Field of the Invention

The field of the invention relates to a process for the preparation of lovastatin. More particularly, it relates to the preparation of lovastatin substantially free of dihydrolovastatin. The invention also relates to pharmaceutical compositions that include the lovastatin substantially free of dihydrolovastatin and use of said compositions for treating hypercholesterolemia.

Background of the Invention

Chemically, lovastatin is (\S,3R,7S,SS,8aR)-8-{2-[(2R,4R)-4-hydτoxy-6- oxotetrahydro-2H-pyran-2-yl]ethyl} -3,7-dimethyl-l,2,3,7,8,8a-hexahydronaphthalen-l-yl (25)-2-methylbutanoate having the structural Formula I. After oral ingestion, the lovastatin, which is an inactive lactone, is hydrolyzed to the corresponding /3-hydroxyacid form. It is a principal metabolite and an inhibitor of the enzyme 3-hydroxy-3- methylglutaryl-coenzyme A (HMG-CoA) reductase and is thus useful as a hypercholesteromic agent. This enzyme catalyzes the conversion of HMG-CoA to mevalonate, which is an early and rate limiting step in the biosynthesis of cholesterol.

FORMULA I

Hypercholesterolemia is known to be a major risk factor for atherosclerosis. Complications of cardiovascular disease, such as myocardial infarction, stroke, and peripheral vascular disease result in many deaths in developed countries. A decrease in plasma LDL levels has been shown to reduce the risk of cardiovascular disease. Statins are by far the most therapeutically effective drugs for reducing the plasma LDL levels. Statins include for example, lovastatin, compactin, pravastatin, simvastatin which are essentially

fermentation based and rosuvastatin, fluvastatin and atorvastatin which are totally synthetic statins.

Lovastatin is one of the first statins described in the literature for example, in U.S. Patent Nos. 4,231,938; 4,294,846 and 4,294,926. Several processes are reported for recovering lovastatin from fermentation broth which suggest use of different solvents for extraction, recrystallization, use of different chromatographic methods for purification of lovastatin, and the like for example, in U.S. Patent No. 4,916,239; 4,231,938; International Publication Nos. (PCT) WO 92/16276; WO 00/63411; WO 03/48149; and European Patent No. 033536. (15',3Λ,75',8 ₁ S',8a/?)-8-{2-[(2/?,4i?)-4-hydroxy-6-oxotetrahydro-2H- pyran-2- yl]ethyl}-3,7-dimethyl-l,2,3,5,6,7,8,8a-octahydronaphthalen- l-yl (25)-2-methylbutanoate, commonly known as dihydro lovastatin (hereinafter referred to as Impurity A) of Formula II is one of the several commonly encountered impurities in lovastatin.

FORMULA II

Typically, the level of Impurity A in lovastatin harvest broth ranges from about 8% to 11% w/w relative to lovastatin. Since impurity is present in such a high amount in the broth, it is very difficult to remove this impurity A in the lovastatin drug product. None of the reported processes provide pure lovastatin with levels of impurity A less than 0.25%. Summary of the Invention

In one general aspect there is provided substantially pure lovastatin having less than 0.25% w/w relative to lovastatin of impurity A of Formula II.

In another general aspect there is provided a pharmaceutical composition that includes a therapeutically effective amount of the substantially pure lovastatin; and one or more pharmaceutically acceptable carriers, excipients or diluents.

In another general aspect there is provided a process for the preparation of substantially pure lovastatin having less than 0.25% w/w relative to lovastatin of impurity A. The process includes:

(a) obtaining a solution of lovastatin in toluene;

(b) recovering lovastatin;

(c) recrystallizing the lovastatin from one or more solvents; and (d) recovering the substantially pure lovastatin by the removal of the solvents.

In another general aspect there is provided a process for removal of impurity A from lovastatin. The process includes dissolving lovastatin in toluene, isolating lovastatin and recrystallizing from one or more solvents.

The process may include further purification of the product obtained. The process may produce the substantially pure lovastatin having less than 0.25% of impurity A. hi particular, it may produce the lovastatin having less than 0.2% of impurity A.

In another general aspect there is provided a method for treating hypercholesteremia in a warm-blooded animal, the method comprising providing a pharmaceutical composition to the warm-blooded animal that includes the substantially pure lovastatin.

The details of one or more embodiments of the inventions are set forth in the description below. Other features, objects and advantages of the inventions will be apparent from the description and claims. Detailed Description of the Invention

The inventors have developed a process for the preparation of substantially pure lovastatin having less than 0.25% of impurity A by HPLC. The process includes obtaining

a solution of lovastatin in toluene; recovering lovastatin; recrystallizing from one or more solvents; and recovering the substantially pure lovastatin by the removal of the solvents.

The inventors also have developed pharmaceutical compositions that contain the substantially pure lovastatin having less than 0.25% of impurity A for example, less than 0.2%, in admixture with one or more solid or liquid pharmaceutical diluents, carriers, and/or excipients.

In general, the solution of lovastatin may be obtained by dissolving lovastatin in toluene. Alternatively, such a solution may be obtained directly from fermentation broth. The solution of lovastatin in toluene may be obtained by heating. It may be heated from about 40°C to about 12O ⁰ C, for example from about 50 ⁰ C to about 100 ⁰ C. It may be heated from about 10 minutes to about 24 hours. More particularly, it may be heated for about 2-3 hours. The solution may be filtered to remove any undissolved foreign particulate matter.

In general, any of the methods known in the art can be employed for isolation of lovastatin from the fermentation broth. In particular, it may be isolated from the fermentation broth in the form of an in-situ aqueous process stream employing the method described in International Publication No. (PCT) patent application WO 02/ 00615.

The lovastatin may be recovered by a technique which includes, for example, distillation, distillation under vacuum, cooling, evaporation, filtration, filtration under vacuum, decantation and centrifugation.

The lovastatin thus recovered may be further purified or additionally purified, by employing commonly practiced recrystallization techniques using solvent / antisolvent mixture to obtain substantially pure lovastatin.

The term "lovastatin substantially free of impurity A" as described herein refers to lovastatin containing less than 0.25% w/w of impurity A relative to lovastatin. The substantially pure lovastatin of the invention has purity greater than 98%.

The term "solvent" includes any solvent or solvent mixture in which lovastatin, is soluble, including, for example, lower alkanol, polar aprotic solvents, and mixtures thereof. Examples of alkanol include those primary, secondary and tertiary alcohols having from one to six carbon atoms. Suitable lower alkanol solvents include methanol,

ethanol, n-propanol, and isopropanol. A suitable polar aprotic solvent includes one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulphoxide, acetonitrile and N-methylpyrrolidone. Mixtures of all of these solvents are also contemplated. In another aspect, a suitable anti-solvent can be added to the clear solution to precipitate the substantially pure lovastatin. The term "anti-solvent" includes any solvent in which lovastatin is insoluble or poorly soluble or practically insoluble or partially soluble, including, for example, cyclopentane, cyclohexane, cycloheptane, hexane, petroleum ether, heptane, water, or mixtures thereof. The resulting pure lovastatin may be formulated into ordinary dosage forms such as, for example, tablets, capsules, pills, solutions, etc. In these cases, the medicaments can be prepared by conventional methods with conventional pharmaceutical excipients.

The lovastatin can be administered for the prevention and treatment of hypocholesterolemia in a warm-blooded animal. For the purpose of this disclosure, a warm-blooded animal is a member of the animal kingdom possessed of a homeostatic mechanism and includes mammals and birds.

The lovastatin is generally administered as part of a pharmaceutical composition with a pharmaceutically acceptable carrier, diluent or excipient and optionally other therapeutic ingredients. The lovastatin may be conventionally formulated into tablets, capsules, suspensions, dispersions, injectables and other pharmaceutical forms. Any suitable route of administration may be employed, such as, for example, peroral or parenteral.

The present invention is further illustrated by the following examples which are provided merely to be exemplary of the invention and do not limit the scope of the invention. Certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

Example 1 :

Fermentation broth (12 L) was acidified to pH 2.0 with sulphuric acid (2N) _. The acidified broth was heated to 5O ⁰ C and was stirred at room temperature for 24 hours. The broth was filtered and the mycelial cake was extracted twice with toluene (6 L + 5 L). The

toluene extract was washed with sodium bicarbonate solution (5% aqueous). The washed toluene extract was concentrated under vacuum at about 50 ⁰ C. The concentrate was cooled to 5 - 10°C and stirred further for 1 hour. The slurry so obtained was filtered and dried to yield 100 gm of lovastatin powder. 80 gm of the lovastatin was dissolved in toluene (16OmL) at +95 °C and the clear homogenous solution was stirred for 10 to 15 minutes and then cooled to +27°C. The slurry was stirred at room temperature for 2 hours and then cooled to +5 ⁰ C and again stirred at this temperature for 2 hours. The cooled slurry was filtered and the wet cake washed with pre-cooled (5°C) toluene (16OmL). The whole operation from the dissolution of lovastatin in toluene till filtration and washing with pre-cooled toluene was repeated again with wet cake. The wet cake was dried under vacuum at temperature +45 ⁰ C ± 2°C, to get lovastatin

Yield: 62 gm Assay: 97.98% w/w Impurity A: 0.56 % w/w (by HPLC)

20 gm of the lovastatin obtained above was further purified via recrystallization in from isopropyl alcohol (14OmL) to yield the substantially pure lovastatin.

Yield: 17.67 gm Assay: 98.23% w/w Impurity A: 0.23% w/w (by HPLC) Example 2:

Lovastatin (20 gm) obtained through example 1; having 97.98 % purity and 0.56 % impurity A (w/w of lovastatin) was taken in dimethylsulphoxide (30OmL) at +26 ⁰ C. The resultant mixture was stirred for 30 minutes to get a clear solution. To the clear solution, deionized water (58mL) was added drop wise till precipitation started. The slurry was stirred for 60 minutes at room temperature and consequently cooled to +1O ⁰ C. The cooled slurry was stirred for 60 minutes and filtered. The wet cake was washed with pre-cooled

deionized water and dried at temperature +45°C under vacuum to get substantially pure lovastatin.

Yield: 17.43 gm Purity: 99.2 % Impurity A: 0.21 % w/w (by HPLC) Example 3:

Lovastatin (20 gm) obtained through example 1; having 97.98 % purity and 0.56 % impurity A was taken in 10OmL of acetonitrile. The slurry was heated to +6O ⁰ C till a clear solution was observed. To the clear solution, 4OmL of deionized water was added at +6O ⁰ C. The temperature of the solution was brought to room temperature (+26 ⁰ C) and stirred for further 60 minutes. The slurry was cooled to +10 ⁰ C and stirred for 60 minutes. The product was filtered and washed with pre-cooled deionized water (4OmL). The wet cake was dried under vacuum at temperature +45°C, till constant weight was obtained.

Yield: 17.33 gm Purity: 98.9 %

Impurity A: 0.12% w/w (by HPLC) Example 4:

An aqueous fermentation broth containing 9.6% of impurity A (w/w, relative to lovastatin) was used for recovery of crude lovastatin by a process as provided in Example 1. Lovastatin (10 gm) containing 3.92% impurity A was added to 5OmL of toluene and the slurry was heated to +95 ⁰ C to get a clear solution. The clear homogenous solution was stirred at + 95 ⁰ C for 10 to 15 minutes and then cooled to room temperature (+ 27°C), when lovastatin lactone crystallized. The slurry was stirred at room temperature for 2 hours. The slurry was then cooled to +5 ⁰ C and stirred at this temperature for 2 hours. The cooled slurry was filtered and the wet cake was washed with pre-cooled (5°C) toluene (20 mL). The whole operation from the dissolution of powder in toluene till filtration and washing with pre-cooled toluene was repeated twice with wet cake. The wet cake was dried under vacuum at temperature + 45 ⁰ C ± 2° C, to give lovastatin.

Yield: 8.3 gm Assay: 96.43 % w/w Impurity A: 0.45 % w/w (by HPLC).

7 gm lovastatin obtained above was further purified by recrystallization from isopropyl alcohol (49mL).

Yield: 6.1 gm Assay: 99.12 % w/w Impurity A: 0.11% w/w

While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.