Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2002-07-23
2004-02-10
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S041000, C549S292000, C560S119000, C560S188000, C560S256000
Reexamination Certificate
active
06689590
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for isolating desired chemical products of reactions conducted in aqueous fermentation broths. The invention further relates to isolation of pravastatin, compactin and lovastatin from a fermentation broth and in particular to isolation of pravastatin made by fermentation of compactin.
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,
The Pharmacological Basis of Therapeutics
879 (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-methyl-glutaryl-coenzyme A reductase enzyme (“HMG-CoA reductase”). HMG-CoA reductase catalyzes the conversion of HMG-CoA to mevalonate, which is the rate determining step in the biosynthesis of cholesterol. Consequently, its inhibition leads to a reduction in the rate of formation of cholesterol in the liver.
Pravastatin is the common medicinal name of the chemical compound [1S-[1&agr;(&bgr;*,&dgr;*)2&agr;,6&agr;,8&bgr;(R*),8a&agr;]]-1,2,6,7,8,8a-hexahydro-&bgr;,&dgr;,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthalene-heptanoic acid. (CAS Registry No. 81093-370.) The molecular structure of pravastatin in free acid form is represented by Formula (Ia) where R═OH. The lactone form is represented by Formula (Ib), with atoms labeled to indicate numbering of the atoms.
Pravastatin, compactin (Formula Ib, R═H), lovastatin (Formula Ib, R═CH
3
), simvastatin, and fluvastatin each possess an alkyl chain that is terminated by a carboxylic acid group closed in a lactone and that bears two hydroxyl groups at the &bgr; and &dgr; positions with respect to the carboxylic acid group. This alkyl chain is the portion of the molecule that binds to HMG-CoA reductase. The carboxylic acid group and the hydroxyl group at the &dgr; position are prone to lactonize as shown in formula (Ib). Lactonizable compounds like the statins may exist in the free acid form or the lactone form or as an equilibrium mixture of both forms. Lactonization causes processing difficulties in the manufacture of statin drugs because the free acid and the lactone forms of the compounds have different polarities. A method of purifying one form is likely to remove the other form along with the impurities resulting in a lower yield. Consequently, great care must ordinarily be exercised when handling lactonizable compounds in order to isolate them in high yield.
Pravastatin exhibits an important therapeutic advantage over other statins. Pravastatin selectively inhibits cholesterol synthesis in the liver and small intestine but leaves cholesterol synthesis in the peripheral cells substantially unaffected. Koga, T. et al.
Biochim. Biophys. Acta
, 1990, 1045, 115-120. This selectivity appears to be due, in part, to the presence of a hydroxyl group at the C-6 position of the hexahydronaphthalene nucleus. The C-6 position is occupied by a hydrogen atom in compactin and a methyl group in lovastatin. Pravastatin is less able to permeate the lipophilic membranes of peripheral cells than the other more lipophilic congeners, Serajuddin et al.,
J. Pharm. Sci
., 1991, 80, 830-34, and the limited mobility of pravastatin is thought to account for its more localized action in the liver and intestine.
According to U.S. Pat. No. 4,346,227, incorporated herein by reference, pravastatin is reported as having been first isolated as a metabolite of compactin by M. Tanaka et al. during a study of compactin metabolism. According to the '227 patent, pravastatin can be obtained by fermentation of compactin using a variety of microorganisms:
Absidia coerulea
IFO 4423 spores,
Cunninghamella echinulata
IFO 4445
, Streptomyces rosochromogenus
NRRL 1233
, Syncephalastrum racemosum
IFO 4814 and
Syncephalastrum racemosum
IFO 4828. After fermentation, pravastatin was separated from the fermentation broth by acidifying the broth to a pH of 3 and extracting pravastatin and other non-hydrophilic organics with ethyl acetate, followed by washing with brine. The pravastatin free acid was lactonized by addition of a catalytic amount of trifluoroacetic acid, then neutralized with dilute sodium bicarbonate, dried over sodium sulfate and evaporated to dryness. The residue was purified by preparative reverse-phase high performance liquid chromatography (“HPLC”). One skilled in the art will appreciate that reverse-phase HPLC is not an economical method of purification for large-scale preparation of a chemical compound.
U.S. Pat. No. 5,942,423 relates to the microbial hydroxylation of compactin to pravastatin using a strain of Actinomadura. The only isolation method presented in the examples is the isolation of minute quantities attendant to analytical scale HPLC analysis of the fermentation broth. According to a more general discussion about isolating pravastatin from the broth, the preferred method of isolation is HPLC.
Commonly-assigned, co-pending PCT Application Serial No. PCT/US00/19384 relates to the microbial hydroxylation of compactin to pravastatin using a strain of
Micromonospora maculata
that is unusually resistant to the antifungal effects of compactin.
U.S. Pat. No. 5,202,029 relates to a process of purifying HMG-CoA reductase inhibitors using HPLC. Following separation of the impurities on the HPLC column, the HMG-CoA reductase inhibitor elutes from the HPLC column as a solute dissolved in the eluent. The eluent is partially evaporated and then water is added to induce crystallization of the HMG-CoA reductase inhibitor.
U.S. Pat. No. 5,616,595 relates to a continuous process for recovering water-insoluble compounds from a fermentation broth by tangential filtration. The fermentation broth is cycled past a filter. The broth becomes increasingly concentrated with each cycle because of loss of water through the filter. Once a desired concentration is reached, the concentrated broth is then slurried with a solvent in which the desired compound is soluble. The slurry is then cycled past the filter. The solution of the desired compound is collected as the filtrate and the desired compound is then isolated from the filtrate and optionally subjected to further purification. The method is said to be applicable to a wide variety of compounds including lovastatin, pravastatin and simvastatin.
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 that separates out is discarded. The organic phase is vacuum distilled at above 40° C., which, in addition to concentrating the solution, promotes lactonization by removal of water. Crystals of lovastatin lactone crystallize upon cooling and are recrystallized to a purity of 90% or greater. Those of skill in the art will appreciate that this method is ill-suited to isolation of the free carboxylic acid or a carboxylate salt form of a statin.
Presently, the most economically feasible method of making pravas
Deak Lajos
Forgacs Iiona
Keri Vilmos
Nagyne Edit Arvai
Szabo Csaba
Biogal Gyogyszergyar Rt.
Srivastava Kailash C.
Weber Jon P.
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