Fermentative preparation process for and crystal forms of cytostatics   

The invention relates to a new biotechnological preparation process that can be used on an industrial scale for the production of epothilones, especially a process for concentrating these compounds in the culture medium, as well as a new strain for the fermentative preparation of these compounds. The invention also relates to new crystal forms of epothilones, especially epothilone B, obtainable by the new processes, their usage in the production of pharmaceutical preparations, new pharmaceutical preparations comprising these new crystal forms and/or the use of these compounds in the treatment of proliferative diseases such as tumours, or in the production of pharmaceutical formulations which are suitable for this treatment.

Of the existing cytotoxic active ingredients for treating tumours, Taxol® (Paclitaxel; Bristol-Myers Squibb), a microtubuli-stabilising agent, plays an important role and has remarkable commercial success. However, Taxol has a number of disadvantages. In particular, its very poor solubility in water is a problem. It therefore became necessary to administer Taxol® in a formulation with Cremophor EL® (polyoxyethylated castor oil; BASF, Ludwigshafen, Germany). Cremophor EL® has severe side effects; for example it causes allergies which in at least one case have led even to the death of a patient.

Although the Taxan class of microtubuli-stabilising anti-cancer agents has been commended as “perhaps the most important addition to the pharmaceutical armoury against cancer in the last decade” (see Rowinsky E. K., Ann. rev. Med. 48 353-374 (1997)), and despite the commercial success of Taxol®, these compounds still do not appear to represent a really great breakthrough in the chemotherapy of cancer. Treatment with Taxol® is linked with a series of significant side effects, and a few primary classes of compact tumours, namely colon and prostate tumours, respond to this compound only to a small extent (see Rowinsky E. K., inter alia). In addition, the efficacy of Taxol can be impaired and even completely neutralised by acquired resistance mechanisms, especially those based on the overexpression of phosphoproteins, which act as efflux pumps for active ingredients, such as “Multidrug Resistance” due to overexpression of the multidrug transport glycoprotein “P-glycoprotein”.

Epothilones A and B represent a new class of microtubuli-stabilising cytotoxic active ingredients (see Gerth, K. et al., J. Antibiot. 49, 560-3 (1966)) of the formulae:

wherein R signifies hydrogen (epothilone A) or methyl (epothilone B).

These compounds have the following advantages over Taxol®:

a) They have better water-solubility and are thus more easily accessible for formulations.

b) It has been reported that, in cell culture experiments, they are also active against the proliferation of cells, which, owing to the activity of the P-glycoprotein efflux pump making them “multidrug resistant”, show resistance to treatment with other chemotherapy agents including Taxol® (see Bolag, D. M., et al., “Epothilones, a new class of microtubuli-stabilizing agents with a Taxol-like mechanism of action”, Cancer Research 55, 2325-33 (1995)). And

c) it could be shown that they are still very effective in vitro against a Taxol®-resistant ovarian carcinoma cell line with modified β-tubulin (see Kowalski, R. J., et al., J. Biol. Chem. 272(4), 2534-2541 (1997)).

Pharmaceutical application of the epothilones, for example for tumour treatment, is possible in an analogous manner to that described for Taxol, see for example U.S. Pat. No. 5,641,803; U.S. Pat. No. 5,496,804; U.S. Pat. No. 5,565,478).

In order to be able to use the epothilones on a larger scale for pharmaceutical purposes, however, it is necessary to obtain appropriate amounts of these compounds.

Until now, the extraction of natural substances by means of myxobacteria, especially the epothilones from the cell strain Sorangium Cellulosum Soce90 (deposited under no. 6773 at the German Collection of Microorganisms, see WO 93/10121) was described in literature. In order to obtain a satisfactory concentration of the natural substances, especially the epothilones, in the culture medium for the subsequent extraction, previously an adsorbate resin based on polystyrene was always added, for example Amberlite XAD-1180 (Rohm & Haas, Frankfurt, Germany).

However, the disadvantage of this process is that, on a large scale, it leads to an abundance of problems. Valves are impaired by the globules of resin, pipes can block, and apparatus may be subject to greater wear due to mechanical friction. The globules of resin are porous and therefore have a large inner surface area (about 825 m2/gram resin). Sterilisation becomes a problem, as air enclosed in the resin is not autoclaved. Thus, the process cannot be practicably carried out on a large scale using resin addition.

On the other hand, without adding resin globules, a satisfactory concentration of epothilones cannot be achieved in the culture medium.

Surprisingly, the requirements for finding a way out of this dilemma have now been found, enabling a satisfactory concentration of natural substances to be obtained from micro-organisms, in particular myxobacteria, which produce epothilones such as epothilone A or B, in particular a concentration of epothilones A and B, in the culture medium, without the addition of resins, and thus enabling production of these compounds, especially epothilones to be carried out on a technical and industrial scale without the above-mentioned disadvantages.

DETAILED DESCRIPTION

One aspect of the invention relates to a process for concentrating epothilones, especially epothilone A and/or B, in particular epothilone B, in a culture medium, in order to produce these compounds on a biotechnological scale, the process comprising microorganisms which produce these compounds, especially myxobacteria (as producers of natural substances), whereby a complex-forming components which is soluble in the culture medium is added to the medium.

A further aspect relates to the corresponding culture medium, which comprises a corresponding complex-forming component and microorganisms, especially myxobacteria, in particular of the genus Sorangium, which are suitable for producing epothilones, especially epothilone A and/or B.

A further aspect of the invention relates to a process for the production of epothilones, especially epothilone A and/or B, especially the two pure compounds, in particular epothilone B, which is characterised in that the epothilones are obtained by working up a culture medium for the biotechnological preparation of these compounds, which comprises as producers of natural substances microorganisms, especially myxobacteria, that produce these compounds, and to which a complex-forming component that is soluble in the culture medium is added, and the subsequent purification and, if desired, separation of the epothilones, for example epothilone A and B.

A fourth aspect of the invention relates to a method of separating epothilones, especially epothilones A and B from one another, which is characterised by chromatography on a reversed-phase column with an eluant comprising a lower alkyl cyanide.

A further aspect of the invention relates to a strain of Sorangium cellulosum obtained by mutagenesis, which under otherwise identical conditions, produces more epothilones than Sorangium cellulosum Soce90.

A further aspect also relates to new crystal forms of epothilone B.

The general terms used hereinabove and hereinbelow preferably have the meanings given hereinbelow:

Where reference is made hereinabove and hereinbelow to documents, these are incorporated insofar as is necessary.

The prefix “lower” always indicates that the correspondingly named radical contains preferably up to a maximum of 7 carbon atoms, in particular up to 4 carbon atoms, and is branched or unbranched. Lower alkyl may be for example unbranched or branched once or more, and is e.g. methyl, ethyl, propyl such as isopropyl or n-propyl, butyl such as isobutyl, sec.-butyl, tert.-butyl or n-butyl, or also pentyl such as amyl or n-pentyl.

A culture medium for the biotechnological preparation of epothilones which contains micro-organisms that produce these compounds, especially myxobacteria, as producers of natural substances, is primarily a medium which comprises a corresponding (naturally occurring or also obtainable by cultivation or in particular by genetic modification) microorganism, especially a myxobacterial strain which is in a position to produce these compounds, in particular a myxobacterium of the genus Sorangium, preferably (in particular for epothilone production) a microorganism of the type Sorangium Cellulosum Soce90 (see WO 93/10121), or a biomaterial derived therefrom or from parts of this myxobacterium, especially a correspondingly derived strain, in particular the strain having the reference BCE33/10, in particular the strain having the reference BCE 63/114 or mutants thereof, and in addition, together with water, preferably other conventional and appropriate constituents of culture media, such as biopolymers, sugar, amino acids, salts, nucleic acids, vitamins, antibiotics, semiochemicals, growth media, extracts from biomaterials such as yeast or other cell extracts, soy meal, starch such as potato starch and/or trace elements, for example iron ions in complex-bound form, or suitable combinations of all or some of these constituents and/or also analogous additions. The corresponding culture media are known to the person skilled in the art or may be produced by known processes (see e.g. the culture media in the examples of the present disclosure, or in WO 93/10121).

One preferred myxobacterium is a strain selected by UV mutagenesis and selection for increased formation of epothilone A and/or B over Sorangium cellulosum Soce90, which is deposited in the DSM under no. 6773, especially the mutant BCE33/10, which was deposited under the number DSM 11999 on 9 Feb. 1998 at the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), and most preferably the mutant having the reference BCE 63/114, which was deposited under number DSM 12539 on 27 Nov. 1998 at the German Collection of Microorganisms and Cell Cultures (DSMZ).

Strain culture and morphological description of strain BCE 33/10 and of strain BCE 63/114: The strain can grow on cellulose as the sole source of carbon and energy with potassium nitrate as the sole source of nitrogen, e.g. on filter paper over ST21 mineral salt agar (0.1% KNO3; 0.1% MgSO4×7 H2O; 0.1% CaCl2×2 H2O; 0.1% K2HPO4; 0.01% MnSO4×7 H2O; 0.02% FeCl3; 0.002% yeast extract; standard trace element solution; 1% agar). On this medium, dark reddish-brown to blackish-brown fruiting bodies are formed. They consist of small sporangioles (ca. 15 to 30 μm diameter) and exist in dense heaps and packs of varying size.

The vegetative bacilli have the shape typical of Sorangium (relatively compact, under the phase contrast microscope dark, cylindrical bacilli with broad rounded ends, on average 3 to 6 μm long and 1 μm thick).

Epothilones are primarily epothilone A and/or B, but also other epothilones, for example epothilones C and D named in International Application WO 97119086 and WO 98/22461, epothilones E and F named in WO 98/22461, and further epothilones obtainable from corresponding microorganisms.

A water-soluble complex-forming component is primarily a water-soluble oligo- or poly-peptide derivative or in particular an oligo- or polysaccharide derivative of cyclic or helical structure, which forms an intramolecular cavity, which because of its sufficiently hydrophobic properties is in a position to bind epothilones, especially epothilone A and/or epothilone B. A water-soluble complex-forming component that is especially preferred is one that is selected from cyclodextrins or (in particular) cyclodextrin derivatives, or mixtures thereof.

Cyclodextrins are cyclic (α-1,4)-linked oligosaccharides of α-D-glucopyranose with a relatively hydrophobic central cavity and a hydrophilic external surface area.

The following are distinguished in particular (the figures in parenthesis give the number of glucose units 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). Especially preferred are δ-cyclodextrin and in particular α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin, or mixtures thereof.

Cyclodextrin derivatives are primarily derivatives of the above-mentioned cyclodextrins, especially of α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin, primarily those in which one or more up to all of the hydroxy groups (3 per glucose radical) are etherified or esterified. Ethers are primarily alkyl ethers, especially lower alkyl, such as methyl or ethyl ether, also propyl or butyl ether; the aryl-hydroxyalkyl ethers, such as phenyl-hydroxy-lower-alkyl, especially phenyl-hydroxyethyl ether; the hydroxyalkyl ethers, in particular hydroxy-lower-alkyl ethers, especially 2-hydroxyethyl, hydroxypropyl such as 2-hydroxypropyl or hydroxy-butyl such as 2-hydroxybutyl ether; the carboxyalkyl ethers, in particular carboxy-lower-alkyl ethers, especially carboxymethyl or carboxyethyl ether; derivatised carboxyalkyl ethers, in particular derivatised carboxy-lower-alkyl ether in which the derivatised carboxy is etherified or amidated carboxy (primarily aminocarbonyl, mono- or di-lower-alkyl-aminocarbonyl, morpholino-, piperidino-, pyrrolidino- or piperazino-carbonyl, or alkyloxycarbonyl), in particular lower alkoxycarbonyl-lower-alkyl ether, for example methyloxycarbonylpropyl ether or ethyloxycarbonylpropyl ether; the sulfoalkyl ethers, in particular sulfo-lower-alkyl ethers, especially sulfobutyl ether; cyclodextrins in which one or more OH groups are etherified with a radical of formula

—O—[alk—O—]n—H

wherein alk is alkyl, especially lower alkyl, and n is a whole number from 2 to 12, especially 2 to 5, in particular 2 or 3; cyclodextrins in which one or more OH groups are etherified with a radical of formula

wherein R1is hydrogen, hydroxy, —O—(alk-O)z-H, —O—(alk(—R)—O—)p-H or —O—(alk(—R)—O—)q-alk-CO—Y; alk in all cases is alkyl, especially lower alkyl; m, n, p, q and z are a whole number from 1 to 12, preferably 1 to 5, in particular 1 to 3; and Y is OR1 or NR2R3, wherein R1, R2 and R3 independently of one another, are hydrogen or lower alkyl, or R2 and R3 combined together with the linking nitrogen signify morpholino, piperidino, pyrrolidino or piperazine; or branched cyclodextrins, in which etherifications or acetals with other sugars are present, especially glucosyl-, diglucosyl-(G2-β-cyclodextrin), maltosyl- or dimaltosyl-cyclodextrin, or N-acetylglucosaminyl-, glucosaminyl-, N-acetylgalactosaminyl- or galactosaminyl-cyclodextrin.

Esters are primarily alkanoyl esters, in particular lower alkanoyl esters, such as acetyl esters of cyclodextrins.

It is also possible to have cyclodextrins in which two or more different said ether and ester groups are present at the same time.

Mixtures of two or more of the said cyclodextrins and/or cyclodextrin derivatives may also exist.

Preference is given in particular to α-, β- or γ-cyclodextrins or the lower alkyl ethers thereof, such as methyl-β-cyclodextrin or in particular 2,6-di-O-methyl-β-cyclodextrin, or in particular the hydroxy lower alkyl ethers thereof, such as 2-hydroxypropyl-α-, 2-hydroxypropyl-β- or 2-hydroxypropyl-γ-cyclodextrin.

The cyclodextrins or cyclodextrin derivatives are added to the culture medium preferably in a concentration of 0.02 to 10, preferably 0.05 to 5, especially 0.1 to 4, for example 0.1 to 2 percent by weight (w/v).

Cyclodextrins or cyclodextrin derivatives are known or may be produced by known processes (see for example U.S. Pat. No. 3,459,731; U.S. Pat. No. 4,383,992; U.S. Pat. No. 4,535,152; U.S. Pat. No. 4,659,696; EP 0 094 157; EP 0 149 197; EP 0 197 571; EP 0 300 526; EP 0 320 032; EP 0 499 322; EP 0 503 710; EP 0 818 469; WO 90/12035; WO 91/11200; WO 93/19061; WO 95/08993; WO 96114090; GB 2,189,245; DE 3,118,218; DE 3,317,064 and the references mentioned therein, which also refer to the synthesis of cyclodextrins or cyclodextrin derivatives, or also: T. Loftsson and M. E. Brewster (1996): Pharmaceutical Applications of Cyclodextrins: Drug Solubilization and Stabilisation: Journal of Pharmaceutical Science 85 (10):1017-1025; R. A. Rajewski and V. J. Stella (1996): Pharmaceutical Applications of Cyclodextrins: In Vivo Drug Delivery: Journal of Pharmaceutical Science 85 (11): 1142-1169).

In the following description of the working up, “epothilone” is understood to be an epothilone which is obtainable from the corresponding microorganism, preferably epothilone C, D, E, F or especially A or in particular epothilone B. If not otherwise stated, where “epothilones” are mentioned, this is intended to be a general term which includes individual epothilones or mixtures.

Working up of the epothilones is effected by conventional methods; first of all, by separating a culture into the liquid phase (centrifugate or filtrate) and solid phase (cells) by means of filtration or centrifugation (tubular centrifuge or separator). The (main) part of the epothilones found in the centrifugate or in the filtrate is then directly mixed with a synthetic resin, for example a resin based on polystyrene as matrix (hereinafter referred to also simply as polystyrene resin), such as Amberlite XAD-16 [Rohm & Haas Germany GmbH, Frankfurt] or Diaion HP-20 [Resindion S. R . L., Mitsubishi Chemical Co., Milan] (preferably in a ratio of centrifugate: resin volume of ca. 10:1 to 100:1, preferably about 50:1). After a period of contact of preferably 0.25 to 50 hours, especially 0.8 to 22 hours, the resin is separated, for example by filtration or centrifugation. If required, after adsorption, the resin is washed with a strongly polar solvent, preferably with water. Desorption of the epothilones is then effected with an appropriate solvent, especially with an alcohol, in particular isopropanol. The solvent phase, especially the isopropanol phase, is then removed from the solvent, preferably by means of prior, simultaneous or subsequent addition of water, in particular in a circulating evaporator, thereby being concentrated if necessary, and the resulting water phase is extracted with a solvent suitable for forming a second phase, such as an ester, for example a lower alkanol lower alkanoate, typically ethyl acetate or isopropyl acetate. The epothilones are thereby transferred into the organic phase. Then the organic phase is concentrated to the extent necessary, preferably to dryness, for example using a rotary evaporator.

Subsequently, further processing takes place using the following steps, whereby the purification step by means of reversed-phase chromatography with elution with a nitrile is an inventive step and is thus compulsory, while the other steps are optional:

molecular filtration (gel chromatography), e.g. on a column of material such as Sephadex LH-20 (Pharmacia, Uppsala, Sweden) with an alcohol such as methanol as eluant;

separation of the epothilones by reversed-phase chromatography after being taken up in a suitable solvent, and elution with a mixture of nitrile/water (compulsory), preferably characterised in that the chromatography is carried out on a column of material, especially a RP-18 material, which is charged with hydrocarbon chains, such as hydrocarbon chains containing 18 carbon atoms, and an eluant comprising a nitrite, especially a lower alkylnitrile, in particular acetonitrile, is used, in particular a mixture of nitrile/water is used, especially a mixture of acetonitrile/water, preferably in a ratio of nitrile to water of about 1:99 to 99:1, primarily between 1:9 and 9:1, e.g. between 2:8 and 7:3, e.g. 3:7 or 4:6.

single or multiple extraction of the residue (especially after evaporation) in a two-phase system consisting of water and a solvent immiscible with water, preferably an ester, in particular a lower alkyl lower alkanoate, such as ethyl acetate or isopropyl acetate;

adsorption chromatography, in particular by adding to a column of silica gel and eluting with an appropriate solvent or solvent mixture, especially a mixture of ester/hydrocarbon, for example lower alkyl alkanoate/C4-C10-alkane, especially ethyl or isopropyl acetate/n-hexane, in which the ratio between the ester and hydrocarbon is preferably in the range 99:1 to 1:99, preferably 10:1 to 1: 10, for example 4:1;

dissolving the residue, which may be obtained after concentration, in an appropriate solvent such as an alcohol, e.g. methanol;

mixing with activated carbon and removal thereof;

recrystallisation, e.g. from appropriate solvents or solvent mixtures, for example consisting of esters, ester/hydrocarbon mixtures or alcohols, especially ethyl or isopropyl acetate: toluene 1:10 to 10:1, preferably 2:3 (epothilone A) or methanol or ethyl acetate (epothilone B);

whereby between each step being employed, the resulting solutions or suspensions are concentrated if necessary, and/or liquid and solid components are separated from one another, in particular by filtering or centrifuging solutions/suspensions. The more precise definitions mentioned below can be preferably used in the above individual steps.

The further working up and purification is preferably carried out

either by direct separation of the epothilones from one another by reversed-phase chromatography after being taken up in an appropriate solvent, for example a nitrile/water mixture, especially an acetonitrile/water mixture (ratio of nitrile to water 1:99 to 99:1, preferably 1:9 to 9:1, especially 3:1), if necessary after filtration or centrifugation, preferably on a silica gel that has been derivatized by hydrocarbon radicals, e.g. a silica gel modified by alkyl radicals containing 8 to 20, especially 18, C-atoms, eluting with an eluant comprising a nitrile, especially a lower alkylnitrile, such as acetonitrile, especially a mixture of the nitrile with water, such as an acetonitrile/water mixture, whereby detection of the interesting fractions is effected in conventional manner, for example by UV detection or (preferably) by on-line HPLC (HPLC with a very small column, the analyses taking less than 1 minute, and detection e.g. at 250 nm), this enabling a particularly exact separation of the fractions containing the desired product to take place; if required, with subsequent concentration, for example by distillation, to remove the nitrile; if desired, with subsequent single or multiple, for example double, extraction of the residue of evaporation in a two-phase system consisting of water and an immiscible solvent, such as ethyl acetate or isopropyl acetate; additional concentration of the organic phase and dissolving of the residue in an appropriate solvent, preferably an ester such as ethyl acetate or isopropyl acetate, if required, filtration or centrifugation, if desired adding to a column of silica gel and eluting with an appropriate solvent or solvent mixture, for example with a mixture of ester/hydrocarbon, e.g. lower alkyl alkanoate/C4-C10-alkane, especially ethyl or isopropyl acetate/n-hexane, in which the ratio of ester to hydrocarbon is preferably in the range 99:1 to 1:99, preferably 10:1 to 1:10, e.g. 4:1; subsequent combining of the fractions containing each desired epothilone, especially epothilone A or epothilone B, and after removing the solvent, for example by distillation, preferably concentrating to dryness; then, dissolving of the residue in an appropriate alcohol, preferably methanol; and if desired, in order to obtain especially high purity, mixing with activated carbon and then separating the activated carbon, for example by filtration; and finally, by recrystallisation as described below under variant 2 (for epothilone B in particular from methanol), separate extraction of the epothilones, especially epothilones A or B. This is the most preferred variant 1, the outstanding characteristic of which is the surprising direct separation by reversed-phase chromatography of the epothilone-containing mixture desorbed by the resin, despite all the impurities in the organic extract;

or (variant 2) first of all exclusion chromatography takes place (molecular filtration) e.g. on a column of material such as Sephadex LH-20 (Pharmacia, Uppsala, Sweden) with an alcohol such as methanol as eluant, and then subsequent separation of the epothilones present in the peak fractions obtained, e.g. epothilone A and B, by reversed-phase chromatography as described above for variant 1; if required twice, if peak fractions of one epothilone contain those of another, for example if those with epothilone A still contain residues of epothilone B; and then separate recrystallisation of each epothilone from appropriate solvents or solvent mixtures, for example from ethyl or isopropyl acetate:toluene 1:10 to 10:1, preferably 2:3 (epothilone A) or methanol or ethyl acetate (epothilone B). This is variant 2 of working up and purification.

With variant 1, highly pure epothilone B may be obtained in a relatively simpler manner (the purity is preferably greater than 97%, especially over 99%).

Variant 1 preferably takes place as follows (whereby preferably the above-mentioned variants can be used instead of the following general definitions): First of all, a culture is separated into the liquid phase (centrifugate or filtrate) and a solid phase (cells) by means of filtration or centrifugation (tubular centrifuge or separator). The (main) part of the epothilones found in the centrifugate or filtrate is then directly mixed with a synthetic resin. After a contact period of preferably 0.25 to 50 hours, the resin is separated, for example by filtration or centrifugation. If required, after adsorption, the resin is washed with a strongly polar solvent, preferably with water. Desorption of the epothilones is then effected with an appropriate solvent, especially with an alcohol, in particular isopropanol. The solvent phase, especially isopropanol phase, is then removed from the solvent, preferably by means of prior, simultaneous or subsequent addition of water, in particular in a circulating evaporator, thereby being concentrated if necessary, and the resulting water phase is extracted with a solvent suitable for forming a second phase, such as an ester, for example a lower alkanol lower alkanoate, typically ethyl acetate or isopropyl acetate. The epothilones are then transferred into the organic phase. Then the organic phase is concentrated to the extent necessary, preferably to dryness, for example using a rotary evaporator. By subsequent reversed-phase chromatography on a silica gel derivatized with hydrocarbon atoms, e.g. a silica gel modified by alkyl radicals containing 18 C-atoms, and eluting with a mixture of a lower alkylnitrile such as acetonitrile with water, the epothilones are directly separated from one another, especially epothilone A and epothilone B; then, concentration takes place by means of distillation, the residue is shaken out once or more, if desired from water with an appropriate solvent that is immiscible with water, preferably an ester such as isopropyl acetate, then the organic phase is again concentrated and the residue of evaporation is dissolved in an ester such as ethyl or isopropyl acetate, filtered if required, the filtrate added to a column of silica gel, and eluted with a mixture of ester/hydrocarbon, e.g. ethyl or isopropyl acetate/n-hexane; subsequently, the fractions containing the epothilone, especially epothilone A or B, are respectively combined and, after removing the solvent by distillation, concentrated, preferably to dryness; the residue is then dissolved in an appropriate lower alkanol, preferably methanol, and in order to obtain especially high purity, mixed with activated carbon and then filtered; finally, the epothilones are extracted by recrystallisation (in the case of epothilone B preferably from methanol).

Variant 2 is effected preferably as follows: After harvest, a culture is separated into the liquid phase (centrifugate) and solid phase (cells) by means of centrifugation (tubular centrifuge or separator). The main part of the epothilones are found in the centrifugate, which is then directly mixed with a polystyrene resin, such as Amberlite XAD-16 [Rohm & Haas Germany GmbH, Frankfurt] or Diaion HP-20 [Resindion S. R. L., Mitsubishi Chemical Co., Milan] (preferably in a ratio of centrifugate: resin volume of ca. 10:1 to 100:1, preferably about 50:1) and stirred in an agitator. In this step, the epothilones are transferred from the cyclodextrin to the resin. After a period of contact of ca. 1 hour, the resin is separated by centrifugation or filtration. Adsorption of the epothilones onto the resin may also be effected in a chromatography column, by placing the resin in the column and running the centrifugate over the resin. After adsorption, the resin is washed with water. Desorption of the epothilones from the resin is effected with isopropanol. The isopropanol phase is then freed of isopropanol preferably by the addition of water in particular in a circulating evaporator, and the resulting water phase is extracted with ethyl acetate. The epothilones are thus transferred from the water phase to the ethyl acetate phase. Then the ethyl acetate extract is concentrated to dryness, for example using a rotary evaporator. An initial concentration of the epothilones is then achieved by means of molecular filtration (e.g. Sephadex LH-20 [Pharmacia, Uppsala, Sweden] with methanol as eluant). The peak fractions from the molecular filtration contain epothilone A and B and have a total epothilone content of >10%. Separation of these peak fractions, which contain epothilone A and B in a mixture, into the individual components, then follows by means of chromatography on a “reversed-phase”, e.g. RP-18 phase (phase which is modified by alkyl radicals containing 18 carbon atoms per chain), with an appropriate eluant, preferably one containing a nitrile such as acetonitrile (this gives better separation than for example alcohols such as methanol). Epothilone A elutes before epothilone B. The peak fractions with epothilone B may still contain small portions of epothilone A, which can be removed by further separation on RP-18. Finally, the epothilone A fraction is crystallised directly from ethyl acetate:toluene=2:3, and the epothilone B fraction from methanol or ethyl acetate.

The invention preferably relates to a process for the concentration of epothilones, especially epothilone A and/or B, in particular epothilone B, in a culture medium for the biotechnological preparation of this (these) compound(s), which contains a microorganism which is suitable for this preparation, especially a Sorangium strain, especially of the type Sorangium Cellulosum Soce90, or a mutant arising therefrom, in particular the strain having reference BCE 33/10, especially the strain having reference BCE 63/114, water and other usual appropriate constituents of culture media, whereby a cyclodextrin or a cyclodextrin derivative, or a mixture of two or more of these compounds is added to the medium, especially one or more, preferably one or two or more cyclodextrins selected from 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), especially α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin; or primarily a cyclodextrin derivative or mixture of cyclodextrin derivatives selected from derivatives of a cyclodextrin, in which one or more up to all of the hydroxy groups are etherified to an alkyl ether, especially lower alkyl, such as methyl or ethyl ether, also propyl or butyl ether; an aryl-hydroxyalkyl ether, such as phenyl-hydroxy-lower-alkyl, especially phenyl-hydroxyethyl ether; a hydroxyalkyl ether, in particular hydroxy-lower-alkyl ethers, especially 2-hydroxyethyl, hydroxypropyl such as 2-hydroxypropyl or hydroxybutyl such as 2-hydroxybutyl ether; a carboxyalkyl ether, in particular carboxy-lower-alkyl ether, especially carboxymethyl or carboxyethyl ether; a derivatised carboxyalkyl ether, in particular a derivatised carboxy-lower-alkyl ether in which the derivatised carboxy is aminocarbonyl, mono- or di-lower-alkyl-aminocarbonyl, morpholino-, piperidino-, pyrrolidino- or piperazino-carbonyl, or alkyloxycarbonyl, in particular lower alkoxycarbonyl, such as preferably lower alkoxycarbonyl-lower-alkyl ether, for example methyloxycarbonylpropyl ether or ethyloxycarbonylpropyl ether; a sulfoalkyl ether, in particular sulfo-lower-alkyl ether, especially sulfobutyl ether; a cyclodextrin in which one or more OH groups are etherified with a radical of formula

—O—[alk-O—]n-H

wherein alk is alkyl, especially lower alkyl, and n is a whole number from 2 to 12, especially 2 to 5, in particular 2 or 3; a cyclodextrin in which one or more OH groups are etherified with a radical of formula

wherein R1 is hydrogen, hydroxy, —O—(alk-O)z—H, —O—(Alk(—R)—O—)p-H or —O—(alk(—R)—O—)q-alk—CO—Y; alk in all cases is alkyl, especially lower alkyl; m, n, p, q and z are a whole number from 1 to 12, preferably 1 to 5, in particular 1 to 3; and Y is OR, or NR2R3, wherein R1, R2 and R3 independently of one another, are hydrogen or lower alkyl, or R2 and R3 combined together with the linking nitrogen signify morpholino, piperidino, pyrrolidino or piperazino; or a branched cyclodextrin, in which etherifications or acetals with other sugars are present, and which are selected from glucosyl-, diglucosyl- (G2-β-cyclodextrin), maltosyl- or dimaltosyl-cyclodextrin, or N-acetylglucosaminyl-, glucosaminyl-, N-acetylgalactosaminyl- and galactosaminyl-cyclodextrin; or a lower alkanoyl, such as acetyl ester of a cyclodextrin. Particular preference is given to a process in which the cyclodextrin and/or the cyclodextrin derivative is added to the culture medium in a concentration of 0.02 to 10, preferably 0.005 to 10, more preferably 0.05 to 5, most preferably 0.1 to 4, for example 0.1 to 2, percent by weight (w/v).

Especially preferred is a process according to one of the two previous paragraphs, in which the cyclodextrin derivative is selected from a cyclodextrin, especially β-cyclodextrin, and a hydroxy lower alkyl-cyclodextrin, especially 2-hydroxypropyl-α-, -β- or -γ-cyclodextrin; or mixtures of one or more thereof; whereby 2-hydroxypropyl-β-cyclodextrin on its own is preferred in particular.

The invention also relates in particular to a culture medium, which comprises a cyclodextrin, a cyclodextrin derivative or a mixture of two or more complex-forming components selected from cyclodextrins and cyclodextrin derivatives, especially a cyclodextrin or cyclodextrin derivative as defined in the third-last paragraph, in particular as in the second-last paragraph, or a mixture of one or more of these compounds, and a microorganism which is suitable for producing epothilones, especially epothilone A and/or B, preferably a strain from the genus Sorangium, especially a strain of Sorangium Cellulosum, e.g. the strain Soce90 or a mutant arising therefrom, in particular the strain BCE 33/10, or especially BCE 63/114.

A further aspect of the invention relates to a process for the production of epothilone A and/or B, especially the two pure compounds, in particular epothilone B, which is characterised in that the epothilones are separated for example by centrifugation into the solid and the liquid phase (centrifugate) by working up a culture medium for the biotechnological preparation of these compounds, as described above, to which has been added a complex-forming component which is soluble in the culture medium, in particular a cyclodextrin, a cyclodextrin derivative or a mixture of two or more cyclodextrins and/or cyclodextrin derivatives; the centrifugate is mixed with a resin, especially a polystyrene resin, or is run through a column filled with such a resin; if necessary, the resin is washed with water; the epothilone(s) is or are desorbed from the resin using a polar solvent, especially an alcohol, primarily a lower alkanol such as isopropanol; if necessary, concentrated by means of prior, simultaneous or subsequent addition of water; an organic solvent which is immiscible with water, for example an ester, such as ethyl acetate, is added, and the epothilone(s) is or are transferred to the organic phase, for example by agitating or stirring; where necessary, the organic phase is concentrated; the epothilones from the organic solution obtained are concentrated through a molecular sieve for compounds of low molecular weight; and then the fractions containing the epothilones, especially epothilone A and/or B undergo separation by a reversed-phase column, preferably eluting with an eluant containing a nitrile, such as acetonitrile (or instead, an eluant containing an alcohol, such as methanol); whereby epothilones A and B are extracted separately, and if desired, can be further concentrated by recrystallisation.

One preferred aspect of the invention relates to a process for the production of epothilone A and/or B, especially the two pure compounds, in particular epothilone B, which is characterised in that the epothilones are separated for example by centrifugation into the solid and the liquid phase (centrifugate) by working up a culture medium for the biotechnological preparation of these compounds, as described above, to which has been added a complex-forming component which is soluble in the culture medium, in particular a cyclodextrin, a cyclodextrin derivative or a mixture of two or more cyclodextrins and/or cyclodextrin derivatives; the centrifugate is mixed with a resin, especially a polystyrene resin, or is run through a column filled with such a resin; if necessary, the resin is washed with water; the epothilone(s) is or are desorbed from the resin using a polar solvent, especially an alcohol, primarily a lower alkanol such as isopropanol; if necessary, the polar solvent is removed by means of prior, simultaneous or subsequent addition of water; the resulting water phase is extracted with a solvent which is suitable for forming a second phase, for example an ester, such as diethyl ester, if necessary, the organic phase is concentrated, preferably to dryness; epothilone A and B are separated from one another directly by reversed-phase chromatography, eluting with an eluant containing a nitrile, especially a lower alkylnitrile, such as acetonitrile, whereby detection is effected in the usual manner, for example by UV detection or preferably by on-line HPLC (HPLC with a very small column, the analyses taking less than 1 minute, and detection e.g. at 250 nm); subsequent concentration, for example by distillation; if desired, the residue is treated from an aqueous solution once or more (for example twice) by extraction with a solvent which is immiscible with water, such as an ester; dissolved in an appropriate solvent, preferably an ester such as ethyl or isopropyl acetate, filtered if necessary, added to a column of silica gel and eluted with an appropriate solvent or solvent mixture, for example with an ester/hydrocarbon mixture; and subsequently, the fractions containing either epothilone A or especially B are separately combined and, after removing the solvent, for example by distillation, concentrated preferably to dryness; then the residue is dissolved in an appropriate alcohol, preferably methanol, then if desired, in order to obtain especially high purity, treated with activated carbon and then filtered; and finally epothilone A or B is obtained by recrystallisation (in the case of epothilone B, particularly from methanol).

A further preferred aspect of the invention relates to a method of separating epothilones, especially epothilones A and B from one another, which is characterised by chromatography on a reversed-phase column with an eluant containing a lower alkyl cyanide, chromatography being carried out on a column material, especially an RP-18 material, which is charged with hydrocarbon chains containing 18 carbon atoms, and employing an eluant containing a nitrile, especially a lower alkylnitrile, in particular acetonitrile, especially a mixture of nitrile/water, in particular a mixture of acetonitrile/water, preferably in a ratio of nitrile to water of ca. 1:99 to 99:1, primarily 1:9 to 9:1, e.g. between 2:8 and 7:3, e.g. 3:7 or 4:6. This separation may follow on to a filtration with a molecular sieve, or is preferably effected directly using the residue after adsorption of the epothilones from the culture medium containing a complex-forming component onto a resin, as described above (“variant 1”). One advantage of separation with an eluant containing a lower alkylcyanide over that using alcohols, such as methanol, is the better separation of epothilones A and B.

The invention relates preferably to a process for the preparation of epothilones, which a) comprises a process for the concentration of epothilones, especially epothilone A and/or B, in particular epothilone B, in a culture medium for the biotechnological preparation of this (these) compound(s), which contains a microorganism which is suitable for this preparation, especially a Sorangium strain, especially of the type Sorangium Cellulosum Soce90, or a mutant arising therefrom, in particular the strain having reference BCE 33/10, especially the strain having reference BCE 63/114, water and other usual appropriate constituents of culture media, whereby a cyclodextrin or a cyclodextrin derivative, or a mixture of two or more of these compounds is added to the medium, especially one or more, preferably one or two or more cyclodextrins selected from a-cyclodextrin (6), β-cyclodextrin (7), γ-cyclodextrin (8), δ-cyclodextrin (9), ε-cyclodextrin (10), ζ-cyclodextrin (11), η-cyclodextrin (12), and θ-cyclodextrin (13), especially α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin; or primarily a cyclodextrin derivative or mixture of cyclodextrin derivatives selected from derivatives of a cyclodextrin, in which one or more up to all of the hydroxy groups are etherified to an alkyl ether, especially lower alkyl, such as methyl or ethyl ether, also propyl or butyl ether; an aryl-hydroxyalkyl ether, such as phenyl-hydroxy-lower-alkyl, especially phenyl-hydroxyethyl ether; a hydroxyalkyl ether, in particular hydroxy-lower-alkyl ether, especially 2-hydroxyethyl, hydroxypropyl such as 2-hydroxypropyl or hydroxybutyl such as 2-hydroxybutyl ether; a carboxyalkyl ether, in particular carboxy-lower-alkyl ether, especially carboxymethyl or carboxyethyl ether; a derivatised carboxyalkyl ether, in particular a derivatised carboxy-lower-alkyl ether in which the derivatised carboxy is aminocarbonyl, mono- or di-lower-alkyl-aminocarbonyl, morpholino-, piperidino-, pyrrolidino- or piperazino-carbonyl, or alkyloxycarbonyl, in particular lower alkoxycarbonyl, such as preferably a lower alkoxycarbonyl-lower-alkyl ether, for example methyloxycarbonylpropyl ether or ethyloxy-carbonylpropyl ether; a sulfoalkyl ether, in particular sulfo-lower-alkyl ether, especially sulfobutyl ether; a cyclodextrin in which one or more OH groups are etherified with a radical of formula

—O—[alk—O—]n—H

wherein alk is alkyl, especially lower alkyl, and n is a whole number from 2 to 12, especially 2 to 5, in particular 2 or 3; a cyclodextrin in which one or more OH groups are etherified with a radical of formula

wherein R1 is hydrogen, hydroxy, —O—(alk—O)z—H, —O—(Alk(—R)—O—)p—H or —O—(alk(—R)—O—)q-alk—CO—Y; alk in all cases is alkyl, especially lower alkyl; m, n, p, q and z are a whole number from 1 to 12, preferably 1 to 5, in particular 1 to 3; and Y is OR1 or NR2R3 , wherein R1, R2 and R3 independently of one another, are hydrogen or lower alkyl, or R2 and R3 combined together with the linking nitrogen signify morpholino, piperidino, pyrrolidino or piperazino;

or a branched cyclodextrin, in which etherifications or acetals with other sugars are present, and which are selected from glucosyl-, diglucosyl-(G2-β-cyclodextrin), maltosyl- or di-maltosyl-cyclodextrin, or N-acetylglucosaminyl-, glucosaminyl-, N-acetylgalactosaminyl- and galactosaminyl-cyclodextrin; or a lower alkanoyl, such as acetyl ester of a cyclodextrin; and

b) comprises a step for separating the epothilones, especially epothilones A and B, from one another, which is characterised by chromatography on a reversed-phase column with an eluant containing a lower alkylcyanide, the chromatography being carried out on a column material, especially an RP-18 material, which is charged with hydrocarbon chains containing 18 carbon atoms, and employing an eluant containing a lower alkylnitrile, especially acetonitrile, in particular a mixture of lower alkylnitrile/water, preferably a mixture of acetonitrile/water, preferably in a ratio of lower alkylnitrile to water of ca. 1:99 to 99:1, primarily 1:9 to 9:1, e.g. between 2:8 and 7:3, e.g. 3:7 or 4:6, whereby if desired, it is possible to use further steps for working up and purification.

The invention also relates in particular to a mutant derived from the strain Sorangium cellulosum Soce90, especially a strain of Sorangium cellulosum which is obtainable by mutagenesis, preferably by one or more UV-induced mutagenesis steps (in particular by UV radiation in the range 200 to 400, especially 250 to 300 nm) with subsequent searching in each step for mutants having increased epothilone production (in particular increased epothilone concentration in the culture medium), this strain under otherwise identical conditions producing more epothilones, in particular more epothilone A and/or B, especially epothilone B, than Sorangium cellulosum Soce90, especially the Sorangium cellulosum strain BCE 33/10, in particular BCE 114.

The invention relates in particular to the individual process steps named in the examples or any combination thereof, the culture media named therein, crystal forms and the strain described therein.

The invention also relates to new crystal forms of epothilone B, especially a crystal form of epothilone B described as modification B and in particular described as modification A.

The crystal forms can be distinguished in particular by their X-ray diagrams. X-ray diagrams taken with a diffractometer and using Cu-Kα1-radiation are preferably used to characterize solids of organic compounds. X-ray diffraction diagrams are used particularly successfully to determine the crystal modification of a substance. To characterize the existing crystal modification A and crystal modification B of epothilone B, the measurements are made at an angle range (2θ) of 2° and 35° with samples of substance that are kept at room temperature.

The X-ray diffraction diagram thus determined (reflection lines and intensities of the most important lines) from crystal modification A (modification A) of epothilone B is characterized by the following table.

2θ Intensity 7.7 very strong 10.6 weak 13.6 average 14.4 average 15.5 average 16.4 weak 16.8 weak 17.1 weak 17.3 weak 17.7 weak 18.5 weak 20.7 strong 21.2 strong 21.9 weak 22.4 weak 23.3 strong 25.9 average 31.2 weak 32.0 average

The invention also relates in particular to a new crystal form of epothilones B, which is characterised by a melting point of more than 120° C., especially between 120° C. and 128° C., in particular 124-125° C. Surprisingly, this value is considerably higher than the values previously described in litreature. The invention relates especially to a crystal form of epothilone B, which is characterised by the X-ray diffraction diagram of the crystal form A and a melting point of above 120°, especially between 120° C. and 128° C., for example between 124° C. and 125° C.

The X-ray diffraction diagram thus determined (reflection lines and intensities of the most important lines) of crystal modification B (modification B) of epothilone B is characterized by the following table.

2θ Intensity

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