Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
2002-01-29
2003-12-02
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S152000, C514S365000, C540S462000, C548S181000, C548S204000, C548S510000, C548S567000, C546S340000
Reexamination Certificate
active
06656711
ABSTRACT:
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.
BACKGROUND TO THE INVENTION
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 &bgr;-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. Nos. 5,641,803; 5,496,804; 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 m
2
/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 microorganisms, 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 OF THE INVENTION
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 als
Hofmann Hans
Küsters Ernst
Mahnke Marion
Memmert Klaus
Mutz Michael
Dohmann George R.
Lilling Herbert J.
Novartis AG
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