KR20030059173A - Pravastatin sodium substantially free of pravastatin lactone and epi-pravastatin, and compositions containing same - Google Patents

Abstract

The present invention provides pravastatin sodium that is substantially free of epiprava, which is a C-6 epimer of pravastatin lactone and pravastatin. The present invention also provides a novel method for recovering high purity pravastatin sodium from fermentation broth. The method includes extracting to form a compound solution, obtaining an ammonium salt of pravastatin from the solution, purifying the ammonium salt of the compound, and converting the salt of the compound to pravastatin sodium.

Description

Pravastatin sodium substantially free of pravastatin lactone and epi-pravastatin, and compositions comprising the same {PRAVASTATIN SODIUM SUBSTANTIALLY FREE OF PRAVASTATIN LACTONE AND EPI-PRAVASTATIN, AND COMPOSITIONS CONTAINING SAME}

Statin drugs are currently the most therapeutically available drugs available to lower LDL levels in the bloodstream of patients at risk for cardiovascular disease. Drugs of this kind include pravastatin, as well as compactin, lovastatin, simvastatin, fluvastatin and atorvastatin.

Pravastatin is a chemical compound [1S- [1α (β ^* , δ ^* ) 2α, 6α, 8β (R ^* ), 8aα]]-1,2,6,7,8,8a-hexahydro-β, δ, 6 -Trihydroxy-2-methyl-8- (2-methyl-1-oxobutoxy) -1-naphthalene-heptanoic acid. (CAS reg. No. 81093-370). The molecular structure of pravastatin is represented by the formula Ia, where R = OH. The lactone form is represented by the formula Ib, and numbers are indicated on the atoms to indicate numbering of the atoms.

Pravastatin, compactin (compound of formula Ia with R = H), lovastatin (compound of formula Ia with R = CH ₃ ), simvastatin, fluvastatin and atorvastatin each have an alkyl chain at the end of the carboxylic acid group, Retains two hydroxyl groups at the β and δ positions. The carboxylic acid group and the hydroxyl group at the δ position are susceptible to lactonation, as shown in formula (Ib). Lactonable compounds such as statins may be present in the free acid form or in the lactone form, or as equilibrium mixtures in both forms. Since the free acid form and the lactone form of the compounds differ in polarity, lactonation makes processing difficult in the production of statin drugs. It is possible to remove one form with impurities by purifying one form, but the yield is low. Therefore, in order to separate them in high yields, great care should generally be taken when handling lactonable compounds.

Currently, the most economically suitable method for preparing pravastatin is to hydroxylate the compactin at the C-6 position by the microorganism. Although enzymatic processes are highly stereoselective, pravastatin sodium, usually obtained after separation from fermentation broth, is contaminated with a significant amount of pravastatin's C-6 epimer (“epiprava”). The C-6 position is bis-allyl, so the C-6 atoms tend to epimerize. To minimize epimerization, pH and other conditions should be carefully controlled during the separation of pravastatin. Existing methods for separating pravastatin from fermentation broth yield pravastatin sodium that is not suitable for separating pravastatin as a sodium salt or contaminated with a significant amount of pravastatin lactone and / or epiprava. The present invention does not require purification by chromatography and meets the needs of the art for an effective method for separating high-purity, high-yield pravastatin sodium from fermentation broth on a preparative scale.

Cross-Reference to Related Applications

This application claims 35 U.S.C. Claims priority to U.S. Provisional Application No. 60 / 238,278, filed October 5, 2000, under 119 (e), hereby incorporated by reference.

Field of invention

The present invention relates to statins, more particularly pravastatin sodium, and to a process for separating them from fermentation broth as enzymatic hydroxylation products of compactin.

The present invention provides pravastatin sodium with little or no epiprava, which is a C-6 epimer of pravastatin lactone and pravastatin. The present invention also provides a process that can be carried out on an industrial scale to produce such substantially pure pravastatin sodium.

Preferred embodiments of this method include extracting pravastatin from an aqueous fermentation broth into an organic solvent, back extracting pravastatin into an aqueous basic solution, and reextracting into an organic solvent to initiate the initial preparation of pravastatin in the fermentation broth. An organic solution is obtained in which pravastatin is concentrated relative to the concentration. Pravastatin can be precipitated as an ammonium salt and then purified by recrystallization of the ammonium salt to obtain pravastatin from the pravastatin concentrated solution. The recrystallized salt is replaced with sodium salt to form pravastatin sodium salt, and any excess sodium ions are removed with an ion exchange resin. Recrystallization, lyophilization or other means can then be used to separate the sodium salt of pravastatin from the solution in a highly pure state.

Detailed description of the invention

The present invention provides pravastatin sodium salt with little or no pravastatin lactone and epiprava, and a downstream method for separating high purity pravastatin sodium from fermentation broth.

Enzymatic hydroxylation of compactin

Enzymatic hydroxylated broth from which pravastatin is isolated is any aqueous broth known for industrial scale fermentation of compactin, for example the method is disclosed in US Pat. Nos. 5,942,423 and 4,346,227. Enzymatic hydroxylation is preferably performed using a bioculture of Steptomyces in a nutrient mixture of compactin and dextrose. If the broth is neutral or basic at the end of fermentation, the acid is added to bring the pH of the broth to about 1-6, preferably 1-5.5, more preferably 2-4. Acids which can be used include hydrochloric acid, sulfuric acid, trifluoroacetic acid or any other protic acid, preferably acids having a pH of less than 1 as a 1M solution in water and the like. Acidifying the fermentation broth converts any pravastatin carboxylate salt in the broth to free acid and / or lactone.

Isolation of Pravastatin Sodium from Substantially Pure

First, pravastatin is obtained from an aqueous fermentation broth in a relatively highly concentrated organic solution in the order of extraction and back extraction steps.

In the first step, pravastatin is extracted from the fermentation broth. C ₁ -C ₄ alkyl esters of C ₂ -C ₄ alkyl formate and C ₂ -C ₄ carboxylic acids can effectively extract pravastatin from an aqueous fermentation broth. Alkyl groups may be straight, branched or cyclic. Preferred esters include ethyl formate, n-propyl formate, i-propyl formate, n-butyl formate, s-butyl formate, i-butyl formate, t-butyl formate, methyl acetate, ethyl acetate, n-propyl acetate, i-propyl acetate, n-butyl acetate, s-butyl acetate, i-butyl acetate, t-butyl acetate, methyl propionate, ethyl propionate, n-propyl propionate, i-propyl Propionate, butyl propionate, methyl butyrate, ethyl butyrate, n-propyl butyrate, i-propyl butyrate, butyl butyrate, methyl isobutyrate, ethyl isobutyrate, propyl isobutyrate and butyl isobutyrate. The inventors have found that ethyl acetate, i-butyl acetate, propyl acetate and ethyl formate are particularly suitable among preferred organic solvents. Most preferred extraction solvent is i-butyl acetate. Other organic solvents may be used instead of esters. Halogenated halocarbons, aromatic compounds, ketones and ethers such as dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, benzene, butyl methyl ketone, diethyl ether and methyl t-butyl ether can be used.

If necessary, back extract pravastatin with a basic aqueous solution of pH about 8.0 to about 9.5. The base is preferably NaOH, NH ₄ OH or KOH, most preferably NaOH. It is desirable to contact the extraction solvent with the basic aqueous solution until substantially no amount of pravastatin in the organic phase is determined by any method including thin layer chromatography or subjective determination that it is sufficiently contacted for complete extraction. In order to optimize the number of times, the back extraction can be performed multiple times. However, one back extraction is very effective when the organic phase is i-butyl acetate. The pravastatin can be concentrated by back extraction with a volume of aqueous base that is smaller than the volume of the organic extract. The back extraction is preferably carried out with a basic aqueous solution of less than 1/3, more preferably less than 1/4 and most preferably less than 1/5 of the volume of the organic extract.

The aqueous solution is preferably acidified with an acid, preferably trifluoroacetic acid, hydrochloric acid, sulfuric acid, acetic acid or phosphoric acid, more preferably sulfuric acid to a pH of about 1.0 to about 6.5, more preferably about 2.0 to about 3.7.

It is preferred to re-extract pravastatin with one of the organic solvents known to be suitable for extracting pravastatin from fermentation broth. The organic solvent may be the same solvent used to extract pravastatin from the fermentation broth, but it is not necessary. In the re-extraction process, preferably less than about 50% (v / v) of the aqueous extract volume, more preferably about 20 to about 33% (v / v), more preferably about 25% (v / v) Pravastatin can be further concentrated by re-extraction with an organic solvent. Pravastatin can be concentrated with 8 L of concentrated organic solution from 100 L of fermentation broth, with 89% yield from the initial organic extract. Although only one extraction is disclosed in a preferred manner for practicing the present invention, those skilled in the art will appreciate that multiple extractions can be performed to obtain purified pravastatin in higher yields. In this preferred manner it is possible to balance the cost of the solvent with the high yield of the product. Variations of the preferred manner in which the yields are further improved by multiple extractions which repeat the above described single extractions do not depart from the spirit of the present invention. Prior to the process of "salting out" to obtain pravastatin from the concentrated organic solution, it is preferred to dry the concentrated organic solution and optionally to bleach with activated charcoal. The drying step may be carried out using conventional drying agents such as MgSO ₄ , Na ₂ SO ₄ , CaSO ₄ , silica, pearlite and the like. It is then preferred to separate the dried and / or decolorized concentrated organic solution by conventional separation methods such as filtration or decanting.

In the next step, pravastatin can be salted from the concentrated organic solution using ammonia or amines. The amines can be primary, secondary or tertiary. Any alkyl or aryl amine that is not hindered enough to prevent ionic interactions between the amine nitrogen and the carboxyl groups of pravastatin can be used. Non-limiting examples of amines include methyl, dimethyl, trimethyl, ethyl, diethyl, triethyl and other C ₁ -C ₆ primary, secondary and tertiary amines, including morpholine, N-methylmorpholine, Isopropyl cyclohexyl amine, piperidine and the like. Regardless of the absence, presence or majority of substituents on nitrogen, salts formed by the reaction of ammonia or amines are referred to below as ammonium salts. This is intended to include salts of amines as well as salts of ammonia.

Ammonium salts may be added alone or in combination with ammonia or amines to induce precipitation of pravastatin ammonium salts. Preferred ammonium salts include NH ₄ Cl, NH ₄ Br, NH ₄ I, (NH ₄ ) ₂ SO ₄ , NH ₄ NO ₃ , (NH ₄ ) ₃ PO ₄ , (NH ₄ ) ₂ S ₂ O ₄ , NH ₄ OAc and the like. Is a salt of ammonia, most preferred is NH ₄ Cl. Ammonium salts and high boiling point liquids and solid amines can be added in a conventional manner as solids, neat liquids or solutions in aqueous or organic solvents, preferably in well ventilated areas. Adding gaseous ammonia requires special equipment for handling corrosive gases. Devices such as pressurized vessels, regulators, valves and lines are widely used. In a particularly preferred embodiment, gaseous ammonia and NH ₄ Cl are added to the concentrated organic solution to obtain pravastatin as the pravastatin salt of ammonia from the concentrated organic solution.

The temperature at which ammonia, amine and / or ammonium salts should be added can be determined by routine experimentation by running the reaction on a small scale and monitoring the exothermicity of the reaction. It is preferable that the solution temperature does not exceed 40 ° C. There is no significant degradation of pravastatin even at temperatures as high as 80 ° C., but many of the organic solvents of the present invention will boil at lower temperatures. If ammonia is used, the preferred temperature range is about -10 to about 40 ° C.

If it appears that it is no longer precipitated, or if it is determined by other means that the consumption of pravastatin is substantially terminated, then the addition should be stopped. If ammonia or volatile amines are used, it is desirable to make vents in the vessel to dissipate excess smoke. The crystals can then be separated by filtration, solvent decantation, solvent evaporation or other methods, with filtration being preferred. The crystals can then be washed, preferably with i-butyl acetate and acetone.

In some cases, after washing the precipitated crystals, it is preferable to purify the pravastatin ammonium salt by recrystallizing one or more times, most preferably three times. In order to purify the pravastatin ammonium salt, it is preferred to dissolve the salt in water. It is desirable to add antisolvents to reduce the polarity of the solution. The antisolvent is preferably a water-soluble organic solvent or a solvent mixture in which the pravastatin salt is hardly dissolved, and i-butyl acetate and acetone are preferable.

The pravastatin salt may be spontaneously recrystallized, or an additional step of adding common ions may be introduced to induce recrystallization. According to a preferred process of purifying pravastatin as ammonium salt, NH ₄ Cl is added to induce recrystallization of the ammonium salt.

Recrystallization may be carried out at about −10 to about 40 ° C., preferably at about 0 ° C. to about 40 ° C. After substantially recrystallizing the pravastatin salt from the solution, the crystals can be separated, for example washed with a 1: 1 mixture of i-butyl acetate and acetone and then dried. Drying can be carried out at room temperature, but preferably at a slightly elevated temperature below 45 ° C., preferably at about 40 ° C. As shown in Examples 3 and 4, recrystallization can be optionally repeated to enhance the effect. One iteration yields about 92% yield.

After purification of pravastatin ammonium salt, it is preferred to replace pravastatin ammonium salt with pravastatin sodium. Dissolving pravastatin ammonium salt in an aqueous solvent, acidifying to pH about 2 to about 4, more preferably about 3.1 with any positive asset, preferably sulfuric acid, and then extracting pravastatin with an organic solvent to liberate pravastatin from the ammonium salt. It is preferable. The organic solvent can be any of the organic solvents mentioned above, but is preferably i-butyl acetate, and in some cases the organic solvent is contacted with an acidified solution until pravastatin transitions to the organic phase substantially completely. The organic phase is preferably separated from the aqueous phase, and optionally washed with water to remove the ammonium residue, followed by back extraction of pravastatin with aqueous sodium hydroxide solution at a pH of about 7.4 to about 13.0. Back extraction should be carried out by lowering the temperature to about 8 to about 10 ℃.

After extraction with aqueous sodium hydroxide, excess sodium cations are removed such that the sodium cation and pravastatin are approximately 1: 1 equivalent using an ion exchange resin insoluble in water. Suitable ion exchange resins are cationic and chelate type resins, with strong and weak acid exchange resins being preferred.

A strong acid cation exchange resins that can be used are sulfonic acid ^- are those having a group (SO ₃ H ^+). Examples include commercially available Amberlite® IR-118, IR-120 252H; Amberlyst® 15, 36; Amberjet 1200 (H) (Rom and Haas); Dowex® 50WX series, Dowex HCR-W2, Dowex 650C, Dowex Marathon C, Dowex DR-2030, and Dowex HCR-S, Ion Exchange Resin (Dow Chemical Company); DIAION SK 102-DIAION SK 116 resin series and Lewatit SP 120 (Bayer). Preferred strong acid cation exchange resins are Amberlite® 120, Dowex 50WX and DIAION SK series.

Weak acid cation exchange resins include those having pendant carboxylic acid groups. Weak acid cation exchange resins include commercially available Amberlite CG-50, IRP-64, IRC 50 and C67, Dowex CCR series, Lewatit CNP series (Bayer) and DIAION WK series (Mitsubishi), among which Amberlite® IRC50, Most preferred are Lewatit CNP80 and DIAION WK 10. Chelated exchange resins are less preferred. Some of the commercially available products are Amberlite® IRC-718 and IRC-467.

A sufficient amount of resin is stirred together with the solution in any method or flask known in the art, including passing the solution comprising pravastatin sodium salt and excess sodium cation through a bed or column of resin. Can be brought into contact with the ion exchange resin. The manner of contact is not important. After removal of excess sodium ions, the pH of the pravastatin sodium solution can be diluted to vary the range, but should be in the range of about 7 to about 10, preferably about 7.4 to about 7.8. If the pH of the pravastatin sodium solution is reduced from a higher pH to a lower pH and there is no further pH reduction at the lower pH, then the removal of excess Na ⁺ ions is substantially complete. After substantially completing the excess ion removal, it is desirable to separate the pravastatin sodium solution from the resin in a conventional manner. The pravastatin sodium solution can be collected from the column or phase as an eluent or separated by filtration, decantation, and the like.

Crystallization may separate pravastatin sodium from pravastatin sodium solution. In order to effectively crystallize, water must first be partially removed, which can be done by vacuum distillation or nano-filtration. Prior to crystallization it is preferred to concentrate the concentration of the pravastatin sodium salt aqueous solution to about 20 to about 50 w / v%. If necessary, the pH of the aqueous pravastatin sodium solution after concentration can be adjusted to about 7 to about 10 with an H ⁺ type ion exchange resin.

The addition of a water soluble organic solvent or organic solvent mixture to the pravastatin sodium solution aids in crystallization. In particular, acetone and acetone / acetonitrile, ethanol / acetonitrile and ethanol / ethyl acetate mixtures are exemplified. One of the most preferred solvent systems for crystallizing pravastatin sodium is 1/3/12 water / acetone / formed by concentrating the pravastatin sodium solution to about 30 w / v% and then adding an appropriate volume of 1/4 acetone / acetonitrile mixture. Acetonitrile mixture. The most preferred crystallization solvent mixture is water-acetone (1:15).

In addition, pravastatin sodium may be separated by lyophilization of an aqueous solution of pravastatin sodium.

Regardless of separation means such as lyophilization, crystallization or other means that do not lower the purity of the product, pravastatin sodium isolated by the practice of the present invention is almost free of pravastatin lactone and epiprava. As demonstrated in the examples below, pravastatin sodium can be separated with less than 0.5% (w / w) pravastatin lactone contaminants and less than 0.2% (w / w) epiprava contaminants. Furthermore, according to a preferred embodiment of the present invention, pravastatin sodium can be separated with less than 0.2% (w / w) pravastatin lactone and 0.1% epiprava. Two of the preferred embodiments are illustrated in Examples 1 and 3.

The high purity pravastatin sodium produced by the methods of the present invention is useful for hypercholesterolemia therapy and can be administered to mammalian patients by any route of administration. The daily oral regimen is the most preferred dosage regimen. In human subjects with normal liver function and normal weight, decreased oral serum cholesterol concentrations are usually observed by oral administration of pravastatin sodium at least 10 mg daily. The dosage of high purity pravastatin sodium is any effective amount. Preferred oral formulations of the present invention comprise about 10 to about 40 mg of pravastatin sodium. Oral formulations include tablets, pills, capsules, troches, sachets, suspensions, powders, lozenges, elixirs and the like. Nearly pure pravastatin sodium can be administered by any route, with the most preferred route of administration being oral.

High purity pravastatin can be administered alone or in combination with pharmaceutical excipients as a composition. Whether administered alone or as a composition, the high purity pravastatin sodium of the present invention may be in the form of a solution or in the form of a powder, granule, coagulant or the like or any other solid form.

The composition of the present invention includes a composition for tableting. The composition for tableting may comprise several or several excipients depending on the tableting method used, the desired release rate and other factors. For example, the compositions of the present invention may contain diluents such as powdered cellulose, microcrystalline cellulose, particulate cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose salts. And other cellulose derived materials such as substituted and unsubstituted cellulose; Starch; Pregelatinized starch; Inorganic diluents such as calcium carbonate and calcium diphosphate and other diluents known in the pharmaceutical industry. Other suitable diluents include sugars and sugar alcohols such as waxes, mannitol and sorbitol, acrylate polymers and copolymers, and pectins, dextrins and gelatins.

Further tableting excipients include binders such as acacia gum, pregelatinized starch, sodium alginate, glucose and other binders used in wet and dry granulation and direct compression tableting processes. Excipients that may be present in the solid compositions of novel forms of pravastatin sodium further include disintegrants such as sodium starch glycolate, crospovidone, low substituted hydroxypropyl cellulose and the like. Further excipients include tableting lubricants such as magnesium stearate, calcium stearate and sodium stearyl fumarate; Spices; Sweeteners; Preservatives; Pharmaceutically acceptable dyes and glidants such as silicon dioxide.

Capsule formulations comprise a solid composition in a capsule composed of gelatin or other encapsulating material. Tablets and powders may be coated. Tablets and powders may be coated with enteric coatings. The enteric coating powder contains materials such as phthalic acid cellulose acetate, hydroxypropylmethyl cellulose phthalate, polyvinyl alcohol phthalate, carboxymethylethylcellulose, copolymers of styrene and maleic acid, copolymers of methacrylic acid and methyl methacrylate, and the like. It can include a coating and can be used with suitable plasticizers and / or thickeners as needed. Coated tablets may be tablets having a coating on the surface of the tablet, or comprising powder or granules with enteric coating.

High purity pravastatin sodium can be administered in injectable formulations as a solute or suspended solid in a sterile solution or suspension. Suitable carriers for sterile injectable formulations are water and oils.

The following examples illustrate the practice of the invention in some embodiments, but these examples should not be construed as limiting the scope of the invention. Other embodiments will become apparent to those skilled in the art upon consideration of the specification and examples. The specification, including examples, is illustrative only and the scope and spirit of the present invention is defined by the following examples.

Example 1

Tablets of Pravastatin

Fermentation broth (100 L) was acidified to pH about 2.5 to about 5.0 by adding sulfuric acid. The acidified fermentation broth was extracted with i-butyl acetate (3 x 50 L). i-butyl acetate extraction yield was found to be 95% by calibrated HPLC analysis compared to internal standard in broth. Concentrated ammonium hydroxide was added to extract the mixed i-butyl acetate phase at pH about 7.5 to about 11.0 with water (35 L). The resulting pravastatin aqueous solution by addition of 5M sulfuric acid was reoxidized to pH about 2.0 to about 4.0 and then back extracted with i-butyl acetate (8 L). The pravastatin solution in the resulting i-butyl acetate was partially dried over perlite and Na ₂ SO ₄ . The pravastatin solution was drained off, filtered to remove the desiccant and bleached on activated carbon (1.7 g). The solution was then filtered to remove activated charcoal and transferred to a flask equipped with a gas inlet.

Then ammonia gas was introduced into the headspace above the solution with rapid stirring. Precipitated crystals of ammonium pravastatin carboxylate salt were collected by filtration and washed sequentially with i-butyl acetate and acetone to obtain pravastatin ammonium salt in approximately 94% purity. Purity is measured by UV absorbance detection at λ = 238 nm and HPLC.

The pravastatin ammonium salt was further purified by crystallization from saturated ammonium chloride solution as follows. Pravastatin salt containing 162 g of active substance was dissolved in water (960 mL) and diluted with acetone (96 mL) and i-butyl acetate (96 mL) at about 35-40 ° C. The solution was cooled to about 30-32 ° C. and further solid NH ₄ Cl was added until no obvious increase in crystal formation led to crystallization of pravastatin ammonium. After addition of ammonium chloride, the solution was cooled to about 0-26 ° C. The pravastatin ammonium crystals were collected by filtration as described above, washed sequentially with i-butyl acetate and acetone, and then dried at about 40 ° C. The resulting pravastatin ammonium salt crystals (155.5 g) were obtained in approximately 98% purity, which was determined by HPLC using the conditions described above.

Further purification of pravastatin ammonium salt was followed. Pravastatin ammonium salt (155.5 g of active substance) was dissolved in water (900 mL). Isobutanol (2 mL) was added, followed by addition of concentrated sodium hydroxide solution to increase the pH to about 10 to about 13.7, and the solution was stirred at room temperature for 30 minutes. Sulfuric acid was added to neutralize the solution to pH about 7, and solid NH ₄ Cl was added to induce pravastatin ammonium crystallization. Crystals (150 g) were collected by filtration and washed with acetone. HPLC detection using the conditions described above revealed that the purity of pravastatin ammonium was about 99.3%.

Pravastatin ammonium was then changed to sodium salt as follows. Pravastatin ammonium salt crystals were dissolved in water (1800 mL). i-butyl acetate (10.5 L) was added. Sulfuric acid was then added to acidify the solution to pH about 2 to about 4, converting pravastatin back to the free acid form. The i-butyl acetate phase containing pravastatin was washed with water (5 x 10 mL). Mixing i-butyl acetate solution in about 900-2700 mL of water with intermittent addition of 8m NaOH until pH reaches about 7.4 to about 13, converting pravastatin to sodium salt and back extracting pravastatin into another aqueous phase It was.

The pravastatin sodium salt solution was then treated with an ion exchange resin to remove excess sodium cations. After separation, the aqueous phase was stirred in IRC 50 in the form of H ⁺ ion exchange resin for 30 minutes at room temperature. Stirring was continued until the pH was about 7.4 to about 7.8.

The solution was then filtered to remove the resin and partially concentrated in vacuo to a weight of 508 g. Acetonitrile (480 mL) was added and the solution was decolorized by stirring on activated charcoal (5 g). Pravastatin sodium was determined by further addition of acetone and acetonitrile to form a 1/3/12 mixture (5.9 L) of water / acetone / acetonitrile while cooling to about −10 to about 0 ° C. in 90% yield. Obtained as. As measured by HPLC using the above conditions, pravastatin sodium of about 99.8% purity was obtained from the fermented active starting material in a total yield of 65%.

Example 2

Recrystallization from the water / acetone / acetonitrile mixture was omitted and the procedure of Example 1 was followed to lyophilize the pravastatin sodium concentrate in water to obtain pravastatin sodium in about 99% purity and about 72% yield.

Example 3

The procedure of Example 1 was followed except that the crystallization of pravastatin ammonium salt was repeated once to further purify the pravastatin ammonium salt to obtain pravastatin sodium in about 99.8% purity and 68.4% yield.

Example 4

The procedure of Example 1 was followed except that the crystallization of pravastatin ammonium salt was repeated twice to further purify the pravastatin ammonium salt to obtain pravastatin sodium in about 99.6% purity and 53% yield.

Example 5

According to the procedure of Example 1, sulfuric acid was added to acidify the fermentation broth (100 L) to a pH of about 2.5 to about 5.0. The acidified fermentation broth was extracted with i-butyl acetate (3 x 50 L). The mixed i-butyl acetate phase, basified to pH about 7.5 to about 11.0 by addition of concentrated ammonium hydroxide, was extracted with water (35 L).

Instead of reoxidizing the aqueous extract as in Example 1 and extracting with i-butyl acetate to obtain a further concentrated organic solution, the aqueous extract was concentrated to 140 g / l under vacuum. The pH of the resulting concentrate was then acidified to between about 4.0 and about 7.5 by addition of 1M HCl.

Ammonium chloride crystals (405 g) were then added to the concentrate, and the pravastatin ammonium salt crystallized at room temperature. The crystals were then filtered off and washed with saturated ammonium chloride solution. The crystals were added to water (1 L) at 40 ° C. After dissolution, the temperature was lowered to 30 ° C. and ammonium chloride (330 g) was added to the solution. The solution was then stirred at room temperature for 15 hours and filtered to recover pravastatin ammonium salt crystals, washed sequentially with i-butyl acetate and acetone, and dried. The crystals produced as in Example 1 were further purified by recrystallization, replaced with sodium salts, and then separated. Pravastatin sodium was obtained in about 99.9% purity and 67.7% yield.

Example 6

The pravastatin sodium salt was determined in HPLC, with 99.8% purity and 64% total yield, from the fermented active starting material, as determined by HPLC, except that the pravastatin sodium salt was crystallized from a 1/15 mixture of water / acetone. A salt was obtained.

Example 7

According to the contents of the first two paragraphs of Example 5, a concentrated aqueous extract (140 g / L) was obtained. The concentrated aqueous extract was divided into two equal parts.

Example 8

According to the procedure of Example 1, pravastatin ammonium salt was separated from the fermentation broth, but the active material was dissolved and crystallized after precipitation with ammonia gas.

Pravastatin i-butyl acetate concentrate (6500 L) was decolorized on activated charcoal (6.5 kg). The solution was filtered to remove activated charcoal and transferred to a vessel equipped with a gas inlet.

The solution contained 183.2 kg of active material.

The pravastatin ammonium salt was precipitated with ammonia gas according to the procedure of Example 1.

Water (1099 L) was added to the vessel in the presence of i-butyl acetate mother liquor to dissolve the precipitated pravastatin ammonium salt.

Ammonium chloride (412 kg) was added to the vessel to crystallize pravastatin ammonium salt. Ammonium chloride was added in 31 portions at 30-32 ° C. for 5 hours. The suspension was stirred at 24-26 ° C. for 1 hour. The crystals were filtered, suspended in i-butyl acetate and filtered, then suspended in i-butyl acetate: acetone (2: 1), filtered, suspended in acetone and filtered. After washing with acetone the crystals were dried in vacuo.

The process yielded pravastatin ammonium salt in about 93% purity as determined by UV detection and HPLC at λ = 238 nm. The crystallized active substance was 168.7 kg.

Example 9

According to the procedure of Example 1, pravastatin ammonium salt was separated from the fermentation broth but crystallized instead of precipitating with ammonia gas.

Pravastatin i-butyl acetate concentrate (4150 mL) was decolorized on activated charcoal (4.15 g). The solution was then filtered to remove activated charcoal and transferred to the flask.

Water (300 mL) was added to i-butyl acetate solution. The pH was adjusted to 9.36 with concentrated ammonia solution (27 mL).

Ammonium chloride (121.5 g) was added to the flask to crystallize pravastatin ammonium salt. Divided into more portions at 30-32 ° C. for 5 hours and ammonium chloride was added. The suspension was stirred for 15 h at 24-26 ° C. The crystals were filtered off, suspended several times, washed and dried.

The process was carried out to obtain pravastatin ammonium salt in about 95% purity as determined by HPLC. The crystallized active substance was 42.7 g.

Example 10

According to the procedure of Example 8, pravastatin ammonium salt was prepared in about 93% purity.

The active substance (10 g) was dissolved in a mixture of water (60 mL): acetone (6 mL): isobutyl acetate (6 mL) at 35-40 ° C. The solution was cooled to 30-32 ° C. Divided into more portions for 5 hours and ammonium chloride (22 g) was added to the solution.

The suspension was cooled to 24-26 [deg.] C. and stirred for 1 hour, then the pravastatin ammonium salt was filtered off and washed sequentially with isobutyl acetate and acetone.

Pravastatin ammonium salt was dried at 40 ° C. Yield 96%. Purity was 97%.

Example 11

According to the procedure of Example 8, pravastatin ammonium salt of about 93% purity was prepared.

The active substance (10 g) was dissolved in a mixture of water (60 mL): acetone (6 mL): isobutyl acetate (6 mL) at 35-40 ° C. The solution was cooled to 30-32 ° C. Divided into more portions for 3 hours and sodium chloride (11.4 g) was added to the solution.

Pravastatin sodium salt was filtered off, washed sequentially with isobutyl acetate and acetone and dried at 40 ° C.

Yield 77%. Purity was 97%.

Example 12

According to the procedure of Example 8, pravastatin ammonium salt was prepared in a purity of about 93%.

The active substance (10 g) was dissolved in a mixture of water (60 mL): acetone (6 mL): isobutyl acetate (6 mL) at 35-40 ° C. The solution was cooled to 30-32 ° C. Lithium chloride (9.3 g) was used for the crystallized stone salt.

The filtered pravastatin lithium salt was washed with isobutyl acetate and dried.

Pravastatin lithium salt was obtained in 96% purity and 89% yield.

Claims (27)

Practically pure pravastatin sodium. 2. The pravastatin sodium of claim 1, containing less than 0.5% pravastatin lactone. 2. The pravastatin sodium of claim 1, containing less than 0.2% epiprava. 2. The pravastatin sodium of claim 1, comprising less than 0.5% pravastatin lactone and less than 0.2% epiprava. 2. The pravastatin sodium of claim 1, containing less than 0.2% pravastatin lactone. 2. The pravastatin sodium of claim 1, containing less than 0.1% epiprava. 2. The pravastatin sodium of claim 1, comprising less than 0.2% pravastatin lactone and less than 0.1% epiprava. a) forming a pravastatin organic solution; b) obtaining solid pravastatin as pravastatin salt; c) purifying pravastatin salt by recrystallization; d) converting the cation of pravastatin salt to pravastatin sodium; And e) separating pravastatin sodium with little pravastatin lactone and epiprava Substantially pure pravastatin sodium prepared by a method comprising a. The method of claim 8, wherein the pravastatin organic solution is pravastatin organic concentrate. The method of claim 8, wherein the cation of pravastatin salt is an amine. The method of claim 10, wherein the amine is selected from the group consisting of ammonium, primary amines, secondary amines and tertiary amines. The method of claim 8, wherein the cation of pravastatin salt is an alkali metal cation. 13. The method of claim 12, wherein the alkali metal cation is selected from the group consisting of lithium, sodium and potassium. The method of claim 8, wherein the solid pravastatin is in crystalline form. The method of claim 8, wherein the solid pravastatin is in the form of an amorphous precipitate. The method of claim 8, wherein the aqueous fermentation broth is extracted with a first organic solvent, pravastatin is back extracted with an aqueous solution at a pH of about 8.0 to about 9.5, the basic aqueous solution is acidified to a pH of about 2.0 to about 3.7, and then acidified. Substantially pure pravastatin sodium, wherein the aqueous solution is extracted with a second organic solvent to form an organic solution of pravastatin. 17. The substantially pure pravastatin sodium according to claim 16, wherein the first and second organic solvents are i-butyl acetate. 9. The substantially pure pravastatin sodium of claim 8, wherein the ammonium salt is purified by one or more crystallizations from a mixture of water and antisolvent. 19. The substantially pure pravastatin sodium of claim 18, wherein the antisolvent is selected from the group consisting of i-butyl acetate and acetone. 19. The substantially pure pravastatin sodium of claim 18, wherein ammonium chloride is added to the mixture of water and antisolvent to induce crystallization of the ammonium salt. 9. The substantially pure pravastatin sodium of claim 8, wherein the ammonium salt is replaced using an acidic or chelate ion exchange resin. 10. The substantially pure pravastatin sodium of claim 8, wherein the pravastatin sodium is separated by recrystallization. 10. The substantially pure pravastatin sodium of claim 8, wherein the pravastatin sodium is separated by lyophilization. A formulation comprising pravastatin sodium and less than about 0.5% pravastatin lactone. A pharmaceutical composition comprising substantially pure pravastatin sodium according to claim 1. A pharmaceutical formulation comprising the pharmaceutical composition of claim 25. A pharmaceutical formulation comprising substantially pure pravastatin sodium according to claim 1.