WO2000018883A1

WO2000018883A1 - TAXANE AND TAXINE DERIVATIVES FROM $i(PENICILLIUM SP.)

Info

Publication number: WO2000018883A1
Application number: PCT/BE1999/000119
Authority: WO
Inventors: Mondher El Jaziri; Billo Diallo
Original assignee: Universite Libre De Bruxelles
Priority date: 1998-09-29
Filing date: 1999-09-15
Publication date: 2000-04-06
Also published as: EP1117760A1; CA2346000A1

Abstract

10-Deacetylbaccatin III and other taxane derivatives are produced by Penicillium sp. This production is potentially enhanced by elicitors present in Taxus sp. extracts. The fungus producing 10-Deacetylbaccatin III is identified as Penicillium aurantiogriseum Dierckx (IHEM 14753). The present invention relates to these fungi, these elicitors, and the process for producing the taxane derivatives, including the culture of the micro-organisms in a medium and the recovery of compounds from the culture. The present invention indicates that taxane derivatives can be produced efficiently within a short time.

Description

TAXANE AND TAXINE DERIVATIVES FROM PENICILLIUM SP ,

Field of the invention

The present invention is related to the use of Penicillium sp. and elicitors isolated from the Taxus extract for the production of 10-Deacetylbaccatin III and other taxane derivatives. Said fungi is a potential important source for taxoid production. Therefore, the present invention concerns also a process for producing said derivatives as well as their use in various prophylactic and/or therapeutical treatments.

Background of the invention

Paclitaxel is a valuable antileukemic and antitumor agent that is now used clinically to treat ovarian and breast cancers in several countries. Limited supply is one of the most important problems in using Paclitaxel as an anticancer agent. At present, the supplies of this compound are inadequate to meet the current or projected demands. Currently, Paclitaxel is mainly produced by extraction from the bark of Taxus brevifolia Nutt . However, its content in this plant is very low, and Taxus is a slow growing plant that is not therefore suitable for cultivation. In order to circumvent these inconveniences, chemical synthesis of Paclitaxel or its hemisynthesis from 10-Deacetylbaccatin III or other related taxanes have been achieved. But the commercial production of Paclitaxel by chemical synthesis is considered to be unfeasible due to the complexity of the taxane skeleton. Hence, the hemisynthesis of Paclitaxel from 10-Deacetylbaccatin III, which can readily be extracted in relatively high yield (0.1%) from Taxus baccata leaves, seems to be presently the best alternative for the production of Paclitaxel. The search for novel processes for producing 10-Deacetylbaccatin III and other related taxanes is therefore worthwhile. In this context, tissue culture of plants of the genus Taxus has been studied world-wide. However, research is still underway and these processes can not yet be developed to substitute for the processes of extraction from plants.

State of the art

A microbial source of Paclitaxel or 10- Deacetylbaccatin and related taxanes would be preferable if it could be easily grown. The production of Paclitaxel and baccatin III by Taxomyces andreanae, an endophytic fungus of Taxus brevifolia, has been reported (US patent 5,322,779). However, the yield of Paclitaxel by this fungus is too low (24-50 ng/litre of culture) for practical use. A novel bacterium, Brevibacterium sp . , has been reported to produce Paclitaxel and other compounds having the taxane skeleton such as baccatin III, 10-Deacetylbaccatin III, etc. (-600 ng of taxane skeleton containing-compounds and -400 ng of Paclitaxel/litre of the culture) (WO patent 95/04154) .

Summary of the invention

The main aim of the present invention is to provide alternative or improved methods for taxane and/or taxine derivatives production, especially for the production of 10-Deacetylbaccatin III and other taxane derivatives, that do not present the drawbacks of the state of the art.

The present invention provides a method for the production of a pharmaceutically active compound, especially 10-deacetylbaccatin III and other taxane or taxine derivatives, by a strain of Penicillium sp.

The present invention is also related to a pure culture of said strain of Penicillium sp. able to produce a diterpene-like compound having a taxane and/or taxine skeleton. Said skeleton is described in the enclosed

Figs . 1 and 2.

Advantageously, the taxane skeleton as represented in Fig. 1 is a taxane skeleton wherein: - Rι_ is an hydrogen atom or an acyl group,

- R₂ is an hydrogen atom or an acyl group,

- R3 is an oxygen atom or the combination of an acetoxyl or hydroxyl group with an hydrogen atom,

- R is an hydrogen atom or an hydroxyl group, - R5 is an hydrogen atom, an acyl group or a glycosyl grou ,

- Ph is a phenyl group, and

- Ac is an acetyl group.

Preferably, said taxane derivative is selected from the group consisting of 10-deacetylbaccatin

III (10-Dab) , baccatin III, Paclitaxel^®, cephalomanine or other known taxanes having preferably active pharmaceutical properties .

According to a second embodiment of the present inventiqn, said diterpene-like compound has a taxine skeleton as represented in Fig. 2. Advantageously, the taxine skeleton as represented in Fig. 2 is a taxine skeleton wherein:

- R_ is an hydrogen atom or an hydroxyl group, and

- R2 , R3 , R4 and R5 are an hydrogen atom or an acyl group . Preferably, said diterpene-like compound is selected from the group consisting of taxine A, taxine B or other known taxines having preferably active pharmaceutical properties .

According to the invention, the preferred Penicillium sp . strains are the ones able to produce the 10-Deacetylbaccatin III and/or taxine B.

Preferably, said strain is the Penicillium aurantiogriseum strain having preferably the deposit number IHEM 14753 that has been deposited at the co-ordinated collection of micro-organisms BCCM (Scientific Institute of Public Health - Louis Pasteur - Mycology IHEM - 14 rue J. Wytsman - B-1050 Brussels (Belgium) ) , said strain having been deposited on September 16, 1998, according to the Budapest Treaty on the International recognition of the deposits of micro-organisms for the purpose of patent procedures .

The present invention concerns also a method for producing said diterpene-like compound comprising the steps of : - culturing in an adequate medium a micro-organism of the genous Penicillium able to produce said diterpene-like compound having a taxane or taxine skeleton, and

- recovering and possibly characterising said diterpene- like compound from the culture medium by various known immunoenzymatic, chromatographic, chemical and/or spectroscopic methods already used, for the analysis and the purification of said compound and possibly of the elicitor(s) present in said medium or present in the Taxus species extract (which are able to enhance the production of by said Penicillium strain or by other known or unknown micro-organisms) .

Advantageously, the method for producing diterpene-like compounds according to the invention comprises the steps of:

- exposing a taxane and/or taxine producing micro-organism according to the invention in a nutrient media capable of supporting growth to said micro-organism, - providing culturing conditions for the media containing said micro-organism, which conditions induce growth and division of said micro-organism, and

- isolating and/or concentrating the desired taxane and/or taxine from said culture media or said microbe. Preferably, said nutrient media contains potential taxoid precursors such as sodium acetate, sodium benzoate or phenylalanine, and an amount of Taxus aqueous extract containing enhancers capable of improving the production of said diterpene-like compounds in the culture media of said microorganism.

A last aspect of the present invention is related to a diterpene-like compound obtained by a Penicillium strain, preferably by the specific Penicillium strain according to the invention above-described. Said diterpene-like compound or taxane or taxine compound could be associated with other taxane or taxine derivatives and could be incorporated in a pharmaceutical composition with an adequate pharmaceutical carrier and/or diluant having various pharmaceutical and/or prophylactic properties, preferably used in the treatment and/or the prevention of ovarian and breast cancer, for the prevention of carcinogenesis and/or the treatment of cancers induced by tumours, especially a malignant tumoral cell including metastases, said compound having a toxic effect upon said tumoral cell or being able to reduce the growth and the dispersion (metastases) of said tumoral cell.

The pharmaceutically adequate carrier (vehicle, excipient or diluant) may vary according to the mode of administration and is possibly combined with an adjuvant in order to improve the therapeutical properties of the pharmaceutical composition or reduce its possible side effects. Suitable pharmaceutically acceptable carriers used in the composition according to the invention are well known by the person skilled in the art and are selected according to the methods generally applied by pharmacists, and may include solid, liquid or gaseous non-toxic pharmaceutically acceptable carriers . The percentage of active product / pharmaceutically acceptable carrier may vary within a very large range limited by the tolerance and the possible side-effects on the patient and by the frequency and the mode of administration.

Known adjuvants that may be used in order to improve the therapeutical properties of the composition according to the invention are usual adjuvants like vitamin C or other molecules that may induce a cellular and/or humoral response against tumoral cells.

Detailed description of the invention

Once the microorganisms according to the invention are obtained in a pure form they are ultimately transferred to an appropriate medium containing potential taxane precursors and to this medium supplemented with aqueous extract of Taxus sp. Two criteria were used to select the most promising microbes: biomass production and 10-Deacetylbaccatin III yield.

The incubation of the fungi is carried out at 24°C in dark conditions for 3 weeks (in 20 ml or 100 ml of medium, respectively in 100 ml or 500 ml flasks; cultures were also made in 1000 ml of medium (in 3 litre flask) to purify taxanes in larger amount.

At the end of the incubation period the fluid (media) is separated from the mycelium and both were lyophilised and then powdered. These powders were extracted with methanol . Parts of the methanol extracts were tested by ELISA for 10-Deacetylbaccatin III equivalent content. The remaining parts are added with equal volume of water and partitioned with dichloromethan. Aliquots of the dichloromethan extracts were analysed by ELISA for 10- Deacetylbaccatin III and for Paclitaxel equivalent content. The ELISA positive extracts were subjected to preparative thin-layer chromatography on silicagel and four fractions were collected for each samples. The fraction showing the highest immunosignal was further analysed by high performance liquid chromatography. Further preparative HPLC experiments yielded 10-Deacetylbaccatin III in sufficient purity and amount for its spectroscopic identification (^XHNMR and MS) .

Evidence of 10-Deacetylbaccatin III production

The fungi were grown on an appropriate medium supplemented with taxane precursors and aqueous extract from Taxus Sp . The cultures were maintained in dark conditions for three weeks. The medium and the grown mycelium were lyophilised and extracted with methanol and partitioned with dichloromethan. The dichloromethan phase was subjected to preparative TLC and HPLC as described above. The fraction containing 10-Deacetylbaccatin III (as identified by HPLC; retention time and UV spectrum) was subjected to spectroscopic analysis (UV, ¹HNMR and MS) and the obtained data were identical to those of the authentic sample of 10-Deacetylbaccatin III. To dispel the notion that the 10- Deacetylbaccatin III isolated from the culture medium might originate from the added aqueous extract from Taxus Sp., the fungi "plugs" used to inoculate the medium was exhaustively extracted and the residue chromatographed. There was no evidence of 10-Deacetylbaccatin III. The aqueous extract from Taxus Sp . was also exhaustively extracted with dichloromethan, and there was no evidence of 10-Deacetylbaccatin III in this dichloromethan extract. The present invention will now be described in more detail with reference to the following examples, which should not be construed to limit the present invention.

Example 1 : Microorganism culture and 10-Deacetylbaccatin III production in flask

Culture of fungi

The pure Penicillium sp . cultures were subcultured in flaskes containing 25 ml of liquid culture medium (neopeptone (lOg/l) , glucose (20g/l) and yeast extract (5g/l) ) . The cultures were kept under shaking (100 rpm) for 3 weeks in dark.

Extraction of 10-Deacetylbaccatin III and the like compounds

The cultured medium and the mycelium were lyophilised and then extracted with methanol. The methanol extract was centrifuged and the supernatant was used for the ELISA analysis as well as for the fractionation and the purification of the taxane derivatives as required. The ELISA positive extracts were added with water and extracted with dichloromet an. The dichloromethan extracts were analysed by TLC and HPLC to purify 10-Deacetylbaccatin III and other taxanes . Determination of 10-Deacetylbaccatin III and the like compounds

Enzyme immunoassay - the dichloromethan extract was analysed by ELISA using a polyclonal antibody against 10-Deacetylbaccatin III and also a polyclonal antibody against Paclitaxel. As a result, it was found that up to 9277 μg of 10-Deacetylbaccatin III equivalent content/g dry wt . and 430 μg of 10-Deacetylbaccatin III equivalent content /g dry wt . were detected in the culture medium.

High performance liquid chromatography - the dichloromethan extract was analysed by high performance liquid chromatography (HPLC) , and the results obtained therefrom were compared with the results of analysis of authentic samples of 10-Deacetylbaccatin III and Paclitaxel. The HPLC conditions were as follows:

- Column: Hibar pre-packed Lichrosorb (RP18 (250x4 mm; 7 mm particle size) .

- Mobile phase: water-acetonitril-methanol (60:30:10). - Flow rate: 1 ml/minute.

- Detection: Absorbance at 227 nm and absorption spectrum at an elution peak with photodiode array detector.

As a result, the extract sample revealed elution peaks corresponding to the elution times of 10- Deacetylbaccatin III at 5.85 minutes and Paclitaxel at 7.3 minutes. Moreover, the absorption spectra over the range of 190-400 nm of the extract sample at those elution peaks were identical to the spectra of 10-Deacetylbaccatin III and Paclitaxel. The agreement of the elution times was confirmed by mixing the extract sample and the 10 -DAB and Paclitaxel standard solutions, subjecting the resulting mixture to HPLC analysis, and finding that the 10-DAB and Paclitaxel elution peaks of the sample and the standards comigrate. Proton nuclear magnetic resonance and mass spectroscopy - the ^ΗNMR and mass spectra of the compounds isolated from the culture medium were identical to those of authentic samples of 10-DAB and Paclitaxel.

Example 2 : Preparation of an aqueous extract of Taxus Sp. and identification of elicitors and /or precursors improving the production of taxanes Fresh Taxus S . bark (cambium and phloem tissue) and leave was extracted with distilled water. This extract was lyophilised and then fractionated using either gel filtration on Sephadex 6-75 or by solvent-partition chromatography. The total extract and its fractions were tested for their potential to improve the production of taxanes by the said microorganisms. It was found that the induction of taxane production is due to several factors present in the Taxus aqueous extract. But these factors seem to be mainly macromolecular components. A strong ELISA signal was also obtained when the dichloromethan fraction of the Taxus aqueous extract was added to the culture medium. This suggested that some taxane precursors are present in this dichloromethan fraction.

According to the present invention, a diterpene-like compound having the taxane skeleton can be produced highly efficiently. The 10-DAB and Paclitaxel contents of a culture of the microorganisms of the present invention belonging to the genus Penicillium are interesting. The 10-DAB content per gram of dry matter of the microorganism culture medium is higher than that of the known plant producing this compound. In addition, the culture period of the microorganisms of the present invention required for the production of 10-DAB and Paclitaxel and the like is several days at most, which is far shorter than that required for the tissue culture of plants of the genus Taxus, i.e. several weeks, and also far shorter than that required for growing and harvesting plants of the genus Taxus, i.e. several years at least.

Claims

1. A Penicillium sp . strain able to produce a diterpene-like compound having a taxane or taxine skeleton.

2. The Penicillium sp . strain according to claim 1, wherein said diterpene-like compound has the following taxane skeleton:

wherein: Rι_ is a hydrogen atom or an acyl group,

R2 is a hydrogen atom or an acyl group,

R3 is an oxygen atom or the combination of an acetoxyl/or a hydroxyl group with a hydrogen atom, R4 is a hydrogen atom or a hydroxyl group, R5 is a hydrogen atom, an acyl group or a glycosyl group,

Ph is a phenyl group, and Ac is an acetyl group.

3. The Penicillium sp . strain according to claim 1 or 2 , wherein said diterpene-like compound having a taxane skeleton is selected from the group consisting of

10-Deacetylbaccatin III, baccatin III, Paclitaxel, cephalomanine and a mixture thereof .

4. The Penicillium sp . strain according to claim 1, wherein said diterpene-like has the following taxine skeleton:

wherein:

Rι_ is a hydrogen atom or a hydroxyl group, and

R2 , R3 , R4 and R5 are hydrogen atoms or acyl groups .

5. The Penicillium s . strain according to claim 4, wherein said diterpene-like compound is selected from the group consisting of taxine A and taxine B.

6. The Penicillium sp. strain according to any one of the preceding claims, which is Penicillium aurantiogriseum.

7. The Penicillium sp . strain according to claim 6, having the deposit number IHEM 14753.

8. A method for producing the diterpene-like compound having a taxane or taxine skeleton, characterised in that it comprises the steps of:

- culturing in an adequate medium the Penicillium sp . strain according to any one of the preceding claims , and

- recovering the diterpene-like compound from the culture of said Penicillium sp . strain.