US2003194787A1PendingUtilityA1
Fermentative preparation process for and crystal forms of cytostatics
Priority: Feb 19, 1998Filed: Jan 8, 2003Published: Oct 16, 2003
Est. expiryFeb 19, 2018(expired)· nominal 20-yr term from priority
Inventors:Hans HofmannMarion MahnkeKlaus MemmertFrank PetersenThomas SchuppErnst KustersMichael Mutz
A61P 35/00A61P 35/04A61P 43/00C07K 2299/00C07D 493/04C12P 17/167C12P 17/181C07D 417/14C12P 17/18C12R 2001/01C12N 1/205
52
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Claims
Abstract
The invention relates to a new process for concentrating epothilones in culture media, a new process for the production of epothilones, a new process for separating epothilones A and B and a new strain obtained by mutagenesis for the production of epothilones, as well as aspects related thereto. New crystal forms of epothilone B are also described.
Claims
exact text as granted — not AI-modified1 . A process for concentrating epothilones in a culture medium for the biotechnological preparation of these compounds, the process comprising microorganisms which produce these compounds, whereby a complex-forming component which is soluble in the culture medium is added to the medium.
2 . A process according to claim 1 for concentrating epothilones in a culture medium for the biotechnological preparation of these compounds, the process comprising myxobacteria as producers of natural substances, whereby a complex-forming component which is soluble in the culture medium is added to the medium.
3 . A process according to claim 1 for concentrating epothilones, in which a culture medium is used for the biotechnological preparation of epothilones, the medium containing a Sorangium strain suitable for the preparation thereof, water and other suitable customary components of culture media,
wherein one or more cyclodextrins are added to the medium, the cyclodextrins being selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, δ-cyclodextrin, ε-cyclodextrin, ζ-cyclodextrin, η-cyclodextrin and θ-cyclodextrin; or a cyclodextrin derivative or a 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, an aryl-hydroxyalkyl ether; a hydroxyalkyl ether; a carboxyalkyl ether; a derivatised carboxy lower alkyl ether in which the derivatised carboxy is aminocarbonyl, mono- or di-lower-alkylaminocarbonyl, morpholino-, piperidino-, pyrrolidino- or piperazino-carbonyl, or alkyloxycarbonyl; a sulfoalkyl 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 and n is a whole number from and including 2 up to and including 12; a cyclodextrin in which one or more OH groups are etherified with a radical of formula
wherein R′ is hydrogen, hydroxy, —O-(alk-O) n —H, —O-(alk(-R)—O—) p —H or —O-(alk(-R)—O—) q -alk-CO—Y; alk in all cases is alkyl; m, n, p, q and z are a whole number from 1 to 12; and Y is OR 1 or NR 2 R 3 , wherein R 1 , R 2 and R 3 , independently of one another, are hydrogen or lower alkyl, or R 2 and R 3 , combined with the binding nitrogen signify morpholino, piperidino, pyrrolidino or piperazino; or a branched cyclodextrin in which etherifications or acetals exist with other sugar molecules, and which are selected from glucosyl-, diglucosyl-(G 2 -β-cyclodextrin), maltosyl- and dimaltosyl-cyclodextrin, or N-acetylglucosaminyl-, glucosaminyl-, N-acetylgalactosaminyl- and galactosaminyl-cyclodextrin; and a lower alkanoyl-, such as acetylester of a cyclodextrin; or mixtures of two or more of the said cyclodextrins and/or cyclodextrin derivatives.
4 . Process according to claim 3 , in which the cyclodextrin and/or the cyclodextrin derivative is added to the culture medium in a concentration of between 0.05 and 10 percent by weight (w/v).
5 . Process according to claim 4 , in which the cyclodextrin and/or the cyclodextrin derivative is added in a concentration of between 0.1 and 2 percent by weight.
6 . A process according to claim 3 , in which the cyclodextrin derivative is selected from a cyclodextrin and a hydroxy lower alkyl cyclodextrin; or mixtures of one or more thereof.
7 . A process according to claim 1 , in which the complex-forming component is 2-hydroxy-propyl-β-cyclodextrin.
8 . A culture medium which comprises a complex-forming component and a microorganism suitable for the production of epothilones.
9 . A culture medium according to claim 8 , in which the complex-forming component used is a cyclodextrin, a cyclodextrin derivative or a mixture of two or more complex-forming components selected from cyclodextrins and cyclodextrin derivatives.
10 . A culture medium according to claim 9 , in which the microorganism is a myxobacterium.
11 . A process for the production of epothilones, in which the epothilones are obtained by working up a culture medium for the biotechnological preparation of these compounds, the medium comprising myxobacteria as producers of natural substances, to which medium is added a complex-forming component which is soluble in the culture medium, and subsequently purifying and, if desired, separating the epothilones from one another.
12 . A process according to claim 11 , wherein the epothilones are obtained by working up a culture medium for the biotechnological preparation of these compounds, the culture medium containing a myxobacterium of the genus Sorangium, and to which medium is added a complex-forming component which is soluble in the culture medium, the culture is separated into the solid and the liquid phase (centrifugate) by centrifugation; the centrifugate is mixed with a resin or is run over 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 with a polar solvent; if necessary this is concentrated with prior, simultaneous or subsequent addition of water; an organic solvent which is immiscible with water is added and the epothilone(s) is or are transferred into the organic phase; the organic phase obtained is concentrated if required; the epothilones from the organic solution obtained are concentrated through a molecular sieve for compounds of low molecular weight; and subsequently the fractions containing the epothilones undergo separation on a reversed-phase column; whereby epothilones A and B are obtained separately and, if desired, can be further concentrated by recrystallisation.
13 . A process according to claim 11 for the production of epothilone A and/or B, wherein the epothilones are obtained by working up a culture medium for the biotechnological preparation of these compounds, to which medium is added a complex-forming component which is soluble in the culture medium, the culture is separated into the solid and the liquid phase (centrifugate) by centrifugation; the centrifugate is mixed with a 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 with a polar solvent; if necessary the polar solvent is removed with prior, simultaneous or subsequent addition of water; the resulting water phase is extracted with a solvent suitable for forming a second phase; the organic phase obtained is concentrated if required, preferably to dryness; epothilone A and epothilone B are separated from one another directly by reversed-phase chromatography whilst eluting with an eluant containing a nitrile; subsequently concentrated; if desired, the residue is treated from an aqueous solution by extracting once or several times with a solvent which is immiscible with water; dissolved in an appropriate solvent, filtered if necessary, added to a silica gel column and eluted with an appropriate solvent or solvent mixture; and subsequently each fraction containing epothilone A or in particular B is separately combined and concentrated by removing the solvent; then the residue is dissolved in an appropriate alcohol, if desired, in order to obtain especially high purity, mixed with activated carbon and then filtered; and finally epothilone A or B is obtained by recrystallisation.
14 . A process for the production of epothilones, which
a) is a process for concentrating epothilones in a culture medium for the biotechnological preparation of these compounds, which contains a microorganism suitable for the preparation thereof, water and other suitable customary constituents of culture media, whereby a cyclodextrin or a cyclodextrin derivative is added to the medium, or a mixture of two or more of these compounds; and b) comprises a step for separating epothilones from one another, which is characterised by chromatography on a reversed-phase column with an eluant containing a lower alkyl-cyanide, whereby chromatography is carried out on column material charged with hydrocarbon chains, and an eluant containing a lower alkylnitrile is used; whereby if desired further working up steps and purification steps are possible.
15 . A method of separating epothilones from one another, which is characterised by chromatography on a reversed-phase column with an eluant containing a lower alkyl-cyanide.
16 . A method according to claim 15 , wherein a column material is used which is charged with hydrocarbon chains containing 18 carbon atoms, and the eluant used is a mixture of water and acetonitrile.
17 . A strain of Sorangium cellulosum , obtained by mutagenesis, which under otherwise identical conditions, produces more epothilones than Sorangium cellulosum Soce90.
18 . A strain according to claim 17 , selected from the strains having the references BCE33/10 and BCE63/114.
19 . A crystal form of epothilone B having the reference modification A, which is characterised by the X-ray diffraction diagram reproduced in the form of a table, obtained using a diffractometer with Cu-Kα 1 radiation.
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
20 . A crystal form of epothilone B having the reference modification B, which is characterised by the X-ray diffraction diagram reproduced in the form of a table, obtained using a diffractometer with Cu-Kα 1 radiation.
2θ
Intensity
6.9
very strong
8.0
weak
8.3
average
10.8
strong
11.5
average
12.4
weak
13.1
strong
15.5
weak
16.2
weak
16.7
average
18.1
average
18.6
average
20.4
weak
20.9
strong
21.3
weak
21.5
very weak
22.5
average
24.2
weak
25.1
average
21 . A pharmaceutical composition which is suitable for administration to a warm-blooded animal for the treatment of a proliferative disease, which contains a quantity of an active ingredient according to one of claims 19 and 20 , which is suitable for the treatment of said disease together with a pharmaceutically acceptable carrier.
22 . Method of treating a warm-blooded animal suffering from a proliferative disease, by administering a dosage of epothilone B which is effective for treating said disease according to one of claims 19 and 20 to a warm-blooded animal requiring such treatment.
23 . Use of a new crystal form of epothilone B according to one of claims 19 and 20 in the production of pharmaceutical preparations.Join the waitlist — get patent alerts
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