Organic compounds -- part of the class 532-570 series – Organic compounds – Carboxylic acid esters
Reexamination Certificate
2003-05-14
2004-02-24
Killos, Paul J. (Department: 1625)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carboxylic acid esters
Reexamination Certificate
active
06696599
ABSTRACT:
FIELD OF THE INVENTION
BACKGROUND OF THE INVENTION
The highest risk factor of atherosclerosis and especially coronary occlusion is the high cholesterol level of the plasma. In the last two decades 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC.1.1.1.34) as the rate limiting key enzyme of the cholesterol biosynthesis was extensively examined. Pravastatin, a compound of Formula I,
and other related compounds (compactin, mevinolin, simvastatin) are the competitive inhibitors of the HMG-CoA reductase enzyme [A. Endo et al., J. Antibiot. 29, 1346-1348 (1976); A. Endo et al., FEBS Lett. 72, 323-326 (1976); C. H. Kuo et al., J. Org. Chem. 48, 1991 (1983)].
Pravastatin was first isolated by M. Tanaka et al. (unpublished results) from the urine of a dog during the examination of the compactin metabolism (Arai, M. et al., Sankyo Kenkyusyo Nenpo, 40, 1-38, 1988). Currently pravastatin is a cholesterol lowering agent with the most advantageous action mechanism in the therapy. Its most important character is tissue selectivity, i.e., it inhibits the cholesterol synthesis at the two main sites of the cholesterogenesis, such as in the liver and in the small intestine, while in other organs the intracellular enzyme limiting effect is hardly detectable, At the same time the cholesterol biosynthesis limiting effect of mevinolin and simvastatin is significant in most of the organs (T. Koga et al., Biochim. Biophys. Acta, 1045, 115-120, 1990).
Pravastatin essentially differs in chemical structure from mevinolin and simvastatin which have more lipophilic character. In the case of the latter compounds the substituent connected to the C-1 carbon atom of the hexahydronaphthalene skeleton is ended in a 6-membered lactone ring, while in the case of pravastatin, instead of the lactone ring, the biologically active, opened dihydroxy acid sodium salt form is present. Another important structural difference is that instead of the methyl group of mevinolin and simvastatin at the C-6-position of the hexahydronaphthalene ring, a hydroxyl group can be found in pravastatin, which results in a further increase in its hydrophilic character.
As a result of the above structural differences pravastatin is able to penetrate through the lipophilic membrane of the peripheral cells only to a minimal extent (A.T.M., Serajuddin et al., J. Pharm. Sci. 80, 830-834, 1991).
Industrial production of pravastatin can be achieved by two fermentation processes. In the first, microbiological stage compactin is prepared, then in the course of a second fermentation the sodium salt of compactin acid as a substrate is converted to pravastatin by microbial hydroxylation at the 6&bgr;-position.
According to published patents, the microbial hydroxylation of compactin can be accomplished to various extents with mold species belonging to different genera, and with filamentous bacteria belonging to the Nocardia genus, with Actinomadura and Streptomyces genera (Belgian patent specification No. 895090, Japanese patent specification No. 5,810,572, U.S. Pat. Nos. 4,537,859 and 4,346,227 and published European patent application No. 0605230). The bioconversion of compactin substrate was published in a 500 &mgr;g/ml concentration using filamentous molds such as
Mucor hiemalis, Syncephalastrum nigricans, Cunninghamella echinulata
and in 2000-4000 &mgr;/ml with Nocardia, Actinomodura and Streptomyces strains belonging to the prokaryotes.
A general problem experienced in the cases of manufacturing the pravastatin with filamentous molds is that due to the antifungal effect of compactin, the microorganisms are not able to tolerate the compactin substrate fed to the culture even at low concentrations (Serizawa et al., J. Antibiotics, 36, 887-891, 1983). The cell toxicity of this substrate was also observed in the hydroxylation with
Streptomyces carbophilus
extensively studied by Japanese researchers (M. Hosobuchi et al., Biotechnology and Bioengineering, 42, 815-820, 1993).
Japanese authors tried to improve the hydroxylating ability of the
Streptomyces carbophilus
strain with recombinant DNA techniques. A cytochrome P-450 monooxygenase system is needed for the hydroxylation of compactin (Matsuoka et al., Eur. J. Biochem. 184, 707-713, 1989). However, according to the authors, in the bacterial cytochrome P-450 monooxygenase system not one but several proteins act in the electron transport, which aggravate the application of the DNA techniques. Development of a cost-effective microbiological hydroxylation method for the manufacture of pravastatin is an extremely difficult, complex task.
The aim of the present invention is to elaborate a new microbial process for the preparation of pravastatin from compactin in industrial scale, which would produce pravastatin at more advantageous conditions than those previously known. During our research work, above all we tried to find a microorganisin strain with a hydroxylase enzyme that can be adapted for the microbial transformation of compactin to pravastatin in a high concentration.
SUMMARY OF THE INVENTION
The present invention relates to a microbial process for the preparation of the compound of formula (I)
from a substrate compound of formula (II),
wherein R stands for an alkali metal or ammonium ion,
comprising the steps of (a) cultivating a strain of
Mortierella maculata
filamentous mold species able to 6&bgr;-hydroxylate a compound of formula (II) on a nutrient medium containing assimilable carbon- and nitrogen sources and mineral salts, (b) feeding the substrate to be transformed into the developed culture of
Mortierella maculata
, (c) fermenting the substrate until the end of bioconversion, (d) separating the compound of formula (I) from the culture broth, and (e) isolating the compound of formula (I).
The present invention also relates to a biologically pure culture of the
Mortierella maculata
n. sp. E-97 strain deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary under accession number NCAIM(P)F 001266 and a biologically pure culture of its mutant, the
Mortierella maculata
n. sp. E-97/15/13 strain deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary under accession number NCAIM(P)F 001267.
REFERENCES:
patent: 4346227 (1982-08-01), Terahara et al.
patent: 4537859 (1985-08-01), Terahara et al.
patent: 5064817 (1991-11-01), Yedgar et al.
patent: 5130130 (1992-07-01), Menez et al.
patent: 850 090 (1983-03-01), None
patent: 0 605 230 (1994-07-01), None
patent: 58-10572 (1983-01-01), None
patent: WO 97/36996 (1997-10-01), None
“The Therapeutic Potential of Cell-Impermeable Inhibitors of Phospholipase A2” by Saul Yedgar,J. Cellular Biochemistry, S17D:178 (1993).
Derwent Abs. Of WO 8806445 (88-271023) Fariss et al, 1987.
Chem. Abs. 82:41565 Woelk et al., J. Neurol (1974) 207(4) 319-26.
Chem. Abs. 11:740 Regelson et al. (WO 8806445) 1987.
Chem. Abs. 11:52434 Valle et al, Abs. EP 505817 (Sep. 1992).
Chem Abs. 112:179707 Abs. JP 01180828 Miyazawa et al. (Jul. 1989).
Chem. Abs. 103:20744 Khan et al., Acta Neurol. Scand. (1985)71(3)212-20.
Endo, A. et al., “ML-236A, ML-236B, and ML-236C, New Inhibitors of Cholesterogenesis Produced by Penicillium Citrinum,” J. Antibiot 29, 1346-1348 (1976).
Endo, A. et al., “Competitive Inhibition of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase by ML-236A and ML-236B Fungal Metabolites, Having Hypocholesterolemic Activity,” FEBS Lett. 72, 323-326 (1976).
Kuo, C.H. et al., Aromatic Hydroxylation. Hydroxybenzaldehydes rom Bromobenzaldehydes via Reaction of in Situ Generated, Lithiated &agr;-Morpholinobenzyl Alkoxides with Nitrobenzene, J. Org. Chem. 48, 1991 (1983).
Arai, M. et al., “Pravastatin Sodium (CS-514), A Novel Cholesterol-Lowering Agent Which Inhibits HMG-CoA Reductase,” Sankyo Kenkyusyo Nenpo, 40, 1-38, 1988.
Koga, T. et al., “Tissue-selective Inhibition of Cholesterol Synthesis in Vivo by Pravastatin Sodium, a 3-Hydroxy-3-methylglutaryl coenzyme A Reductase Inhibitor,” Biochim. Biophys. Acta, 1045, 115-1120, 1990.
A.T.M., Serajuddin et al.,
Albrecht Karoly
Ambrus Gabor
Andor Attila
Barta Istvan
Bidlo nee Igloy Margit
Institute for Drug Research Ltd.
Killos Paul J.
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