WO2004087935A2 - Fermentation process for the preparation of pravastatin - Google Patents
Fermentation process for the preparation of pravastatin Download PDFInfo
- Publication number
- WO2004087935A2 WO2004087935A2 PCT/IB2004/001003 IB2004001003W WO2004087935A2 WO 2004087935 A2 WO2004087935 A2 WO 2004087935A2 IB 2004001003 W IB2004001003 W IB 2004001003W WO 2004087935 A2 WO2004087935 A2 WO 2004087935A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- compactin
- pravastatin
- concentration
- formula
- substantially pure
- Prior art date
Links
- 0 CC[C@](C)C(O[C@@](C1)[C@]([C@](CC)CCC[C@](C[C@](C*)O)O)C(C)=C[C@]1O)=O Chemical compound CC[C@](C)C(O[C@@](C1)[C@]([C@](CC)CCC[C@](C[C@](C*)O)O)C(C)=C[C@]1O)=O 0.000 description 2
Classifications
-
- 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
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/02—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
- C07C69/22—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety
- C07C69/33—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety esterified with hydroxy compounds having more than three hydroxy groups
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2602/00—Systems containing two condensed rings
- C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
- C07C2602/14—All rings being cycloaliphatic
- C07C2602/26—All rings being cycloaliphatic the ring system containing ten carbon atoms
- C07C2602/28—Hydrogenated naphthalenes
Definitions
- the field of invention relates to a fermentation process for the preparation of substantially pure pravastatin.
- the process provides a method of producing pravastatin by microbial hydroxylation of compactin (ML-236B) by maintaining a concentration of compactin at not less than 300 ⁇ g /ml during the process.
- the process produces substantially pure pravastatin with reduced related impurities.
- the invention also relates to pharmaceutical compositions that include the substantially pure pravastatin.
- Hy ercholesterolemia or elevated plasma cholesterol level has long been recognized as a major risk factor for atherosclerotic disease, and specifically for coronary heart disease.
- the biosynthesis of cholesterol is a major contributing factor to hypercholesterolemia.
- HMG-CoA reductase catalyzes the conversion of HMG-CoA to mevalonate. It was expected that plasma cholesterol could be reduced as a result of inhibition of HMG-CoA reductase because more than 70% of the total input of body cholesterol is derived from de novo synthesis in humans.
- Pravastatin, simvastatin, lovastatin, mevastatin, atorvastatin. fluvastatin, cerivastatin and derivatives and analogues thereof are known as HMG-CoA reductase inhibitors, and are used as antihypercholesterolemic agents.
- HMG-CoA reductase 3-hydroxy-3-methylglutaryl coenzyme A reductase
- compactin ML-236B
- HMG-CoA reductase 3-hydroxy-3-methylglutaryl coenzyme A reductase
- pravastatin sodium was chosen as a candidate for development.
- Pravastatin sodium was chosen because of its stronger and more tissue-selective activity than the prototype compound.
- pravastatin is by the microbial hydroxylation of compactin at C-6 position.
- Microbial hydroxylation of compactin to pravastatin can be accomplished to various extents with molds belonging to different genera, such as Mucor Rhizopus, Syncephalastrum, Cunninghamella, Mortierella and with filamentous bacteria belonging to different genera, such as Nocardia,
- pravastatin The most common problem encountered in the commercial production of pravastatin is that most microorganisms are not able to tolerate the compactin substrate fed even at low concentrations due to its cytotoxic effect. (Biotechnol. Bioeng., 42:815-820, 1993]. Taking into account the efficient manufacture of the active ingredient on an industrial scale, it is important to have a strain that is able to tolerate high concentrations of compactin, as well as, pravastatin.
- the purity of the active ingredient is an important factor for the manufacturing of a safe and effective pharmaceutical product.
- Several structurally related products are sometimes produced during the fermentation processes, and often only one product is desired from a process, thereby requiring a method to control the ratio of these different analogues.
- a commercial process typically requires either exclusive or predominant production of one product. While the nature of polyketide synthase permits controlled biosynthesis of a single chemical entity, a significant number of polyketide synthases generate related products (J. rnd. Microbiol. Biotechnol., 27; 368-377, 2001). However, the known methods of producing pravastatin are ill-suited for controlling the formation of these analogues.
- a process for producing substantially pure pravastatin includes culturing microorganisms under conditions capable of converting compactin to pravastatin by maintaining the concentration of compactin at a level of not less than about 300 ⁇ g /mL during the process.
- the process is accomplished through the use of fermentation techniques known in the art, for example, the repeated fed-batch culture technique.
- the process may include periodically adding quantities of compactin in the culture broth during the fermentation to maintain the concentration of compactin at not less than about 300 ⁇ g ImL during the process.
- the concentration of compactin is maintained at a level within the range of about 300-900 ⁇ g /mL. This embodiment may result in an about 14- fold decrease in the amount of Impurity B of Formula III and an about 7-fold decrease in the amount of the compound of Formula IV.
- the compactin used in this process may be in the form of a solution.
- the compactin solution may include soluble salts of compactin, for example, the sodium salt of compactin.
- the compactin may be one or any of at least substantially purified compactin, semi-purified compactin and an intermediate compound produced during the synthesis of compactin.
- the microorganisms may be any species of the genus Streptomyces.
- the microorganism may be Streptomyces carbophilus. i other embodiments, the microorganism may be a Streptomyces carbophilus strain, variant or a mutant thereof.
- the conditions capable of converting compactin to pravastatin include the fermentation production medium containing glucose at a concentration level of about 15- 23 (g/L), Soya bean meal at a concentration level of about 25-38 (g/L), cottonseed meal at a concentration level of about 2-4 (g/L), com steep liquor at a concentration level of about 5-8 (g/L), sodium chloride at a concentration level of about 5-6 (g/L) and calcium carbonate at a concentration level of about 2-3 (g/L).
- the conditions capable of converting compactin to pravastatin may also include maintaining the temperature of the production medium at about 18 °C to about 50°C. In another embodiment, the temperature is maintained at about 25 °C to about 30 °C.
- the conditions capable of converting compactin to pravastatin may also include the maintenance of the pH of the production medium from about 5 to about 10. In another embodiment, the pH is maintained from about 6 to about 8.5. In yet another embodiment, the pH may be maintained from about 7.3 to about 8.0.
- the conditions capable of converting compactin to pravastatin may also include agitation at about 100 to about 600 rpm. In one embodiment, the agitation may be at about 100 to about 350 rpm.
- the percentage conversion of compactin to pravastatin is at least about 50%> w/w as determined by HPLC.
- the percentage conversion may be at least about 65 to about 75% w/w, or at least about 70%> w/w as determined by HPLC.
- substantially pure pravastatin containing not more than about 0.12% w/w of Impurity B and not more than about 0.6% w/w of 3"-hydroxy-pravastatin.
- a pharmaceutical composition that includes substantially pure pravastatin containing not more than about 0.12% w/w of Impurity B and not more than about 0.6%> w/w of 3"-hydroxy-pravastatin, and pharmaceutically acceptable excipients.
- a method of treating hypercholesterolemia includes administering to a patient in need of treatment for hypercholesterolemia a pharmaceutical composition that includes substantially pure pravastatin containing not more than about 0.12% w/w of Impurity B and not more than about 0.6% w/w of 3"-hydroxy-pravastatin 5 and other pharmaceutically acceptable excipients.
- the present invention provides an efficient process for the preparation of substantially pure pravastatin of Formula I by microbial hydroxylation of compactin of Formula II.
- concentration of compactin at not less than 300 ⁇ g /ml throughout the process, the quantities of Impurity B and 3"-(S)-hydroxy pravastatin, of Formula III and IV respectively, are substantially reduced.
- Compactin is provided and contacted with whole cells of Streptomyces sp. under the conditions in which the microorganism converts compactin to pravastatin. The process may be carried out subsequent to or during the growth of the microorganism to be employed.
- the compactin substrate is placed in contact with the microorganisms and is converted to pravastatin.
- Compactin may be provided in the form of a solution comprising the sodium salt of compactin, and may be the purified, crude or intermediate stage of compactin.
- substantially pure pravastatin as used herein is defined as pravastatin or a pharmaceutically acceptable salt thereof having a purity of not less than 99.3% w/w wherein the impurity B and 3"-hydroxypravastatin are not present at not more than about 0.12% w/w of Impurity B and not more than about 0.6% w/w of 3"-hydroxy-pravastatin as determined by HPLC.
- the conversion of compactin to pravastatin can be done using fermentation techniques known in the art; for example, those of the types useful for large-scale industrial fermentation process, such as batch, fed-batch or continuous culture techniques. For example, agitated liquid submerged culture techniques can be used.
- the growth of the microorganism may be achieved through the use of an appropriate medium containing nutrients, such as carbon, nitrogen sources and trace elements, which are added to the culture medium.
- nutrients such as carbon, nitrogen sources and trace elements, which are added to the culture medium.
- Suitable assimilable carbon sources include one or more of glucose, glycerol, maltose, dextrin, starch, sucrose etc.
- Suitable nitrogen sources include one or more of soybean meal, peptones, cottonseed meal, com steep liquor, meat extract, yeast extract, ammonium sulfate, ammonium nitrate etc.
- Suitable inorganic salts include one or more of sodium chloride, phosphates, calcium carbonate etc.
- Suitable seed medium may include glucose at concentrations of about 16-25 (g/L), Soya bean meal at concentrations of about 4-6 (g/L), peptone at concentrations of about 4- 6 (g/L), potassium phosphate at concentrations of about 0.08-0.13 (g/L) and calcium carbonate at concentrations of about 4-6 (g/L).
- the seed medium used can comprise glucose at concentrations of about 20 (g/L), Soya bean meal at concentrations of about 5(g/L), peptone at concentrations of about 5 (g/L), potassium phosphate at concentrations of about 0.1 (g/L) and calcium carbonate at concentrations of about 5 (g/L).
- Suitable production medium may include glucose at concentrations of about 15-23 (g/L), Soya bean meal at concentrations of about 25-38 (g/L), cottonseed meal at concentrations of about 2-4 (g/L), com steep liquor at concentrations of about 5-8 (g/L), sodium chloride at concentrations of about 5-6 (g/L) and calcium carbonate at concentrations of about 2-3 (g/L).
- the production medium used may include glucose at concentrations of about 18 (g/L), Soya bean meal at concentrations of about 30 (g/L), cottonseed meal at concentrations of about 3 (g/L), com steep liquor at concentrations of about 6 (g/L), sodium chloride at concentrations of about 6 (g/L) and calcium carbonate at concentrations of about 2.4 (g/L).
- the incubation temperature can be about 18°C to about 50°C, for example, about
- Suitable pH of the culture broth may range from about 5 to about 10, for example, about 6.0 to 8.5, or for example, about 7.3 to about 8.0.
- the process may be carried out under aerobic conditions, such as by means of aeration and/or agitation.
- the fermentation broth may be agitated at about 100 to 600 rpm, for example, at about 100 to about 350 rpm or for example, at about 150 to 300 rpm.
- the amount of compactin at the onset of the process can be, for example, in the range from about 300 ⁇ g/mL to about 1800 ⁇ g/mL of the culture medium, or for example, in the range of about 300 ⁇ g/mL to about 900 ⁇ g/mL.
- the resulting substantially pure pravastatin recovered from processes described herein contains not more than about 0.12%> w/w of Impurity B and not more than about 0.6%) w/w of 3"-hydroxy-pravastatin as determined by HPLC.
- the substantially pure pravastatin can be formulated into a dosage form with other pharmaceutically acceptable excipients.
- Optional excipients include, but are not limited to, one or more of colorants, diluents, lubricants, binders, disintergrants, and glidants.
- Suitable dosage forms include one or more of tablets, capsules, dispersions, and liquids.
- Any percentage of conversion of compactin to pravastatin can be practiced according to processes described herein, for example at least about 50%> w/w, or for example, at least about 60%w/w, or for example, about 65-75% w/w.
- Streptomyces carbophilus The seed medium was inoculated with a slant culture at 28°C for 2 days. 400 ml of mature seed culture was added to a 20 L production fermenter.
- a seed medium containing glucose 20 (g/L), Soya bean meal 5 (g/L), peptone 5 (g/L), potassium phosphate 0.1 (g/L) and calcium carbonate 5 (g/L) was inoculated with spores of Streptomyces sp. from a slant culture, and cultured at 500 rpm, 28°C for 2 days, to give a seed culture. 400 ml of this seed culture was added to 20-L production fermenter containing glucose 18 (g/L), soya bean meal 30, (g/L) cottonseed meal 3 (g/L), com steep liquor 6 (g/L), sodium chloride 6 (g/L) and calcium carbonate 2.4 (g/L).
- the medium was pre-sterilized at 121°C for 30 minutes. After the growth of the microorganism, an initial shot of compactin was added to the fermenter to a level of 500 ⁇ g/ml and further cultured. Compactin was further added when the concentration of compactin level was less than 50 ⁇ g/ml. The rate of compactin bioconversion was monitored by HPLC. The percentage of conversion was calculated on the basis of compactin added and pravastatin produced. Concentrations (w/w) of Impurity B and 3"S hydroxy pravastatin were also determined by HPLC.
- Example 2 Bioconversion of Compactin to Pravastatin - Controlled Compactin Concentration Level The procedure of Example 1 was followed but the concentration of compactin was maintained between 300-900 ⁇ g/ml throughout the batch as determined by HPLC. Compactin was added when the concentration reached the lowest value in the range.
- concentration levels of impurity B and 3"S hydroxy pravastatin were in the range of 0.08 to 0.12% w/w and 0.4 to 0.6% w/w respectively. These values represent about 14-fold and 7-fold lower impurity levels, respectively, as compared to the pravastatin obtained in Example 1 ( Figures 1 and 2).
- Example 2 The resultant product of Example 2 was purified using an industrial scale recovery process detailed in PCT Patent Application WO 01/44144. The purity of pravastatin obtained is about 99.4%> w/w as determined by HPLC.
- Example 3 Kinetics of Compactin Bioconversion
- This example illustrates the kinetics of the conversion of compactin to pravastatin.
- the medium and culture conditions employed were the same as in Example 1.
- FIGURE 1 A first figure.
- pravastatin is obtained with very low quantities of related compounds, Impurity B and 3"S hydroxy pravastatin, when the concentration of compactin was maintained between 300 and 900 ⁇ g/mL.
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP04725114A EP1613760A2 (en) | 2003-04-01 | 2004-04-01 | Fermentation process for the preparation of pravastatin |
CA002521276A CA2521276A1 (en) | 2003-04-01 | 2004-04-01 | Fermentation process for the preparation of pravastatin |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IN559DE2003 | 2003-04-01 | ||
IN559/DEL/2003 | 2003-04-01 |
Publications (2)
Publication Number | Publication Date |
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WO2004087935A2 true WO2004087935A2 (en) | 2004-10-14 |
WO2004087935A3 WO2004087935A3 (en) | 2005-01-13 |
Family
ID=33104991
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Application Number | Title | Priority Date | Filing Date |
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PCT/IB2004/001003 WO2004087935A2 (en) | 2003-04-01 | 2004-04-01 | Fermentation process for the preparation of pravastatin |
Country Status (3)
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EP (1) | EP1613760A2 (en) |
CA (1) | CA2521276A1 (en) |
WO (1) | WO2004087935A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1953233A1 (en) * | 2007-02-02 | 2008-08-06 | LEK Pharmaceuticals d.d. | Fermentation process for preparing pravastatin |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998045410A1 (en) * | 1997-04-10 | 1998-10-15 | Yungjin Pharmaceutical Ind. Co., Ltd. | A new microorganism streptomyces exfoliatus yj-118 and a method for producing pravastatin sodium by using the strain |
WO2000017182A1 (en) * | 1998-09-18 | 2000-03-30 | Lek Pharmaceutical And Chemical Company D.D. | PROCESS FOR OBTAINING HMG-CoA REDUCTASE INHIBITORS OF HIGH PURITY |
US20010026934A1 (en) * | 1995-06-07 | 2001-10-04 | Arnold Demain | Conversion of compactin to pravastatin by actinomadura |
US20020081675A1 (en) * | 1999-02-03 | 2002-06-27 | Antonia Jekkel | Microbial process for preparing pravastatin |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP3463875B2 (en) * | 2001-08-06 | 2003-11-05 | 三共株式会社 | How to purify pravastatin |
-
2004
- 2004-04-01 WO PCT/IB2004/001003 patent/WO2004087935A2/en not_active Application Discontinuation
- 2004-04-01 EP EP04725114A patent/EP1613760A2/en not_active Withdrawn
- 2004-04-01 CA CA002521276A patent/CA2521276A1/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20010026934A1 (en) * | 1995-06-07 | 2001-10-04 | Arnold Demain | Conversion of compactin to pravastatin by actinomadura |
WO1998045410A1 (en) * | 1997-04-10 | 1998-10-15 | Yungjin Pharmaceutical Ind. Co., Ltd. | A new microorganism streptomyces exfoliatus yj-118 and a method for producing pravastatin sodium by using the strain |
WO2000017182A1 (en) * | 1998-09-18 | 2000-03-30 | Lek Pharmaceutical And Chemical Company D.D. | PROCESS FOR OBTAINING HMG-CoA REDUCTASE INHIBITORS OF HIGH PURITY |
US20020081675A1 (en) * | 1999-02-03 | 2002-06-27 | Antonia Jekkel | Microbial process for preparing pravastatin |
Non-Patent Citations (4)
Title |
---|
DATABASE WPI Section Ch, Week 200250 Derwent Publications Ltd., London, GB; Class B05, AN 2002-471387 XP002301127 -& JP 2002 128739 A (SANKYO CO LTD) 9 May 2002 (2002-05-09) * |
GRAHEK R ET AL: "Chromatographic purification of some 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors" JOURNAL OF CHROMATOGRAPHY, ELSEVIER SCIENCE PUBLISHERS B.V. AMSTERDAM, NL, vol. 918, no. 2, 25 May 2001 (2001-05-25), pages 319-324, XP004239132 ISSN: 0021-9673 * |
HOSOBUCHI M ET AL: "APPLICATION OF COMPUTER TO MONITORING AND CONTROL OF FERMENTATION PROCESS: MICROBIAL CONVERSION OF ML-236B NA TO PRAVASTATIN" BIOTECHNOLOGY AND BIOENGINEERING, INTERSCIENCE PUBLISHERS, LONDON, GB, vol. 42, no. 7, 1993, pages 815-820, XP002036625 ISSN: 0006-3592 cited in the application * |
PARK JOO-WOONG ET AL: "Bioconversion of compactin into pravastatin by Streptomyces sp." BIOTECHNOLOGY LETTERS, vol. 25, no. 21, November 2003 (2003-11), pages 1827-1831, XP008033522 ISSN: 0141-5492 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1953233A1 (en) * | 2007-02-02 | 2008-08-06 | LEK Pharmaceuticals d.d. | Fermentation process for preparing pravastatin |
WO2008092950A1 (en) | 2007-02-02 | 2008-08-07 | Lek Pharmaceuticals D.D. | Fermentation process for preparing pravastatin |
Also Published As
Publication number | Publication date |
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EP1613760A2 (en) | 2006-01-11 |
CA2521276A1 (en) | 2004-10-14 |
WO2004087935A3 (en) | 2005-01-13 |
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