate of 20 l, however, is mixed with only 18 HP

Pre-culture in shake flask

Using as source 500 ml supported culture, 4×450 ml of medium G52 (in the Erlenmeyer flask 2 l) was inoculated with 50 ml of this culture and incubated for 4 days in a mixer with an offset of 50 mm at a speed of 180 rpm and at 30°C.

Intermediate culture: 20 liter and 100 liter

20-liter culture: 18 l environment G52 in a fermenter having a total volume of 30 l, inoculated with 2 l of pre-culture. Cultivation continued for 3-4 days under the following conditions: 30°C, 250 rpm, 0.5 l of air per 1 liter of liquid medium per minute, the excess pressure of 0.5 bar, without pH control.

100-liter culture: 90 l environment G52 in a fermenter having a total volume of 150 l, inoculated with 10 l 20 l pre-culture. Cultivation continued for 3-4 days under the following conditions: 30°C, 150 rpm, 0.5 l of air per 1 liter of liquid medium per minute, the excess pressure of 0.5 bar, without pH control.

Main culture: 10-, 100 -, or 500-liter

10-liter culture: the Ingredients to obtain 10 l of medium 1V12 sterilized in 7 l of water, then add 1 liter of sterile 10%solution of 2-(hydroxypropyl)-β-cyclodextrin (SE-#x003B2; -CD) and inoculated with 2 l of 20-liter intermediate culture. Duration of cultivation of principal crops is 6-7 days under the following conditions: 30°C, 250 rpm, 0.5 l of air per 1 liter of liquid medium per minute, the excess pressure of 0.5 bar, the pH control using H2SO4/KOH to a pH of 7.6±0,5 (i.e. without control when the pH value of 7.1 to 8.1).

100-liter culture: the Ingredients to obtain 100 liters of medium 1V12 sterilized in 70 liters of water, then add 10 l of sterile 10%solution of 2-(hydroxypropyl)-β-cyclodextrin and inoculated with 20 l 20 l intermediate culture. Duration of cultivation of principal crops is 6-7 days under the following conditions: 30°C, 200 rpm, 0.5 l of air per 1 liter of liquid medium per minute, the excess pressure of 0.5 bar, the pH control using H2SO4/KOH to a pH of 7.6±0.5 in. The sequence of crops for 100-liter fermenter schematically below:

500-liter culture: the Ingredients to obtain 500 l environment 1V12 sterilized in 350 l of water, then add 50 l of sterile 10%solution of 2-(hydroxypropyl)-β-cyclodextrin and inoculated with 100 l 100 l intermediate culture. Duration of cultivation of principal crops is 6-7 days under the following conditions: 30°C, 120 rpm, 0.5 l of air per 1 liter of liquid medium is min, the excess pressure of 0.5 bar, the pH control using H2SO4/KOH to a pH of 7.6±0.5 in.

Analysis of products

Sample preparation

50 ml of sample is mixed with 2 ml polystyrene Amberiite XAD16 (firm Rohm and Haas, Frankfurt, Germany) and shaken at 180 rpm for 1 h at 30°C. the Polystyrene is then filtered using a nylon sieve with a pore size of 150 μm, washed with a small amount of water and then mixed with the filter in the tube type Nunc volume of 15 ml.

Elution of the product from polystyrene

In a test tube with filter and polystyrene add 10 ml of isopropanol (>99%). After this closed tube is shaken for 30 min at room temperature on a rotary mixer type Rota-Mixer (firm Labinco BV, the Netherlands). Then 2 ml of fluid is removed by centrifugation and add the supernatant with a pipette in the column for GHUR.

GHUR-analysis
Column:Waters-Symetry C18, 100×4 mm, and 3.3 μm WAT066220+pre-column 3,9×20 mm WAT054225
Solvents:And: 0,02%phosphoric acid
B: acetonitrile (purity for IHVR)
Gradient:41% B from 0 to 7 min
100% B from 7.2 to 7.8 min min
Ȋ 41% In 8 to 12 min
The temperature of thermostat:30°
Detection:250 nm, detection by UV-DAD
The volume of injected sample:10 ál
Retention time:aptilon: 4,30 min; epothilone In: 5,38 min

Example 2A: the Impact of adding cyclodextrin and cyclodextrin derivatives on the concentration obtained epothilones

All investigated cyclodextrine derivatives obtained from the company Fluka, Buchs, Switzerland. Experiments are performed in shake flasks with a volume of 200 ml containing 50 ml of culture. As controls use a bulb with resin (polystyrene) Amberlite XAD16 (firm Rohm & Haas, Frankfurt, Germany) as adsorbent and without the addition of binders. After incubation for 5 days using HIVR can be defined the following titles epothilones.

AdditiveNo. of productConc. [%, wt./about]1Epothilone And [mg/l]Epothilone In [mg/l]
Amberlite XAD16 (about./about.)2,0%vol.9,2the 3.8
2-hydroxypropyl-β-cyclodextrin563320,12,71,
2-hydroxypropyl-β-cyclodextrin"0,5the 4.73,3
2-hydroxypropyl-β-cyclodextrin"1,0the 4.73,4
2-hydroxypropyl-β-cyclodextrin"2,0the 4.74,1
2-hydroxypropyl-β-cyclodextrin"5,01,70,5
2-hydroxypropyl-α-cyclodextrin563300,51,21,2
2-hydroxypropyl-α-cyclodextrin"1,01,21,2
2-hydroxypropyl-α-cyclodextrin"5,02,52,3
β-cyclodextrin287070,11,61,3
β-cyclodextrin"0,53,62,5
β-cyclodextrin"1,04,83,7
β-cyclodextrin"2,04,82,9
β-cyclodextrin"1,10,4
methyl-β-cyclodextrin662920,50,8<0,3
methyl-β-cyclodextrin"1,0<0,3<0,3
methyl-β-cyclodextrin"2,0<0,3<0,3
2,6-di-ortho-methyl-β-cyclodextrin399151,0<0,3<0,3
2-hydroxypropyl-γ-cyclodextrin563340,10,3<0,3
2-hydroxypropyl-γ-cyclodextrin"0,50,90,8

AdditiveNo. of productConc. [% wt./about]1Epothilone And [mg/l]Epothilone In [mg/l]
2-hydroxypropyl-γ-cyclodextrin"1,01,10,7
2-hydroxypropyl-γ-cyclodextrin"2,02,60,7
2-hydroxypropyl-γ-cyclodextrin"5,05,01,1
without adding0,50,5

1)Except Amberlite (vol.%), all percentages are % wt./about.

Some of the investigated cyclodextrins (2,6-di-ortho-methyl-β-cyclodextrin, methyl-β-cyclodextrin) in the investigated concentrations have no effect or an adverse effect on the production of epothilone. As can be seen from the examples, the addition of 1-2% 2-hydroxypropyl-β-cyclodextrin increases the production of 6-8 times compared to production without the use of cyclodextrins.

Example 2B: Fermentation 10-liter culture with addition of 1% 2-hydroxypropyl-β-cyclodextrin

The fermentation is carried out in a 15-liter glass fermenter. The medium contains 10 g/l of 2-(hydroxypropyl)-β-cyclodextrin obtained from Wacker Chemie, Munich, Germany. The change in product yield in the fermentation process are presented in table 2. Fermentation finished in 6 days and the product is processed.

Table 2
The change in product yield during a 10-liter fermentation
The process of cultivation (days)Epothilone A (mg/l)Epothilone In (mg/l)
0 00
100
20,50,3
31,82,5

The process of cultivation (days)Epothilone A (mg/l)Epothilone In (mg/l)
43,05,1
53,75,9
63,6the 5.7

Example 2B: Fermentation 100-liter culture with addition of 1% 2-hydroxypropyl-β-cyclodextrin

The fermentation is carried out in a 150-liter fermenter. The medium contains 10 g/l of 2-(hydroxypropyl)-β-cyclodextrin. The change in product yield in the fermentation process are presented in table 3. The fermentation is finished after 7 days and produce handling product.

Table 3
The change in product yield during a 100-liter fermentation
The process of cultivation (days)Epothilone A (mg/l)Epothilone In (mg/l)
000
100
20,30
30,91,1
41,52,3
51,63,3
61,83,7
71,83,5

Example 2G: Fermentation 500-liter culture with addition of 1% 2-hydroxypropyl-β-cyclodextrin

The fermentation is carried out in 750-liter fermenter. The medium contains 10 g/l of 2-(hydroxypropyl)-β-cyclodextrin. The change in product yield in the fermentation process are presented in table 4. The resulting fermentation product is collected after 7 days and produce handling.

Table 4
The change in product yield in the process 500-liter fermentation
The process of cultivation (days)Epothilone A (mg/l)Epothilone In (mg/l)
000
100
200
30,60,6
41,72,2
53,14,5
63,15,1

Example 2E: WITH anicely example fermentation 10-liter culture without the addition of the adsorbent

The fermentation is carried out in a 15-liter glass fermenter. The environment does not contain any cyclodextrin or other adsorbent. The change in product yield in the fermentation process are presented in table 5. The resulting fermentation product, do not collect and do not handle.

Table 5
The change in product yield during a 10-liter fermentation without adsorbent
The process of cultivation (days)Epothilone A (mg/l)Epothilone In (mg/l)
000
100
200
300
40,70,7
50,71,0
60,81,3

Example 3: Processing epothilones: allocation of 500-liter main crop

Output 500-liter primary culture as described in example 2G, 450 l, share it with cleansing separator Westfalia type SA-20-06 (speed 6500 rpm) in liquid phase (centrifugate+flush water=650 l) and solid phase (cells approximately 15 kg). The main part of aptilo is s found in centrifugate. Obtained after centrifugation of the cell mass contains less than 15% of the assigned amount epothilones, and are not further processed. Centrifugal displacement of 650 l then placed in the tank with agitator volume of 4000 l, is mixed with 10 liters of Amberlite XAD16 (volume ratio centrifugal: resin (polystyrene) 65:1) and stirred. After contact with the resin for about 2 hours, it is removed by centrifugation using a centrifuge with sink type Heine (the capacity of the basket 40 l, the rotation speed 2800 rpm). The resin is removed from the centrifuge and washed with 10-15 l of deionized water. Desorption carried out twice by stirring the resin in glass tanks with agitator volume of 30 l and 30 min, each time using 30 l of isopropanol. Department isopropanole phase from the resin is performed using a vacuum filter. Then the isopropanol is removed from the United isopropanolic phases, adding 15-20 liters of water in a vacuum evaporator with circulation (by Schmid-Verdampfer), and the resulting aqueous phase displacement of approximately 10 l of extracted three times, using each time for 10 l of ethyl acetate. The extraction was performed in glass vessels with a volume of 30 l with stirring. An ethyl acetate extract was concentrated to a volume of 3-5 l in a vacuum evaporator with circulation (by Schmid-Verdampfer) and then concentrated to dryness on a rotary evaporator (type Büchi). In the floor is out of 50.2 g of an ethyl acetate extract. An ethyl acetate extract was dissolved in 500 ml of methanol, the insoluble fraction is filtered through a folded filter and the solution make a 10-pound column of Sephadex LH 20 (Pharmacia, Uppsala, Sweden) (column diameter 20 cm, the filling level of about 1.2 m). Elution is carried out, using as eluent methanol. Epothilone a and B mainly present in fractions 21-23 (fraction size 1 liter). These fractions are concentrated to dryness in vacuo on a rotary evaporator (total weight of 9.0 g). Then these cefalexina fraction of the maximum content epothilones (9.0 g) was dissolved in 92 ml of a mixture acetonitrile:water:methylene chloride (50:40:2), the solution is filtered through a folded filter and contribute to the RP-column (device type Prepbar 200, the company Merck; 2.0 kg LiChrospher RP-18, Merck, grain size of 12 μm, the diameter of the column 10 cm, level 42 cm; Merck, Darmstadt, Germany). Elution carried out with a mixture of acetonitrile:water (3:7) (flow rate 500 ml/min; retention time epothilone And about 51-59 min; retention time epothilone In approximately 60-69 min). Fractionation is controlled using a UV detector at a wavelength of 250 nm. Fractions are concentrated to dryness in vacuo using a rotary evaporator Büchi. Weight epothilone And the peak fraction is 700 mg, which according to the obtained using GHUR data (external standard) corresponds to 75.1%. MA is sa epothilone B in the peak fraction 1980 mg, according to the obtained using GHUR data (external standard) corresponds to 86,6%. In conclusion, the fraction epothilone A (700 mg) crystallized from 5 ml of a mixture of ethyl acetate:toluene (2:3) to give 170 mg epothilone And the net kristalliset [content according to GHUR (area %) is 94,3%]. Crystallization epothilone (1980 mg) is carried out using 18 ml of methanol, getting 1440 mg epothilone B in the form of pure kristalliset [content according to GHUR (area %) is 99,2%], tPL(epothilone), for example, 124-125°C.

The table below shows data1H-NMR analysis for epothilone In:

NMR at 500 MHz; solvent: DMSO-d6; chemical shift δ frequent./million relative to TMS (s means singlet, d means doublet, m means multiplet):

δ (multiplicity)The integral (number N)
7,34 (s)1
6,50 (s)1
5,28 (d)1

δ (multiplicity)The integral (number N)
5,08 (d)1
4,46 (d)1
4,08 (m)1
3,47 (m)1
3,11 (m)1
and 2.83 (dd)1
2,64 (s)3
2,36 (m)2
2,09 (s)3
2,04 (m)1
1,83 (m)1
1,61 (m)1
1,47-1,24 (m)4
1,18 (s)6
1,13 (m)2
1,06 (d)3
0,89 (d+s overlap)6
∑=41

According to this example (example 3) epothilone To receive in the form of crystalline modification And characterized by x-ray modifications (see General part of the present description).

Example 4: Crystalline modification In epothilone

50 mg epothilone (obtained, for example, according to the above method) suspended in 1 ml isopropanol and shaken for 24 h at 25°C. the Product is filtered and dried. After drying in high vacuum get epothilone B in the form of white crystals. For crystalline modification of the product is characterized by x-ray modifications (see General part of the present description).

The following specific embodiments of form part of the invention:

a) method of separation epothilones primarily epothilones a and b, to the which differs that it is carried out by chromatography on a column of reversed phase using an eluent containing (ness.)alkylene;

b) the method according to paragraph a), characterized in that the material used column carries the hydrocarbon chains containing 18 carbon atoms, and the eluent is a mixture of water and acetonitrile;

in) strain Sorangium cellulosum obtained by mutagenesis, in which otherwise identical conditions produces a greater number epothilones than strain Sorangium cellulosum Soce90;

g) strain under item (C), designated as VSE/10;

d) crystalline form epothilone In marked modification And characterized by the presence of reginorum obtained using a diffractometer with Cu-Kα1the quality of the radiation source, which is presented in the following table:

18,5
Intensity
7,7very strong
10,6weak
13,6average
14.4Vaverage
15,5average
16,4weak
16,8weak
17,1weak
the 17.3weak
17,7weak
weak
20,7strong
of 21.2strong
of 21.9weak

Intensity
22,4weak
23,3strong
25,9average
31,2weak
32,0average

e) crystalline form epothilone In marked modification In other than the presence of reginorum obtained using a diffractometer with Cu-Kα1the quality of the radiation source, which is presented in the following table:

Intensity
6,9very strong
8,0weak
8,3average
10,8strong
11,5average
12,4weak
13,1strong
15,5weak
16,2weak
16,7average
18,1with ednea
18,6average
20,4weak
20,9strong
21,3weak
a 21.5very poor
22,5average
24,2weak

Intensity
25,1average

W) of the pharmaceutical composition, which contains the active substance under item (d) or (e) in combination with a pharmaceutically acceptable carrier;

C) a method of treating a warm-blooded animal suffering from a proliferative disease, by introducing epothilone In under item (d) or (e) in a dose effective for the treatment of this disease, to a warm-blooded animal that is in need of such treatment;

and the use of crystalline form under item (d) or (e) for the treatment of a proliferative disease;

K) the application of a new crystalline form epothilone In under item (d) or (e) in the preparation of pharmaceutical compositions, where the crystalline form of this type is mixed with one or more carriers.

1. A method for concentrating epothilones in an aqueous culture medium containing nutrients, suitable for cultivation producer EP is milonov Sorangium cellulosum, characterized in that the culture medium add at least one complexing component is able to be dissolved in culture medium selected from the group including

a) α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, δ-cyclodextrin, ε-cyclodextrin, ζ-cyclodextrin, η-cyclodextrin and θ-cyclodextrin;

b) cyclodextrine derivative or a mixture cyclodextrine derivatives selected from derivatives

(1) a cyclodextrin in which one or more up to all of the hydroxy groups are converted with the formation of simple Olkiluoto ether, arylhydroxylamine ether, hydroxyalkyl ether, carboxycellulose ether, derivatizing carboxy(ness.)Olkiluoto ether, in which derivational carboxypropyl is aminocarbonyl, mono - or di-(ness.)alkylaminocarbonyl, morpholino-, piperidino-, pyrrolidino or piperazinylcarbonyl or alkoxycarbonyl, sulfoalkyl ether;

(2) a cyclodextrin in which one or more Oh-groups etherification using a radical of the formula

-O-[Alk-O]n-H

where Alk denotes an alkyl, and n denotes an integer from 2 to 12 inclusive;

(3) a cyclodextrin in which one or more Oh-groups etherified the Vano using a radical of the formula

where R' denotes hydrogen atom, hydroxy, -O-(Alk-O)z-H, with Alk in all cases refer to alkyl, m, n, and z represent an integer from 1 to 12, a Y indicates QR1or NR2R3where R1, R2and R3independently from each other represent a hydrogen atom or (ness.)alkyl, or R2and R3together with the attached nitrogen atom represent morpholino, piperidino, pyrrolidino or piperazine derivatives, or

(4) a branched cyclodextrin, which contains ether group or acetals formed with other sugar molecules, and which are selected from glucosyl-, diglycosyl(G2-β-cyclodextrin), maltose or demultiplication or N-acetylglucosaminyl, glucosaminyl-, N-acetylgalactosamine or galactosaminidase, and (ness.)alkanoyl, such as complex acetyloxy ether cyclodextrin,

C) a mixture of two or more of these cyclodextrins and/or tikilometrovoy derivatives

and the prefix "(ness.)" means that the radical contains up to 7 carbon atoms.

2. The method according to claim 1, characterized in that the cyclodextrin and/or cyclodextrine derivative added to the culture medium at a concentration of from 0.05 to 10% (wt./vol.).

3. The method according to claim 2, characterized in that the cyclodextrin and/and and cyclodextrine derivative added to the culture medium at a concentration of from 0.1 to 2% (wt./vol.).

4. The method according to claim 1, characterized in that the complexing component is cyclodextrine derivative selected from cyclodextrin and hydroxy(nissalke)cyclodextrin or mixtures comprising one or more of these compounds.

5. The method according to claim 1, characterized in that the complexing component is a 2-hydroxypropyl-β-cyclodextrin.

6. The culture medium for growing Sorangium cellulosum producer epothilones, which includes water and a suitable combination of all or some of the components selected from the group comprising biopolymers, sugar, amino acids, salts, nucleic acids, vitamins, antibiotics, synthetic chemicals, growth factors, extracts of biological materials, soy flour, starch and minerals and other components and/or similar additives, characterized in that it further includes a complexing component in an amount of from 0.02 to 10 wt./about.

7. The culture medium according to claim 6, characterized in that the complexing component is a cyclodextrin, cyclodextrine derivative or a mixture of two or more complexing component selected from cyclodextrin and cyclodextrine derivatives.

8. The method of obtaining epothilones, characterized in that the prepared water culture is inuu Wednesday, designed for biotechnological production of epothilones using a microorganism suitable for the production of epothilones, mainly myxobacteria, to the culture medium add complexing component is able to be dissolved in culture medium and representing a cyclodextrin, cyclodextrine derivative or a mixture of two or more complexing component selected from the cyclodextrin or cyclodextrine derivatives, followed by cultivation, cleaning and, if necessary, by separation epothilones.

9. The method according to claim 8, characterized in that the biotech receiving epothilones carried out using myxobacteria of the genus Sorangium.

10. The method according to claim 9, characterized in that the culture medium after culturing mycobacteria of the genus Sorangium subjected to separation into solid and liquid (centrifugal) phase by centrifugation, further centrifugal mixed with resin and dispersed on a column filled with the same resin, if necessary, the resin is washed with water, epothilone(s) is obtained by desorption from the resin with a polar solvent, if necessary, the phase of the solvent is concentrated by the prior, simultaneous or subsequent addition of water, add organic solvent which is not miscible with water, and epothilone(s) transferred organicheskoi phase, the organic phase is optionally concentrated, epothilone obtained from the organic solution, concentrate using molecular sieves for compounds with low molecular weight and then containing epothilone fraction is subjected to separation on a column of reversed phase, resulting in a gain separately epothilone a and b, which optionally can be further concentrated by recrystallization.

11. The method of obtaining epothilones, characterized in that it envisages the following stages:

a) concentration epothilones in culture medium intended for biotechnological production of epothilones using a microorganism that is suitable for receiving, containing water and other appropriate components of the culture media with the addition of complexing component cyclodextrin or cyclodextrines derivative or a mixture of two or more of these compounds, and

b) separation epothilones using chromatography on a column of reversed phase using eluent containing (ness.) alkylene, and chromatography is carried out on the material of the column, which carries the hydrocarbon chain, and apply eluent containing (NISS.-)alkynylaryl, and if necessary, carry out additionally the e stage of processing and purification stages.

12. The method of separation epothilones primarily epothilones a and b, characterized in that exercise chromatography of the product obtained using the method according to claim 1, where the chromatography is carried out on a column filled with the material of the column with reversed phase and eluent comprising (ness.)alkylene.

13. The method according to item 12, characterized in that the material quality speakers use the material that carries the hydrocarbon chains containing 18 carbon atoms, and as the eluent using a mixture of water/acetonitrile.

14. Crystalline form epothilone In marked modification and characterized by x-ray obtained with a diffractometer using Cu-Kα1as a radiation source and having the following parameters:

td align="center"> weak
2θ the reflection angle in degreesThe intensity of the most important lines
7,7very strong
10,6weak
13,6average
14.4Vaverage
15,5average
16,4weak
16,8weak
17,1weak
the 17.3weak
17,7
18,5weak
20,7strong
of 21.2strong
of 21.9weak
22,4weak
23,3strong
25,9average
31,2weak
32,0average

15. Pharmaceutical composition for treating a proliferative disease of warm-blooded animal and/or human that contains the active substance in 14 in combination with a pharmaceutically acceptable carrier.

16. A method of treating a proliferative disease of warm-blooded animal and/or human, wherein the patient is administered aptilon, characterized in 14 in effective dose.



 

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SUBSTANCE: invention relates to new biologically active compounds. Invention describes a compound of the formula (1):

wherein R1 means carbon chain with 2-30 carbon atoms that can be linear, branched, saturated and can be substituted once or twice with the following groups: -OH, =O, -(C1-C6)-alkyl or the group:

; R2 means (C1-C6)-alkyl; E means phosphorus atom (P); each X1, X2 and X3 means independently of one another -O- in its all stereochemical forms and mixtures of these forms taken in any ratio, and its physiologically acceptable salts also. Also, invention describes a method for preparing cyclipostins and the strain Streptomyces HAG 004107, DSM 13381 as a producer of cyclipostins. Invention provides preparing new compounds possessing valuable biological properties.

EFFECT: improved preparing method, valuable properties of substances.

12 cl, 9 tbl, 21 ex

FIELD: agriculture, biotechnology.

SUBSTANCE: claimed method includes blending of waste from poultry industry or stockraising with microbial materials providing solid phase fermentation of obtained mixture. Consortium based on strains of bacterium Baccilus subtilis, fungi Trichosporon cutaneum, Trichoderma viride Sp., Fusarium sambucinum and yeast S.cerevisiae diastaticus taken in equal ratio is used as composting stimulator. Consortium is introduced into composting substrates in form of liquid suspension or dehydrated biomass in concentration of 0.005-0.05 % based on composting mixture, conditioned at temperature of 20-65°C and humidity of 35-50 % for 4-7 days depending on retreating waste composition. Dolomite chips are added into compost in amount of 5 wt.% based on composting mixture.

EFFECT: effective method for compost production.

2 cl, 7 ex

FIELD: microbiology.

SUBSTANCE: invention relates to microorganism culture, producing high viscous polysaccharide, and uses thereof in oil producing industry. Strain Azotobacter vinelandii can produce exopolysaccharide in amount of 16 g/l and convert medium to gel. Said gel possesses necessary regulating properties for oil extrusion from depleted oil wells.

EFFECT: bacterium culture useful in oil producing industry.

3 tbl, 4 ex

FIELD: organic chemistry, biochemistry, pharmacy.

SUBSTANCE: invention relates to new anellated carbamoyl azaheterocycles of the general formula (1)

or (2) possessing the inhibitory effect on protein kinase activity, a focused library comprising these compounds, and pharmaceutical composition based on thereof. In the general formula (1) or (2) R1 represents hydrogen atom or optionally substituted (C1-C6)-alkyl; R2 and R3 represent independently of one another hydrogen atom, inert substitute, optionally substituted (C1-C6)-alkyl, optionally substituted (C3-C8)-cycloalkyl, optionally substituted phenyl, optionally substituted aryl, optionally substituted heterocyclyl; R4 represents optionally substituted (C1-C6)-alkyl, optionally substituted (C3-C8)-cycloalkyl, optionally substituted phenyl, optionally substituted aryl, optionally substituted heterocyclyl; A and B in common with carbon and nitrogen atoms joined to the form an optionally substituted and optionally condensed azaheterocycle; D and F in common with carbon atoms joined form an optionally substituted and optionally condensed phenyl or aryl, optionally substituted and optionally condensed azaheterocycle. K and L in common with carbon and nitrogen atoms joined to them form an optionally substituted azaheterocycle. Also, invention related to methods for preparing compounds of the general formulae (1) or (2).

EFFECT: improved preparing methods.

10 cl, 2 sch, 25 tbl, 7 ex

FIELD: organic chemistry, chemical technology, medicine.

SUBSTANCE: invention relates to a novel method for preparing 14β-hydroxy-1,4-carbonate-desacetylbaccatin III and intermediate substances used in preparing new derivatives of taxane and possessing an antitumor activity. Method involves the following stages: a) protection of hydroxyls at positions 7 and 10 in 10-desacetylbaccatin III wherein R and R1 are taken among hydrogen atom, (C1-C10)-alkyl or aryl, (C1-C10)-alkyl- or aryl-carbonyl, trichloroacetyl, (C1-C4)-trialkylsilyl; preferably, when R and R1 are similar then they represent trichloroacetyl; when they are different then, preferably, R represents trichloroacetyl and R1 represents acetyl; or R represents triethyl or trimethylsilyl and R1 represents acetyl; b) two-stage oxidation to yield a derivative oxidized to carbonyl at position 13 and hydroxylated at position 14; c) carboxylation of vicinal hydroxyls at positions 1 and 14 to yield 1,14-carbonate derivative; d) reduction of carbonyl at position 13; e) removal of protective groups at positions 7 and 10. Also, invention relates to intermediate substances. Invention provides preparing intermediate substances used in synthesis of taxane.

EFFECT: improved preparing method.

8 cl, 8 ex

FIELD: organic chemistry, medicine, oncology, pharmacy.

SUBSTANCE: invention relates to a new pentacyclic compound derivative of taxane represented by the formula:

wherein R1 represents dimethylaminomethyl group or morpholinomethyl group; R2 represents halogen atom or alkoxy-group comprising from 1 to 6 carbon atoms, or its salt eliciting an antitumor effect, and to a medicine agent based on its. Invention provides preparing new derivatives of taxane eliciting the valuable biological effect.

EFFECT: valuable medicinal properties of compound.

13 cl, 1 dwg, 4 tbl, 16 ex

FIELD: organic chemistry, pharmacology.

SUBSTANCE: invention relates to new aromatic diketone derivatives of formula I

(R4, R5, R6, and R7 are independently H, OH, X-alkyl, wherein X represents oxygen; K is group of formula II

or III

;

L is group of formula IV

;

or K and L together form group of formula VI ,

wherein R1 and R3 are independently H or alkyl; R2 is H or alkyl; X1-X7 are independently O, NH; and ring "cyclus" together with carbon atom labeled with letters c and d represents anthraquinone, hydroquinone or phenyl, optionally substituted with one or more hydroxyl, alkoxyl, or alkyl groups), as well as pharmaceutically accepts salts thereof, ethers, esters, tautomers, stereomers and mixtures in any ratio. Derivatives of present invention are glucose-6-phosphatetranslocase inhibitors. Also disclosed are method for production of derivatives, pharmaceutical composition containing the same, and uses thereof as drugs, in particular for treatment of diabetes mellitus.

EFFECT: new compounds and pharmaceutical composition for treatment of diabetes mellitus.

20 cl, 4 tbl, 6 ex

FIELD: organic chemistry, biochemistry, medicine, pharmacy.

SUBSTANCE: invention relates to new bis-tetrahydrofuranbenzodioxolyl sulfonamide compounds of the formula (I): , its salts, stereoisomers and racemates that are effective inhibitors of protease activity. Also, invention relates to pharmaceutical preparations, methods for inhibition of retrovirus proteases, in particular, to resistant retrovirus proteases, to many drugs, methods for treatment and prophylaxis of infection or disease associated with retrovirus infection in mammals and to methods for inhibition of retrovirus replication. Invention provides preparing new derivatives of bis-tetrahydrofuranbenzodioxalyl sulfonamides eliciting the valuable pharmaceutical properties.

EFFECT: valuable medicinal properties of compound and composition, improved treatment method.

16 cl, 2 dwg, 3 tbl

FIELD: organic chemistry, chemical technology, medicine, oncology, pharmacy.

SUBSTANCE: invention relates to new derivative of taxane of the formula (I):

that elicits strong antitumor effect. Also, invention relates to intermediates substances, a method for preparing compound of the formula (I), a method for preparing 1,14-β-hydroxy-1,14-carbonate-baccatin III-derivatives substituted with isoserine residue at position 3 and to pharmaceutical composition based on compounds of the formula (I). Invention provides preparing new derivative of taxane that elicits higher activity and reduced toxicity as compared with paclitaxel.

EFFECT: improved preparing method, enhanced and valuable medicinal properties of compound.

10 cl, 7 tbl, 6 ex

FIELD: color-forming compositions and recording material.

SUBSTANCE: claimed composition includes developer containing urea-urethane compound and colorless or light colored leuco dye. Recording material based on this composition also is proposed.

EFFECT: color-forming compositions with improved image conservation ability and increased image intensity.

21 cl, 14 tbl, 153 ex

The invention relates to new derivatives epothilone formula I, where the bond indicated by a wavy line indicates that the bond “a” is either CIS-or TRANS-form; (I) R2absent or represents oxygen; “a” denotes a single or double bond; “b” is absent or represents a simple bond; and “C” is absent or represents a simple bond, provided that when R2denotes oxygen, then “b” and “C” both represent a simple bond and a represents a simple bond; if R2no, the “b” and “C” both are absent and “a” represents a double bond; and if “a” represents a double bond, R2“b” and “C” are absent; R3denotes a radical selected from the group comprising hydrogen; (ness.)alkyl, especially methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl; -CH2F; -CH2-OH; R4and R5independently of one another denote hydrogen; R1denotes a radical of the structure (a-d); (II) if R3means (ness.)alkyl, especially methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl; -CH2F; CH2-HE; and other symbols except for the R1have the values listed above in their characters except for the R1have the above values, R1can also represent a fragment of formula (j); or a salt of the compounds of formula I, if there is a salt-forming group
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