Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-06-23
2001-08-14
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S125000
Reexamination Certificate
active
06274360
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for converting compactin to pravastatin using an agent derived from the filamentous bacterium Actinomadura, to methods for lowering cholesterol levels in mammals, and to Actinomadura and to Actinomadura hydroxylase.
BACKGROUND OF THE INVENTION
One of the major causes of atherosclerosis and coronary disease is attributed to high blood cholesterol levels. It has been estimated that at least about 50% of total body cholesterol is derived from de novo cholesterol synthesis. A major rate-limiting step in the cholesterol biosynthetic pathway is catalyzed by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase. Compactin and pravastatin have been reported to be competitive inhibitors of HMG-CoA reductase, and the presence of either one can result in inhibition of cholesterol biosynthesis.
Microbial hydroxylation of compactin can produce hydroxylated forms of compactin, e.g., pravastatin. Some hydroxylated forms are reportedly more effective than compactin as competitive inhibitors of HMG-COA. It has been reported that this hydroxylation can be effected to differing degrees by many different genera of fungi, and from the bacteria Nocardia and
Streptomyces roseochromogenus
and
Streptomyces carbophilus.
See, e.g., U.S. Pat. No. 5,179,013; U.S. Pat. No. 4,448,979; U.S. Pat. No. 4,346,227; U.S. Pat. No. 4,537,859; Canadian Patent No. 1,150,170; Canadian Patent No. 1,186,647; Serizawa et al., J. Antibiotics 36:887-891 (1983).
A problem with using fungi for the production of pravastatin is that they generally do not tolerate increases in the amount of compactin added to the culture medium, presumably due to the anti-fungal activity of compactin. Serizawa et al., J. Antibiotics 36:887-891 (1983).
The cytochrome P450 system has been shown to be required for the hydroxylation of compactin to pravastatin in
Streptomyces carbophilus.
Matsuoka et al., Eur. J. Biochem. 184:707-713 (1989). Problems with the use of such an enzyme is that it is a complex of proteins rather than a single protein, making recombinant DNA manipulations difficult, and that compactin, which is an inducer of the cytochrome P450 system, is very expensive.
SUMMARY OF THE INVENTION
It is an object of the invention to provide an effective and relatively inexpensive method for converting compactin to pravastatin.
It is another object of the invention to use Actinomadura, a filamentous bacterium, to convert compactin to pravastatin.
It is yet another object of the invention to use an Actinomadura hydroxylase to convert compactin to pravastatin.
It is yet another object of the invention to use an Actinomadura constitutive hydroxylase that does not require compactin as an inducer, to convert compactin to pravastatin.
Still another object of the invention is to use pravastatin, derived from an Actinomadura hydroxylase that converts compactin to pravastatin, to treat a mammal so as to lower the mammal's blood cholesterol level.
According to the invention, a method for converting compactin to pravastatin is provided. Compactin is provided and contacted with an agent, e.g., a hydroxylation enzyme, e.g., a hydroxylase that is, e.g., constitutive and cytochrome P450 system-independent, derived from Actinomadura, under conditions in which the agent converts compactin to pravastatin. In certain embodiments, the pravastatin is isolated.
Preferably, the compactin is provided by providing, e.g., a microorganism, e.g., a fungus or bacterium, that produces compactin, or a cell free extract of a microorganism that produces compactin, or cell free culture media from a pregrown culture of a microorganism that produces compactin, or a solution comprising compactin, or semi-purified compactin, or substantially purified compactin.
In certain embodiments, the compactin is contacted with the agent, e.g., by contacting whole cells of Actinomadura with the compactin, or by contacting a cell free extract of Actinomadura with the compactin, or by contacting cell free culture media from a pregrown culture of Actinomadura with the compactin, or by contacting a solution having the Actinomadura agent with the compactin, or by contacting semi-purified or substantially purified Actinomadura agent with the compactin.
Variations include, e.g., contacting, e.g., a culture, e.g., a pregrown culture or a starting culture, or a cell free extract, of the microorganism that produces compactin, or semi-purified or substantially purified compactin, with, e.g., a culture, e.g., a pregrown culture or a starting culture, or a cell free extract, of Actinomadura, or semi-purified or substantially purified Actinomadura agent.
Another aspect of the invention is a cell free extract derived from Actinomadura, having an agent, e.g., a hydroxylase, that converts compactin to pravastatin.
Another aspect of the invention is a hydroxylase, e.g., semi-purified or substantially purified, from Actinomadura that converts compactin to pravastatin, wherein the hydroxylase is a constitutive enzyme, wherein the activity of the hydroxylase is stimulated by any of ATP, ascorbic acid or Mg
++
, but not by Fe
++
or Fe
+++
, and wherein the hydroxylase is cytochrome P450 system-independent.
Another aspect of the invention is purified Actinomadura ATCC 55678 having an agent for converting compactin to pravastatin.
Yet another aspect of the invention is a method for treating a mammal to lower the blood cholesterol level of the mammal. Pravastatin, derived from compactin by contacting the compactin with an agent, e.g., a hydroxylase, derived from Actinomadura that converts compactin to pravastatin, is provided. The pravastatin is administered to a mammal in need of such treatment to cause a lower blood cholesterol level in the mammal.
The above and other objects, features and advantages of the present invention will be better understood from the following specification.
DETAILED DESCRIPTION
This invention provides a method for converting compactin to pravastatin. Compactin is provided and contacted with an agent derived from Actinomadura under conditions in which the agent converts compactin to pravastatin. In certain embodiments, the pravastatin is isolated.
Compactin (also known as mevastatin, ML-236B, and CS-500) is meant to include, e.g., the acid form (also known as ML-236B carboxylic acid), the lactone form (also known as ML-236B lactone), and salts and esters thereof. The lactone form of compactin may be represented by the formula (I):
A preferred compactin is the sodium salt of compactin.
Pravastatin (also known as eptastatin, mezalotin, pravachol, CS-514, and SQ-31000) is meant to include, e.g., the acid form, the lactone form, and salts and esters thereof The 3&bgr;-hydroxy lactone form of pravastatin may be represented by the formula (II):
Other forms of hydroxylated compactin include compounds in which the hydroxyl group is added at other positions of the compactin molecule, e.g., at position 6.
The compactin can be provided in any way which enables the agent to act upon it. For example, compactin can be provided by providing a microorganism, e.g., a fungus or bacterium, that produces compactin. Microorganism is meant to include, e.g., microbial cells which are intact, immobilized or permeabilized. Production of compactin by the microorganism is meant to include the microorganism using its own natural gene or genes, or fragments thereof, to produce the compactin, and/or the microorganism using a foreign gene or genes, or fragments thereof, to produce the compactin. The foreign gene can be introduced into the microorganism by standard molecular cloning techniques known in the art, or by any other means which will result in expression of the compactin producing gene or genes, or fragments thereof, in the microorganism. In certain embodiments, the compactin is provided by using a cell free extract of a microorganism that produces compactin. The cell free extract can be prepared by a variety of methods known in the art, e.g., by physical or chemical means, so as to rupture the cells. Such metho
Davis Joseph
Demain Arnold L.
Peng Yulin
Yashphe Jacob
Lilling Herbert J.
Massachusetts Insitute of Technology
Testa Hurwitz & Thibeault LLP
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