WO2007029082A2 - Procede de fermentation ameliore pour la preparation d'ascomycine - Google Patents

Procede de fermentation ameliore pour la preparation d'ascomycine Download PDF

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Publication number
WO2007029082A2
WO2007029082A2 PCT/IB2006/002409 IB2006002409W WO2007029082A2 WO 2007029082 A2 WO2007029082 A2 WO 2007029082A2 IB 2006002409 W IB2006002409 W IB 2006002409W WO 2007029082 A2 WO2007029082 A2 WO 2007029082A2
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WO
WIPO (PCT)
Prior art keywords
ascomycin
compound
process according
homologous
fermentation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2006/002409
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English (en)
Other versions
WO2007029082A3 (fr
Inventor
Parveen Kumar
Sanjeev Kumar Sharma
Hitesh Kumar Malviya
Ramanchandran Balasubramanian
Ashubhai Dalal
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Ranbaxy Laboratories Ltd
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Ranbaxy Laboratories Ltd
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Publication date
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Publication of WO2007029082A2 publication Critical patent/WO2007029082A2/fr
Publication of WO2007029082A3 publication Critical patent/WO2007029082A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/18—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms

Definitions

  • the present invention relates to an improved fermentation process for the preparation of ascomycin.
  • Ascomycin or FK-520 as exemplified in Formula I, is a well-known antifungal antibiotic, which inhibits filamentous fungi, for example, Penicillium chrysogenum and acid-fast bacteria. Ascomycin is also useful as a starting material for the synthesis of pimecrolimus, which is used for the treatment of atopic eczema.
  • Ascomycin (FK-520) is usually produced by microbial fermentation, wherein during this fermentation process the homologous compound FK-523 is formed.
  • Asocmycin (FK-520) must then be separated and purified from FK-523.
  • the presence of FK-523 in ascomycin is undesirable as it has decreased immunosuppressive activity than ascomycin.
  • FK-523 has a methyl group at C-21 resulting from the substitution of a propionate for a butyrate during polyketide synthesis.
  • the separation and purification of ascomycin from the FK-523 compound produced during fermentation is often complicated, the two compounds have almost the same number of carbon atoms and physical properties, such as solubility and affinity to solvents.
  • U.S. Patent No. 3,244,592 discloses the isolation of ascomycin from Streptomyces hygroscopicus subspecies ascomyceticus (ATCC 14891, MA 6475) and its use as an antifungal antibiotic.
  • U.S. Patent No. 4,894,366 discloses a method for the production of ascomycin and FK-523 by fermentation using Streptomyces subspecies yakushimaensis No. 7238 (FERM BP-928), wherein the ascomycin is isolated and purified from the fermentation broth by conventional methods.
  • Hatanaka et al. J. Antibiotics 41, 1592-1601, (1988) disclose a method for the production of ascomycin (FK-520) as an immunosuppressant from Streptomyces subspecies yakushimaensis No. 7238 (FERM BP-928, MA6531) and its subsequent purification by extraction from the fermentation broth followed by a crystallization step.
  • the prior art methods target two approaches to control the level of FK-523 homolog impurity in the ascomycin produced by the fermentation technique.
  • the first approach is the separation of FK-523 impurity from ascomycin by selective extraction from the fermentation broth and purification by a crystallization step.
  • the second approach is by valine feeding during fermentation or employment of a valine- overproducing Streptomyces strain. FK-523 levels of between 2% to 12% are observed in the ascomycin depending upon the quantity of valine fed.
  • the process includes incubating an ascomycin producing microorganism in a nutrient medium at a temperature of between about 24°C to about 32 0 C and agitating at a rate of between about 2.0 to about 4.0 m/s to produce ascomycin.
  • Embodiments of the process may include one or more of the following features.
  • the incubation temperature may be between about 26°C to about 30 0 C, or it may be 28°C.
  • the agitation rate may be between about 2.0 and about 2.8 m/s.
  • the agitation rate may be 2.4 m/s.
  • the ascomycin producing microorganism may be an ascomycin hyperproducer or mutant thereof.
  • the ascomycin producing microorganism may also be a Streptomyces species or mutant thereof.
  • the ascomycin producing microorganism may be Streptomyces hygroscopicus subspecies ascomyceticus (ATCC 14891).
  • the ascomycin producing microorganism is grown in a nutrient medium.
  • the nutrient medium may include one or more of soya bean meal, dextrose, dextrin, milk, cotton seed meal, casein enzyme hydro lysate, CaC12 and CaCO3.
  • the ascomycin produced may include less than about 6.0% of the homologous ascomycin compound FK-523.
  • the ascomycin may include less than about 5.0% of the homologous ascomycin compound FK-523.
  • an ascomycin compound in another general aspect there is provided an ascomycin compound.
  • the ascomycin compound includes less than about 6.0% of the homologous ascomycin compound FK-523.
  • Embodiments of the compound may include one or more of the following features.
  • the ascomycin may include less than about 5.0% of the homologous ascomycin compound FK-523.
  • a method of inhibiting the growth of filamentous fungi or acid-fast bacteria in a mammal in need thereof includes administering a pharmaceutical dosage form comprising an ascomycin compound wherein the ascomycin compound comprises less than about 6.0% of the homologous ascomycin compound FK-523.
  • Embodiments of the method may include one or more of the following features.
  • the ascomycin compound may include less than about 5.0% of the homologous ascomycin compound FK-532.
  • the present inventors have surprisingly found that ascomycin having significantly reduced levels of the FK-523 homolog impurity may be produced by altering certain physico-chemical parameters, such as incubation temperature and agitation rates during fermentation.
  • the present process is cost effective, commercially scalable without compromising the overall yield of ascomycin.
  • the present process offers a simpler means of controlling the FK-520 impurity during the fermentation stage itself and results in marked improvement over the prior art methods for the production of ascomycin by fermentation.
  • the present process uses the ascomycin hyper producer strain Streptomyces hygroscopicus subspecies ascomyceticus (ATCC 14891). It is to be understood that the production of ascomycin by the present process is not limited to the use of the particular microorganism described herein.
  • the present invention also includes the use of any mutants, which are capable of producing ascomycin, including natural mutants and artificial mutants, which can be produced from ascomycin producing microorganisms or mutants thereof by conventional methods known in the art.
  • Table 1 represents a comparison between the prior art methods for production of ascomycin and the present invention with respect to the fermentation conditions and level of FK-523 contamination in ascomycin.
  • the present inventors have surprisingly observed that during the fermentative production of ascomycin, FK-523 formation may be reduced from about 20% to 4% using an incubation temperature of from about 24 0 C to about 32 0 C and an agitation rate from about 2.0-4.0 m/s.
  • a first aspect of the present invention provides a fermentation process for the production of ascomycin, wherein the process includes incubating ascomycin producing microorganism in a nutrient medium at a temperature of about 24°C -32°C and agitation rate of 2.0-4.0 m/s.
  • the ascomycin producing Streptomyces strain is incubated in a nutrient medium for between about 180 to about 240 hours at 24 0 C to 32 0 C and is agitated at a rate of between about 2.0 to about 4.0 m/s.
  • the level of ascomycin and FK-523 homolog impurity in fermentation broth is determined using a slight modification of the HPLC method reported in Biotechnology and Bioengineering 59: 595-604, 1998.
  • Ascomycin may be isolated from the fermentation mixture either using the whole broth or using the mycelium and the filtrate obtained therefrom.
  • the incubation temperature may also be maintained at between about 26 0 C to about 3O 0 C and the agitation rate may be set at between about 2.0 to about 2.8 m/s. For example, the incubation temperature may be maintained at about 28 0 C and the agitation rate at about 2.4 m/s.
  • Streptomyces hygroscopicus subspecies as corny ceticus mutant was grown and maintained on yeast extract malt extract agar slants. The surface of the slant was scraped and used to inoculate a 250 ml shake flask containing 35 ml of a sterilized seed medium containing dextrose (1.0 g/L), dextrin (10 g/L), milk (2 g/L), cotton seed meal (2.5 g/L), casein enzyme hydrolysate (5 g/L), MgSO 4 JH 2 O (0.05 g/L), FeSO 4 JH 2 O (0.025 g/L), NaCl (0.5 g/L), CaCl 2 (0.02 g/L), ZnSO 4 JH 2 O (0.01 g/L), MnSO 4 JH 2 O (0.005 g/L) and phosphate buffer (2ml /L; pH 7.0).
  • dextrose 1.0 g/L
  • dextrin 10
  • the pH of the seed medium was adjusted to 7.2.
  • the flask was incubated for 48 hours at 28 0 C on a rotary shaker.
  • An aliquot of the resulting seed culture was used to inoculate a shake flask containing presterilized production medium consisting of dextrin (140 g/L), cotton seed meal (14 g/L), soya bean meal (10 g/L), glycerol (10 g/L), soya peptone (10 g/L), polyethylene glycol (12.5 g/L), potassium phosphate (0.8 g/L), and calcium carbonate (1.5 g/L).
  • the pH was adjusted to 7.2.
  • the flask was incubated at 24°C on a rotary shaker.
  • FK 523 content 18% (by HPLC).
  • Example 1 The strain propagation and inoculum preparation methods were same as described in Example 1. 10 ml of the inoculum obtained in Example 1 was added to a 10 L bioreactor containing seed medium described in Example 1, except that the milk powder and cottonseed meal concentration was 3 g/L each and additionally calcium carbonate (0.5 g/L) was added. The medium was sterilized at about 121 0 C for about 30 minutes.
  • the seed culture was used to inoculate 20 L production bioreactor containing dextrin (30 g/L), cottonseed meal (10 g/L), soya bean meal (10 g/L), glycerol (10 g/L), soya peptone (10 g/L), polyethylene glycol (12.5 g/L), potassium phosphate (0.8 g/L) and calcium carbonate (1.5 g/L).
  • dextrin solution was fed during fermentation when the pH rose beyond 7.6-7.8.
  • the fermentation was run at about 24 0 C.
  • the agitation rate was increased to between about 3.2 to about 4.0 m/s during growth phase.
  • the agitation rate was then decreased during the production stage to between about 2.4 to about 2.8 m/s.
  • strain propagation, inoculum preparation and initial fermentation batch conditions were the same as described in Example 1 and 2, except that the agitation rates during growth and production phase was between about 2.7 to about 3.3 m/s and between about 2.0 to about 2.4 m/s respectively.
  • Example 1 The procedure of Example 1 was followed for microorganism propagation and fermentation, except that the production shake flasks were incubated at about 28 0 C.
  • Example 1 The procedure of Example 1 was followed for microorganism propagation and fermentation, except that the production shake flasks were incubated at 3O 0 C.
  • Example 1 The procedure of Example 1 was followed for microorganism propagation and fermentation, except that the production shake flasks were incubated at 32°C.
  • Example 1 The strain propagation and inoculum preparation methods were same as described in Example 1. 100 ml of inoculum obtained in Example 1 was added to 100 L seed fermenter containing seed medium comprising the components as described in Example 2. After 48 hours of incubation, 40 L of seed was used to inoculate 400 L production medium comprising components as described in Example 2. The fermentation was run at 28 0 C. A dose of dextrin solution was fed during fermentation when pH rose beyond 7.6-7.8. The agitation rate during growth and production phase was between about 2.7 to about 3.3 m/s and between about 2.0 and about 2.4 m/s respectively.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé de fermentation amélioré pour la préparation d'ascomycine.
PCT/IB2006/002409 2005-09-05 2006-09-01 Procede de fermentation ameliore pour la preparation d'ascomycine Ceased WO2007029082A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN2363/DEL/2005 2005-09-05
IN2363DE2005 2005-09-05

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WO2007029082A2 true WO2007029082A2 (fr) 2007-03-15
WO2007029082A3 WO2007029082A3 (fr) 2007-04-26

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010026873A1 (fr) * 2008-09-02 2010-03-11 財団法人微生物化学研究会 Nouveau composé céramidastine, son procédé de fabrication et son utilisation

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107245460B (zh) * 2017-05-18 2020-01-03 福建省微生物研究所 一种高产子囊霉素的吸水链霉菌诱变菌株及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3244592A (en) * 1962-06-09 1966-04-05 Arai Tadashi Ascomycin and process for its production
US4894366A (en) * 1984-12-03 1990-01-16 Fujisawa Pharmaceutical Company, Ltd. Tricyclo compounds, a process for their production and a pharmaceutical composition containing the same

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010026873A1 (fr) * 2008-09-02 2010-03-11 財団法人微生物化学研究会 Nouveau composé céramidastine, son procédé de fabrication et son utilisation
JP2010059069A (ja) * 2008-09-02 2010-03-18 Microbial Chem Res Found 新規化合物セラミダスチン、その製造方法及びその用途
US8263369B2 (en) 2008-09-02 2012-09-11 Microbial Chemistry Research Foundation Compound ceramidastin, method for producing the same, and use of the same

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