Title of Invention

A PROCESS FOR BIOTRANSFORMATION OF TAXOIDS TO 10 DEACETYLBACCATIN-III

Abstract

A process for biotransformation of taxoids into 10-deacetylbaccatin-lll by cultivating the strain of Flavobacterium dehydrogenans in a medium containing yeast extract 2-4%, potassium dihydrogen phosphate 0.1 to 0.2% & disodium hydrogen phosphate 0.2 to 0.3% by known methods, incubating the mixture of taxoid in organic solvent with the above culture broth for the period in the range of 24 120 hours at a temperature of in the range of 29 to 30°C on a rotatory shaker at 200-220 rpm extracting the culture broth with the organic solvent to obtain 10-deacetylbaccatin-lll.

Full Text

The present invention relates to a process for biotransformation of the taxoids to 10-deacetylbaccatin-lll. The present invention particularly relates lo a process for the preparation of 10-deacctylbaccatin-III by the biotransformation of the taxoids of plant origin or synthetic in nature, individually or in combination. The taxoids such as cephalomannine, 10-deacetyltaxol, 10-deacetylcephalomannine, baccatin-lll, 10-deacetylbaccatin-lll, 7-epitaxol, taxol-C and xylosides of taxol congeners and the like including taxol itself are present in the bark of plants such as Taxus wallichiana. The taxoids rich fraction or the individual taxoid pure form is biotransformed to 10-decetylbaccatin-lll with Flavobacterium dehydrogenans. The organism has been isolated and duly deposited in RRL repository and designated as RRL-189. 10-deacetylbaccatin-lll is a key precursor of taxol, a neutral alkaloid found in plains of Taxus spp. and useful against refractory ovarian and breast cancer. Paclitaxel (Taxol) is a complex polycyclic diterpene, which occurs as a minor component in various species of Taxus. To obtain taxol from bark of Taxus brevifolia and Taxus wallichiana on a commercial scale, a large number of trees must be felled, Taxol was first isolated in 1971 by Wall and his collaborators, and was shown lo possess antileukemic and tumor inhibitory properties (Wani, M.C. et al. J. Am. Chem. Soc, 93, 2325, 1971). It is effective against refractory ovarian cancer and breast cancer for the deaths of 60,000 women every year. Taxol exhibits a mode of action unprecedented among cancer chemotherapeutic agents. Taxol targets microtubule formation but in a unique fashion. Unlike known antimicrotubule agents, which block microtubule production, taxol promotes tubulin polymerization and microtubule against depolymerization. It prevents cell division without affecting DNA, RNA or protein synthesis (Veronika, N.K. et. al. J. Pharm. Biomed, Anal, 14, 997 1996). Earlier method of production of paclitaxel involved extraction of the bark of the pacific yew, Taxus brevifolia. However demand for paclitaxel exceeds the supply from this source. Semisynthesis from 10-deacetylbaccatin-lll (10-DAB) is now the most important method for production of paclitaxel. (Nanduri, V. B. et. al. Biotech Bioeng, 48, 547, 1995). Docetaxol (Taxotere), another new chemotherapeutic agent used for the treatment of cancer is also prepared from 10-deacetylbaccatin-lll (10-DAB) (Guenard, D. et. al. Ace. Chem. Res. 26, 160, 1993, Adeline, M. T. et. al. J. Liq. Chrom. and Rel. Technol,. 20, 3135, 1997). 10-DAB is the most important intermediate not only for the production of taxol but also for other related anticancer drugs. The chemical methods for deacylation of Taxol congeners to 10-DAB-lll comprises of methanolysis which gives mixtures of taxoid products along with low yield of Baccatin-lll (Wani, M.C. et al. J. Am. Chem. Soc. 93, 2325, 1971; Miller, R.W. et al J.Org. Chem. 41, 1469,1981). Treatment of Taxol with ZnBr2 in methanol yields a mixture of 10-deacetyl taxol and 7-epi- 10-deacetyltaxol but 7-epi- 10-deacetyltaxol is formed in largest amount (Samamayake, G; J. Org. Chem. 56, 5114, 1991). Treatment of Taxol and its protected derivatives with a variety of lewis acids also resulted in the production of C-7 epimerization of taxoids. When the C-7 hydroxyl group was protected deacetylation did not occur (Chem, S. et al Tetrahedron, 49, 2805, 1993). However site specific enzymatic hydrolysis of taxanes have been reported by Nanduri et al Biotech. & Bioengg., 48: 547-550, 1995 and Hanson et. al. J. Biol. Chem., 269 (35): 22145-22149, 1994 respectively in which the microorganism used is Nocardiodes albus. However in our invention we have used Flavobacterium dehydrogenans, which is different from the microorganism reported earlier. The main object of the present invention is to provide a process for the biotransformation of taxoids into 10-DAB-lll which deviates the drawbacks as detailed above. Another object of the present invention is to increase the percentage of 10-DAB-lll in taxoids of synthetic or plant origin, in which the concentration of 1 O-DAB-III is minimal by submerged fermentation using a strain of Flavobacterium dehydrogenans RRL-189. The organism is already known and has been earlier used for biotransformation of steroids. In the present invention, this micro-organism has been used for the first time for the biotransformation of taxoids. Still another object of the present invention is to provide an economic method for the conversion of taxoids into useful product i.e. 10-DAB-lll. Accordingly, the present invention provides a process for biotransformation of taxoids into 10-deacetylbaccatin-lll characterized by the steps of cultivating the strain of Flavobacterium dehydrogenans in a medium containing yeast extract 2-4%, potassium dihydrogen phosphate 0.1 to 0.2% & disodium hydrogen phosphate 0.2 to 0.3% by known methods, incubating the mixture of taxoid in organic solvent such as herein described with the above culture broth for the period in the range of 24 120 hours at a temperature of in the range of 29 to 30°C on a rotatory shaker at 200-220 rpm extracting the culture broth with the organic solvent such as herein described to obtain 10-deacetylbaccatin-lll. In an embodiment of the present invention, medium used for cultivating Flavobacterium dehydrogenans has been described which may contain yeast extract 2-4%, potassium dehydrogen phosphate 0.1-0.2% and disodium hydrogen phosphate 0.2-0.3%. In another embodiment of the invention solvent used for dissolving taxoids may be such as dimethyl formamide, methanol and propylene glycol. In yet another embodiment organic solvent used for extraction may be such as n-hexane, ethyl acetate, methylene chloride, chloroform, and carbon tetrachloride or their mixtures. The purification of the product may be effected by conventional chromatography. Characteristics of Flavobacterium dehydrogenans Cells vary from cocobacilli to slender rods non-sporulating gram positive. Growth on solid media is pigmented yellow, orange, red or brown and tint may vary with media and temperature. Colour most pronounced on potato, gelatin or milk containing media and at lower temperature (15-20°C), often light is required for maximum pigmentation. Pigments not soluble in media and have not been characterized but generally presumed to be carotenoids. Colonies \ typically transluscent, smooth and entirely or occasionally opaque. Incubation temperature below 30°C is preferable and growth may be inhibited by higher temperature (above 37°C). In the drawing accompanying this specification the scheme for deacylation of taxol (paclitaxel) of the formula 1 and taxol congeners such as cephalomannine, 10-deacetycephalomanine, 10-deacetytaxol, 7-xylosyltaxol and baccatin-lll of the formula 2 present in the extract to 10-deacetybaccatin-lll of the formula 3 has been suggested which is accomplished by submerged fermentation using a strain of Flavobacterium dehydrogenans RRL-189. We herein report the use of microorganism which is able to hydrolyse taxol congeners and other taxanes. The strain exhibiting an enzyme activity for fast deacylation of C-13 side chain in the taxol congeners has comparatively lesser activity for hydrolysis of C-10 acetyl of baccatin-IIL; The enzymes in the microorganism transformed paclitaxel and baccatin-III to 10-DAB-III. These observations formed the basis of studies for enzymatic enhancement of 10-DAB-III by; biotransformation of taxoid fraction of Taxus extracts. The enzymes were also found to be active on extracts and semipurified fractions isolated from bark and foliage biomass. Accordingly the present invention provides a process of enhancement of 10-DAB-III in • biotransformation of taxoids in the extract of Taxus spp. by Flavobacterium dehydrogenans RRL- 189 which comprises the following steps: The organism, a developed strain of Flavobacterium dehydrogenans RRL-189, is inoculated in a sterilized medium having pH 6.0-6.5 and containing yeast extract 1-2%, glucose 0.5-1.5%, peptone 0.2-0.5%, agar 1.5-2% and incubated for 1-3 days. Cell suspension from agar slant is transferred in a preculture medium having pH 6.0-6.5 and containing yeast extract 1-2%, glucose 0.5-1.5%, peptone 0.2-0.5%. The inoculated medium was incubated and shaken continuously for a period of 24 to 72hrs when the optimum growth was obtained at 25-30° ± 1°C. The preseed 5-10% was inoculated into the production medium containing yeast extract 2-4 %, potassium dihydrogen phosphate 0.1-0.2 % and disodium hydrogen phosphate 0.2-0.3 % at pH 6.5-6.8. After 24 hrs of incubation the taxoid extracts dissolved in methanol were added and the reaction mixture stirred on a rotary shaker for 3-5 days at 200-220 rpm.The reaction was monitored by chromatographic techniques such as TLC, HPLC using different solvent systems at different wavelengths and by UV and HPLC scanning using diode array detector. In the embodiment of the present invention is described in detail a process of enhancement of 10- deacetylbaccatin-III in biotransformation of taxoids in extract of Taxus spp. using a strain of Flavobacterium dehydrogenans RRL-189. The following examples are given by the way of illustrations of the present invention and should not be construed to, limit the scope of the present invention. The yields shown in the examples are on the basis of weights and not on the basis of percentage conversion (theoreticals) to 10-DAB which will obviously be higher in view of its lower molecular weight. Example-I Three days old culture of Flavobacterium dehydrogenans RRL-189 from agar slants was inoculated in a sterilized preculture medium having pH 6.5 with yeast extract 1%, glucose 1%, peptone 0.5% ande agar 2%. The inoculated medium was incubated at 29° C on a rotary shaker at 220 rpm. Production medium contained yeast extract 3%, potassium dihydrogen phosphate 0.17% and disodium hydrogen phosphate 0.22% with pH at 6.8. The medium was distributed in 100 ml quantities in 500 ml Erlenmeyer flasks and plugged. Sterilization of medium was done at 15 psi for 15 minutes. 5% of the 24 hrs old preseed was added in each production flask and was incubated at 29° C on a rotary shaker at 220 rpm. After 24 hrs of incubation pure baccatin-III (lOOµg/ml) dissolved in methanol was added. Incubation of the reaction mixture was continued for 5 days. At the end of the incubation the culture broth was extracted with dichloromethane and the extract evaporated in a rotary evaporator. The product was analysed by TLC and HPLC. The yield of 10-deacetylbaccatin-III was 34.96% (w/w). Example-II Three days old culture of Flavobacterium dehydrogenans RRL-189 from agar slants was inoculated in a sterilized preculture medium containing yeast extract 3%, glucose 1%, peptone 0.5%, agar 2% with pH at 6.5. The inoculated medium was incubated at 29° C on a rotary shaker at 220 rpm. Production medium contained yeast extract 3%, potassium dihydrogen phosphate 0.17% and disodium hydrogen phosphate 0.22% with pH at 6.8. The medium was distributed in 100 ml quantities in 500 ml Erlenmeyer flasks and plugged. Sterilization of medium was done at 15 psi for 15 minutes. 5% of the 24 hrs old preseed was added in each production flask and was incubated at 29° C on a rotary shaker at 220 rpm. After 24 hrs of incubation pure paclitaxel (lOOµg/ml) dissolved in methanol was added. Incubation of the reaction mixture was continued for 5 days. At the end of the incubation the culture broth was extracted with dichloromethane and the extract evaporated in a rotary evaporator. The product was analysed by TLC and HPLC. The yield of 10-deacetyl baccatin-III was 54.37% (w/w) ( 85% theoretical). Example-Ill A fraction was isolated by repeated chromatography and crystallisation of taxoid fraction. It was confirmed to be a mixture of three compounds (M-O-T) viz. taxol (1.23%), 10-deacetybaccatin-III (11.78%) and baccatin-III (87.11%) by HPLC when cospiked with authentic standards. Three days old culture of Flavobacterium dehydrogenans RRL-189 from agar slants was inoculated in a sterilized preculture medium containing yeast extract 1%, glucose 1%, peptone 0.5% and agar 2% with pH at 6.5. The inoculated medium was incubated at 29° C on a rotary shaker at 220 rpm. f Production medium contained yeast extract 3%, potassium dihydrogen phosphate 0.17% and disodium hydrogen phosphate 0.22% with pH at 6.8. The medium was distributed in 100 ml quantities in 500 ml Erlenmeyer flasks and plugged. Sterilization of medium was done at 15 psi for 15 minutes. 5% of the 24 hrs old preseed was added in each production flask and was incubated at 29° C on a rotary shaker at 220 rpm. After 24 hrs of incubation M-O-T (Img/ml) dissolved in methanol was added. Incubation of the reaction mixture was continued for 5 days, At the end of the incubation the culture broth was extracted with dichloromethane and the extract evaporated in a rotary evaporator. The product was analysed by TLC and HPLC. Analysis of biotransformed product revealed that of the 87.11% of baccatin-III present in the M-O-T, 63.62% was biotransformed to 10-deacetybaccatin-III while 18.55% remained unconverted and the balance forming other products. All the above studies were carried on the fractions obtained by partitioning the broth in benzene. Example-IV Taxoids rich-fractions from Taxus wallichiana were isolated by partitioning methanol extract in water and dichloromethane. Dichloromethane solubles were fractionated into high and low molecular weight compounds using gel permeation chromatography. The taxomeres were eluted with organic solvents and further resolved by column chromatography using silica gel as stationary phase with gradient increase of acetone in w-hexane. The taxoid rich fractions containing taxol congeners were analysed by HPLC and designated as 'Substrate A'. Three days old culture of Flavobacterium dehydrogenans RRL-189 from agar slants was inoculated in a sterilized preculture medium having pH 6.5 with yeast extract 1%, glucose 1%, peptone 0. 5%, agar 2%. The inoculated medium was incubated at 29° C on a rotary shaker at 220rpm. Production medium contained yeast extract 3%, potassium dihydrogen phosphate 0.17% and disodium hydrogen phosphate 0.22% with pH at 6.8. The medium was distributed in 100 ml quantities in 500 ml Erlenmeyer flasks and plugged. Sterilization of medium was done at 15 psi for 15 minutes. 5% of the 24 hrs old preseed was added in each production flask and was incubated at 29° C on a rotary shaker at 220 rpm. After 24 hrs of incubation 'Substrate A' (Img/ml) dissolved in methanol was added. Incubation of the reaction mixture was continued for 5 days. At the end of the incubation the culture broth was extracted with dichloromethane and the extract evaporated under vacuum. The product was analysed by TLC and HPLC. 'Substrate A' containing 14.4% 10-DAB-III yielded a product 10-DAB-III, content of which was enhanced to 42.3%, a 2.9 fold increase in 10-DAB-III content. Example-V Taxoids rich fractions were isolated by partitioning aqueous methanol extract in dichloromethane. Dichloromethane solubles were fractionated into high and low molecular weight compounds using gel permeation chromatography. The taxomeres which were eluted with organic solvents were enriched by column chromatography using silica gel as stationary phase with gradient increase of acetone in n-hexane. The taxoid rich fractions containing taxol congeners were analysed by HPLC and designated as 'Substrate B'. Three days old culture of Flavobacterium dehydrogenans RRL-189 from agar slants was inoculated in a sterilized preculture medium of pH 6.5 containing yeast extract 1%, glucose 1%, peptone 0.5%, agar 2%, The inoculated medium was incubated at 29° C on a rotary shaker at 220 rpm. Production medium contained yeast extract 3%, potassium dihydrogen phosphate 0.17% and disodium hydrogen phosphate 0.22% with pH at 6.8. The medium was distributed in 100 ml quantities in 500 ml Erlenrneyer flasks and plugged. Sterilization of medium was done at 15 psi for 15 minutes. 5% of the 24 hrs old preseed was added in each production flask and was incubated at 29° C on a rotary shaker at 220 rpm. After 24 hrs of incubation 'Substrate B' (Img/ml) dissolved in methanol was added. Incubation of the reaction mixture was continued for 5 days. At the end of the incubation the culture broth was extracted with dichloromethane and evaporated on a rotary evaporator. The product was analysed by TLC and HPLC. 'Substrate B' with 7.5% 10-DAB-III yielded a product with 19.7% 10-DAB-III, a 2.6 fold increase in 10-DAB-III content. The salient features of the present process. 1. The Example I illustrates the deacetylation of baccatin-III to 10-DAB-III in about 35% yield (w/w) corresponding to 37.7% of theoretical which confirms the presence of 10-deacetylase enzyme in the culture. 2. The Example II illustrates the deacylation of paclitaxel to 10-DAB-III in about 54% yield (w/w) corresponding to 85% of theoretical after 96 hr fermentation confirming thereby the presence of taxolase, an enzyme which can deacylate at C-13. 3. The Example III illustrates conversion of baccatin-III in a fraction to 10-DAB-III on biotransformation. The fraction containing 87.11% of baccatin-III and 11.66% of 10- DAB-III yielded a product with 63.75% 10-DAB-III. 4. The Example IV and V illustrates 2.6 to 2.9 fold enhancement of 10-DAB-III in content of taxoid fractions (containing also 10-DAB-III) on biotransformation. Advantages of the process: 1. Biotransformation of crude taxoid mixture isolated from T. wallichiana can be achieved with the help of this microbial process and the yield of 10-DAB can be increased to 3- fold. 2. The organism used in this invention has got more capability for C-13 deacylation as compared to C-10 deacetylation of Taxol congeners and this microbial process has an edge over the chemical methods which often results in the formation of complex mixtures of taxoids along with epimerization at C-7 substituents. We claim : 1. A process for biotransformation of taxoids into 10-deacetylbaccatin-lll characterized by the steps of cultivating the strain of Flavobacterium dehydrogenans in a medium containing yeast extract 2-4%, potassium dihydrogen phosphate 0.1 to 0.2% & disodium hydrogen phosphate 0.2 to 0.3% by known methods, incubating the mixture of taxoid in organic solvent such as herein described with the above culture broth for the period in the range of 24 1 20 hours at a temperature of in the range of 29 to 30°C on a rotatory shaker at 200-220 rpm extracting the culture broth with the organic solvent such as herein described to obtain 10-deacetylbaccatin-lll. 2. A process as claimed in claim 1 wherein organic solvent used is methyl alcohol, dimethyl formamide or propylene glycol. 3. A process as claimed in claims 1-2 wherein the organic solvents used for extraction is ethyl acetate, methylene chloride, hexane and chloroform or their mixtures. 4. A process for biotransformation of taxoids into 10-deacetylbaccatin-lll substantially as herein described with reference to the examples.

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1191-del-1999-abstract.pdf

1191-del-1999-claims(cancelled).pdf

1191-del-1999-claims.pdf

1191-del-1999-complete specification (granted).pdf

1191-del-1999-correspondence-others.pdf

1191-del-1999-correspondence-po.pdf

1191-del-1999-description (complete).pdf

1191-del-1999-form-1.pdf

1191-del-1999-form-2.pdf

1191-del-1999-form-3.pdf

1191-del-1999-form-4.pdf

1191-del-1999-form-9.pdf