Liquid purification or separation – With means to add treating material – Chromatography
Reexamination Certificate
2001-01-25
2002-05-14
Rotman, Alan L. (Department: 1625)
Liquid purification or separation
With means to add treating material
Chromatography
C210S656000, C435S125000, C435S129000, C435S130000, C435S132000, C549S289000, C549S292000, C549S299000, C560S129000, C560S130000
Reexamination Certificate
active
06387258
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for purifying compounds from a fermentation broth. More specifically, this invention relates to isolation of statins from a fermentation broth in a crystalline form.
BACKGROUND OF THE INVENTION
Complications of cardiovascular disease, such as myocardial infarction, stroke, and peripheral vascular disease account for half of the deaths in the United States. A high level of low density lipoprotein (LDL) in the bloodstream has been linked to the formation of coronary lesions which obstruct the flow of blood and can rupture and promote thrombosis. Goodman and Gilman's,
The Pharmacological Basis of Therapertics
879 (Joel G. Hardman et al., eds. 9th ed. 1996). Reducing plasma LDL levels has been shown to reduce the risk of clinical events in patients with cardiovascular disease and in patients who are free of cardiovascular disease but who have hypercholesterolemia. Scandinavian Simvastatin Survival Study Group, 1994; Lipid Research Clinics Program, 1984a, 1984b.
Statin drugs are currently the most therapeutically effective drugs available for reducing the level of LDL in the blood stream of a patient at risk for cardiovascular disease. This class of drugs includes, inter alia, compactin, lovastatin, simvastatin, pravastatin and fluvastatin. The mechanism of action of statin drugs has been elucidated in some detail. They disrupt the synthesis of cholesterol and other sterols in the liver by competitively inhibiting the 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme (“HMG-CoA reductase”). HMG-CoA reductase catalyzes the conversion of HMG-CoA to mevalonate, which is the rate limiting step in the biosynthesis of cholesterol. Consequently, its inhibition leads to a reduction in the rate of formation of cholesterol in the liver.
Compactin is the common medicinal name of the chemical compound 2-methylbutanoic acid 1,2,3,7,8,8a-hexahydro-7-methyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthalenyl ester of the formula:
Compactin, also called mevastatin, was the first statin drug shown to be a HMG-CoA reductase inhibitor. Compactin has been purified from
Penicillium citrinum
and
Penicillium adametzioides.
(See U.S. Pat. Nos.3,983,140; 4,049,495 and; 5,691,173 which are incorporated herein by reference.)
Lovastatin is the common medicinal name of the chemical compound 2-methylbutanoic acid 1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-(tetrahydro4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthalenyl ester of the formula:
Lovastatin, also called mevinolin, differs from compactin only in the presence of a methyl group and can be isolated from
Aspergillus terreus.
(See U.S. Pat. Nos. 4,294,926; 4,420,491; 4,319,039; and 4,294,896 which are incorporated herein by reference.) Lovastatin has also been isolated from several other microorganisms. (See British Patent No. GB 2,046,737; German Patent No. 44 02 591; Canadian Patent No. 2,129,416; and Hungarian Patent No. HU 208,997.)
Compactin and lovastatin, as well as other statins, exist as open ring hydroxy-acids and in the lactone form as shown:
The equilibrium between lactone and hydroxy-acid makes purification difficult because the free acid and the lactone forms of the statin compounds have different polarities. A method of purifying one form is likely to remove the other form, thereby decreasing overall yield. Consequently, great care must ordinarily be exercised when purifying statin compounds in order to isolate them in high yield.
U.S. Pat. No. 5,202,029 relates to a process for purifying the lactone form of statin compounds using HPLC. The crude fermentation broth (e.g., lovastatin, simvastatin, pravastatin, fluvastatin and mevastatin) is dissolved in an organic solvent and eluted through a HPLC column. The statin elutes from the column as a solute dissolved in the eluant. The eluant is partially evaporated and subsequently water is added to induce crystallization. The primary disadvantage for industrial scale production is the great expense of the chromatography columns.
U.S. Pat. No. 5,616,595 relates to a continuous process for recovering a wide variety of water-insoluble compounds from a fermentation broth by tangential filtration. The process may be applied to the lactone form of lovastatin, pravastatin and simvastatin. The process comprises cycling a fermentation broth through a filter which retains the insoluble compound. The compound is dissolved in a solvent and the solution filtered. The solution of the desired compound is collected as the filtrate and the desired compound may then be subjected to further purification. Because of the insolubility of the compounds in water, the process requires solvent for solubilization and multiple filtration membranes are required. The repeated filtration renders the process very costly for large scale manufacture.
A process for isolating lovastatin in the lactone form is described in U.S. Pat. No. 5,712,130. In this process, lovastatin is extracted from a fermentation broth with butyl acetate. The resulting solution is then centrifuged and an aqueous phase is discarded. The organic phase is, vacuum distilled at above 40° C., which, in addition to concentrating the solution, promotes formation of the lactone by removal of water. Crystals of lovastatin lactone form upon cooling and are recrystallized to a purity of 90% or greater. However, prior to use as a pharmaceutical the crystalized lovastatin must be further purified, which decreases the overall yield and adds additional expenses to the procedure.
OBJECTS AND SUMMARY OF THE INVENTION
The known methods of isolating a statin from a fermentation broth do not achieve a pharmaceutically acceptable level of purity with a process economical on an industrial scale, or require chromatographic separation to achieve high purity. An object of the present invention is to meet a need in the art for a simple, fast, high yield process for isolation of statins from a fermentation broth at a pharmaceutically acceptable level of purity (i.e., not less than 98.5%).
Another object of the present invention is a process for isolating a statin compound from a fermentation broth, wherein the statin compound comprises a carboxylic acid capable of forming a lactone and a fused bicyclic ring. The process comprises the steps of: extracting the statin compound from the fermentation broth by contacting the fermentation broth with an extraction solvent, and extracting the statin compound from the fermentation broth into the extraction solvent, wherein the extraction solvent is a hydrophobic organic extraction solvent; separating the hydrophobic organic organic extraction solvent from the fermentation broth; concentrating a solution of the hydrophobic organic extraction solvent containing the extracted statin compound; and purifying the extracted statin compound by crystallization.
Another object of the present invention is a process for isolating a statin compound from a fermentation broth, wherein the statin compound comprises a carboxylic acid capable of forming a lactone and a fused bicyclic ring, the process comprising the steps of:
(a) pretreating the fermentation broth under alkaline conditions to remove non-polar impurities;
(b) extracting the statin compound from the fermentation broth into a hydrophobic organic extraction solvent;
(c) separating the hydrophobic organic extraction solvent from the fermentation broth;
(d) concentrating a solution of the hydrophobic organic extraction solvent containing the extracted statin compound;
(e) washing the concentrated solution of the hydrophobic organic extraction solvent with an aqueous solution containing a base to purify the lactone; and
(g) purifying the extracted statin compound by crystallization.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is a process for obtaining highly purified crystals of statin compounds from a fermentation broth, as illustrated by the purification of compactin and lovastatin. One of ordinary skill in the art will recognize that the process of the present inven
Deak Lajos
Forgacs Iiona
Keri Vilmos
Biogal Gyogyszergyar Rt.
Covington Raymond
Kenyon & Kenyon
Rotman Alan L.
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