CA2581031A1 - Pravastatin sodium substantially free of pravastatin lactone and epi-pravastatin, and compositions containing same - Google Patents

Abstract

The present invention provides pravastatin sodium substantially free of pravastatin lactone and epiprava. the C-6 epimer of pravastatin. The present invention further provides a novel process for recovering pravastatin sodium from a fermentation broth in such high purity. The process includes the stages of forming an solution of the compound by extraction, obtaining an ammonium salt of pravastatin from the solution, purifying the ammonium salt of the compound and transposing the salt of the compound to pravastatin sodium.

Description

WO 02/030415 PCTlUS01131230 PRAVASTATIN SODIUM SUBSTANTIALLY FREE OF PRAVASTATIN LACTONE
AND EPI-PRAVASTATIN, AND COMPOSITIONS CONTAINING SAME

CROSS-REFEi;ENCE TO REL TED APPI:.ICA ION
The pmsent application claiats priority under 35 U.S.C. 119(e) to U.S.
Ptovisiona!
Application No. 601238,276, filed October 5, 2000, which is incorporated herein by reference.

FIELD OE THE INVENTiON
The present invention relates to statins and more particularly to pravastatin sodium and processes for isolating it as a product of ettzymatie hydroxylation ofcompactin from a fermentation broth.

B~ CrRO[JM) OF THfi 1IWENTION
Statin drugs are currently the most therapeutically affective drugs available for reducing the level of LDL in the blood stream of a patient at risk for eardiovascular disease. This class of drugs includes pravastatin as well as compactin, lovastatin, simvastatin, fluvastatin and atorvastatin.
Pravastatin is the common medicinal name of'the chemical compound [1S-[la((3',S')2a,6a,80(R'),8aaj)-f,2,6,7,8,8a-hexahydra-p,E,6-trihydroxy-2-methyi-8-(2-methyl-I-oxobutoxy)-1-naphthalene-heptanoic acid. (CAS Registry No. 81093-370.) The molecular structure of pravastatin is represented by'Formula (Ia) where R-OH.
The lactone fotm is represented by Formula (Ib), with atoms labeled to indicate numbering of the atoms.

SUBSTITUTE SHEET (RULE 26) HO C~H HO, s'a .."AH i ~la) ~ O H (lb) CH9 CHg CHsr,re.l~s~~z CHg R Rse.~s a~i s pravastatin, compactin (Formula Ia, R-k1), lovastatin {Formula Ya, R=CH,), simvastatin, fluvastatin and atorvastatin each possess an alkyl chain that is terminated by a carboxylic acid group and that bears two hydroxyl groups at the P and 6 positions with respect to the carboxylic acid group. The carbox'tiylle acid group and the hydroxyl group at the 8 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 fotms. Lactonization causes processing difi'i,culties 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 isoiate them in high yieid.
Presently, the most economically feasible meibod ofmiaiGing pravastatin is by microbiat hydroxylation of compactin at the C-6 position. A]though enrymatic processes are highly stereoselective, it is common for pravastatin sodium obtained after isolation from a Sarsnenation broth to be contaminated with a significant amount of the C-6 epitner of pravastatin ("epiprava"). The C-6 position i's bis-allylic and, hence, the C-6 atom Is prone to epimerize. Cateflul control of pH and other conditions during isolation of pravastatin is required tn order to minimize epimerization. Known methods of isolating pravastatin from a fermentation brotb either are ill-suited for isolating pravastatin as its sodium salt or produce pravastatin sodium contaminated with significant amounts of pravastatin lactone and/or epiprava. The present invention meots a need in the art for an efficient method of isolating pravastatin sodium from a fermentation broth in high purity, in high yield, on a preparative scale and without the need for cbromatographio purification.

SUBSTlTUTE SHEET (RULE 26) SU Mp-RY O TXIL IiVVENTIQI,~
The present invention provides pravastatin sodium substantially free of pravastatin lactone and epiprava, the C-6 epimer of pravastatin. The invention further provides a process that can be practiced on an industrial scale for producing such substantiaily pure pravastntin sodium.
A prefarred tmbodiment of the process involves extraotion of pravastatin from an aqueous fermentation broth into an organic solvent, back-extraction ofpravastatin into a basic aqueous solution and a re-extraction into an orpnic solvent, resulting in an organic solution that is enriched in pravastatin relative to the initial concentration of pravastatin in the fermentation broth. The pravastatin may be obtained from the enriched solution by precipitation as its ammor-ium salt and then purification by recrystallization of the amnionium salt. The recrystallized salt is then transposed to form pravastatin sodium salt and any excess sodium ions are scavenged with an ion exchange resin. The sodium salt of pravastatin may then be isolated in a highly pure state from solution by recrystallization, lyophilization or other means.

DETAII..ED DESC1tIPTiON OF T.HE 2MTTON
The present invention provides pravastatin sodium substantially free of pravastatin lactone and epiprava and a downstream process for isolating pravastatin sodium fivm a fermentation broth in such high purity, Enz~vrnatic Fiydroxylation of Comrractiel The enzymatic hydroxylation broth from which pravastatin is isolated can be any of the aqueous broths known for industrial scale fermentation of compactin, such as the methods described in U.S. Patents Nos. 5,942,423 and 4,346,227. prefe,crably, the enzymatic hydroxylation is conducted using a living culture of Steptontycea, with a nutrient mixture of compactin and dextrose, If the broth Is neutral or basic upon eompletion of the fermentation, then an acid is added to it to bring the broth to a pH of between about I and 6, preferably between I and 5.5 and more preferably between 2 and 4. Acids that may be used include hydrochloric acid, sulfuric acid, trifluoroacetic acid or any other protic acid, SUBSTtTUTE SHEET (RULE 26) preferably one having a pH of Iess than i as a I M solution in water.
Acidification of the formentation broth converts any pravastatin carboxylate salts in the broth to the free acid and/or lactonc.

Tsolatinn of 5ubstanti llv Pure Pravastatin Sodium Pravastatin is 6rst obtained from an aqueous fermentation broth in a relatively highly concentrated organic solution by a sequence of extraction and back-extraotion steps.
In the first step, pravastatin is extracted from the fermentation broth. C,r-C4 alkyl forrnat+es and C,-C{ atkyt esters of Cz-C4 carboxylic acids are capable of efficient extraction of pravastatin from an aqueous fermentation broth. THe alkyl group may be linear, brancbed or cyclic. Preferred esters include ethyl formate, n-propyl formatc, i-propyl formate, n-butyl formate, s-butyl formate, i-butyl formate, t-butyl formate, methyl aoetate, ethyl acetate, n-propyl aeetate,.i-propyl aaetate, n-butyl acetate, s-butyl acetate, i-butyl acetate, r butyi acetate, methyt propionate, ethyl propionate, n-propyl propionate, i-propyl propionate, butyl propionate, methyl butyrate, ethyl butyrate, n-propyl butyrate, i-propyl butyrate, butyl buryrates, metbyl isobutyr:te, ethyl isobutyrate, propyl isobutyrates and butyl isobutyrates. Of these preferred organic solvents we have found that ethyl acetate, i-butyl acetate, propyl acetate and ethyl forrnate are especially well suited.
Thc most preferred extraction solvent is i-butyl acetate. Other organic solvents may be substituted for the esters. Halogenmd haloeatbons, arvmatic compounds, ketones and ethers may be used, such as dichtoromethane, ehlorofrnm, carbon tetrachloride, 1,2-dichloroethane, benzene, butyl methyl katone, diethyl ether and methyl t-butyl ether.
Pravastatin is optionally back-extracted into a basic aqueous solution of pH
from about 8.0 to about 9.5. The base is prcferably NaOH. NH4QH or KOH, most preferably NaOH. 't'he extraction solvent is preferabily contacted with the basic aqueous solution until the amount of pravastatin in the organic phase has been substantially depleted as determined by thin layer chromatography or any other method lncluding the subjective judgment that sufficient contacting has occurred for complete extraction.
Multiple back-extn+ot9ons may be performed for optimal recovery. However, a single back-extraction is highly efficient when the organic phase is i-butyl acetate. Back-extraction may be used to SUBSTITUTE SHEET (RULE 26) concentrate the pravastatin by using a volusxle of aqueous base that Is less than the volumc of the organic extract. Preferably, the back-extraction is conducted with a volume of basic aqueous solution that is less than one third of the volume of the organic extract, more preferably less than one fourth and most preferably, about one fifth the volume of the organic extract.
The aqueous solution is preferabily acidified with an aoid, preferably trifluoroaeetic acid, hydrochloric acid, sulfnrie 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.
Pravastatin Is preferabily re-extracted into one of the organic solvents previously described as suitable for extracting pravastatin from the fermentation broth, 'I-he organic solvent nsay be, but need not be, the same solvent used to extract pravastatin from the fermentation broth. In this re-extraction, further enrichment of pravastatin may be accomplished by re-extracting into an amount of organic solvent that is preferably less than i 5 about 50 % (v/v) of the aqueous extract, more preferably from about 33%
(v/v) to about 20"/0 (v/v) and still rr-ore preferably about 25% (v/v) the volume of the aqueous extract.
Pravastatin may be concentrated from 100 L of fermentation broth to 8 L of enriched organic solution in 89% yield from the initial organic extract. It will be appreciated by those skilled in the art that a higher yield of purified pravastatin may be attained by performing multiple extractions where only a single extraction has been described in tbis preferred mode for practicing the invention. This preforred mode achieves a balance of solvent economy and high product yield. Deviations from this preferncd mode which furtber enhance the yield by repeated extractions where only one has been described above do not necessarily depart from the spirit of the invention. Befora proceeding to obtain pcavastatin from the enriched organic solution by "salting out," the enriched organic solution is preferably dried, which may be done using a conventional drying agent such as MgSO4, Na2SO., CaSO4t silica, perlite and the like, and optionally deeolorized with activated carbon. A dried and/or decolorized enriched organic solution is preferablly then separated conventionally, as for instance by filtration or decanting.
In the next step, pravastatin may be salted Out fl=om the enriched organic solution with ammonia or an amine. The amirte may be a primary, secondary or tertiary amine.

SUBSTITUTE SHEET (RULE 26) WO 02/030415 PC'Y'/USU2/31234 Any alkyl or aryl amine that is not so hindered as to prevent ionic interaction between the amine nitrogen and the carboxyl group of pravastatin may be used. The amirtes include, but are not limited to, nnethyl, dimethyl, trimethyl, ethyl, diethyl, triethyl and other C1-Ca primary, secondary and tertiary amines; and further include morpholi>ie, N-S methylmorpholine, isopropyl cyclohexyl amine, piperidine and the like.
Regardless of the absence, presence or muitiplicity of substitution on nitrogen, a salt fonnad by reaction of ammonia or an amine is hereafter referred to as an ammonium salt. Its meaning is intended to encompass salts of amines as well as a salt of amnionia.
Precipitation of the anunonium salt of pravastatin also may be induced by addition of an amnzoniurn salt either alone or in combination with the ammonia or amine. The preferred ammonium salts are the following salts of ammonia: NH4CI, NkiBr, NHI, (NH4)2SO4, NI-14NO3, (NHõ)yPOõ (NH.)=Sz04 and NH4OAc, the most preferred being NI-14CI. Ammonium salts and high boiling liquid and solid amines may be added by conventional means, preferably in an area with good vwtilation, either as solids, neat -liquids or solutions in aqueous or organio solvent. A.ddition of gaseous ammonia requires special equipment for handlin$ caustic gases. Such equipment, including pressure vessels, regulators, valves and lines are widely available. In an especially preferred embodiment, pravastatin is obtained from the enriched organic solution as the pravastatin salt of ammonia by addition of gaseous ammonia and NH4Cl to the enriched organic solution.
The temperature at which the ammonia, amine andlor ammonium salt should be added can be determined by routine experimentation by conducting the reaction on a small scale and monitoring the exothermicity of the reaction. Preferably, the solution tamperature is not allowed to exoeed 40 C. Although temperatures as 5igh as 80 C may be experienced without signif'icant decomposition of pravastatin, many organic solvents of this invention will boil at a lower temperature. When ammonia is used, the preferred temperature range is from about -10 C to about 40 C.
Preferably, once precipitation appears to cease or once consumption of pravastatin is determined to be substantially complete by other tneans, the addition should be ceased.
Whcn ammonia or a volatile amine is used, the vessel is preferably vented to disperse excess fumes. The crystals may then be isolated by filtration, decantation of the solvent, SUBSTITUTE SHEET (RULE 26) WO 02/030435 pCT/YlS01/31230 evaporation of the solvent or other such method, preferably filtration. The crystals may then be washed, preferrably with i-butyl acetate and acetone.
After optionally washing the pr cipitated arystals, the pravastatin ammonium salt is preferablly purified by one or more, or most pretbrably three, recrystaliizations. To purify the pravastatin ammonium salt, the salt is preferablly dissolved in water. The polarity of the solution is preforablly decreased by addition of an anti-solvent. The anti-solvent is preferablly a water-soluble organic solvent or solvent mixture in whiclt the pravastatin salt is poorly soluble, i-butyl acetate and aaetone being preferred.
'Y'lte pravastatin salt may be allowed to recrystallize spontaneously, or may be induced to recrystallize by taking the further step of adding a common ion.
Aocording to the preferred process wherein pravastatin is purified as its ammonia salt, NH~Ci is added to induoe recrystallization of the ammonium salt.
The reorystallization may be performed at between about -I0 C and about 40 C, preferably between about 0 C and about 40 C. After the pravastatin salt has been substantially rocrystallized from the solution, the crystals are isolated and may be washed, for example with a 1:1 mixture of i-butyl acetate and acetone and then dried.
Drying may be conducted at ambient temperature but is preferably conducted at mildly elevated temperaturo of less than 45 C and preferably about 40 C. The recrystallization may optionally be repeated to good effect as shown in Examples 3 and 4. Each repetition occurs in about 92% yield.
After purification of the pravastatin anunonium salt, the pravastatin ammonium salt is prefarablly transposed to pravastatia sodium. Pra.vastatin is preferablly liberated from the ammonium salt by dissolving in an aqueous solvent, aoidifying with any protic acid, but prefcrably sulfuric acid, to a pH=of about 2 to about 4, more preferably about 3.1, and extracting pravastatin with an organic solvent. The organic solvent, which may be any of the organic solvents listed above but preferably is f-butyl acetate, is optionally contacted with the acidified solution until pravastatin is substantially compietely transfered to the organic phase. The organic phase is preferablly separated from the aqueous phase and, after optionally' washing with water to remove anunonium residues, the pravastatin is preferablly back-extracted with aqueous sodium hydroxide solution at a pH of from about SUBSTITUTE SHEET (RULE 26) wo 02/030419 PCT/USD1/31230 7.4 to about 13Ø The back-extraction is preferably conducted at a reduced temperature of about 8 to about 10 C.
After extraction into aqueous sodium hydroxide, excess sodium cations are scavenged to attain a near 1: 1 equivalence of sodium cation and pravastatirt using a water insoluble ionic exchange resin. Suitable ion exchange resins are the cationic and chelate type resins, the preferred being strong and weak acid exchange resins.
Among the strong acid cationic exchenge resins which may be used are those having sulfonic acid (SO,V) groups. These include the commercial products Amberlite IR-118, IR-120252H; Amberlysi' 15, 36; Ambet'jet 1200(H) (Rohm atidHaas);Dowaxl ] 0 50WX series, Dowex HCIt-W2, Dowex 650C, Dowex Marathon C. Dowex DR-2030, and Dowex HCR-S, ion exchange resins (Dow Chemical Co.); DIAION SIC 102 to DIAION
SK 116 resin series and Lewatit SP 120 (Bayer).;fhe preferred strong acid cationic exchange crsins are Amberliteo 120, Dowex 50WX and DIAION SK series.
Weak acid cationic exchange resins include those which have pendant carboxylic acid groups. Weak acid cationic exchange resins include the commarcial products Amberlitc Cc3-50, IRP-64, IRC 50 and C67, Dowex CCR series, Lewatit CNP series (Bayer) and DIAION WK serlas (Mitsubisbi), of these, the most preferred are Arnberlitam IRC50, Lewatit CNP 80 and DIAION WK 10. Less preferred are the chelata type exchange resins. Some of the commercial varieties that are available include Amberlite IRC-7 18, and TRC-467.
The solution containing pravastatin sodium salt and excess sodium cations may be contacted with the ion exchange resin by any method known to the art, including passage of the solution through a column or bed ofthe resin or by stining a sufficient quantity of the resin In a flask with the solution. The mode of contaot is not critical, After scavenging of the excess sodium ion, the pH of a pravastatin sodium solution should be in the range of about of 7 to about 10, preferably about 7.4 to about 7.8, although the p14 will vary with dilution. Reduction in the pH of the pravastatin sodium solution from a higher pH to a lower pH and then leveling oft'of the pH at the lower level is an indication of substantial completion of scavenging excess Na' ions. After scavcnging is substantially complete, the pravastatin sodium solution is preferablly separated from the resin in a oonventional SUBSTITUTE SHEET (RULE 26) manner. it may either be coll.ected as the eluent from a column or bed or may be separated by fiitration, decantation and the like.
Pravastatin sodium may be isolatad from the pravastatin sodium solution by crystallization. Eflicient crystallization may first require partial removal of the water, which can be conducted by vacuum distilladon or nano-filtration. Prcferably, the aqueous pravastatin sodium salt solution is concentrated from about 20 to about 50 w/v% before crystallizing. If necessary, after ooncentration the aqueous pravastatin sodium solution can be adjusted to a pH of between about 7 and about 10 wlth an ion exchange resin in H*
form.
Addition of a water-soluble organio solvent or organic solvent mixture to the pravastatin sodium solution will assist the crystallization. In particular, there may be mentioned acetone and acetone/acetonitrile, ethanol/acetonitrile and ethanol/ethyl aoetate mixtures. One of the most prefernd solvent system for crystallizing pravastatin sodium is a 1/3/12 water/acetone/acetonitrile rnixtat'o fbrmed by concentrating the pravastatin sodium solution to about 30 w/v% and then adding an appropriate volume of 1/4 aeetone/acetonitrile mixture. The nwst preferred crystallization solvent mixture is water-acetone (1;15).
Pravastatin sodium also may be isolated by lyophitization of the aqueous pravastatin sodium solution.
Whether isolated by lyophilization or crystallization or other means that does not diminish the purity of the product, the pravastatin sodium that is isolated in the practice of the present inventive process is substantially free of pravastatin lactone and epiprava. As demonstrated in the examples that follow, pravastatin sodium may be isolated with less than 0,5% (w/w) contamination by pravastatin lactone and less than 0.2% (wlw) oontamination by bpiprava. Pravastatin sodium further may be isolated with less than 0.2% (w/w) pravastatin lactone and 0. i96 epiprava by adhering to the preferred embodiments ofthe invention, two of which are exemplified in Examples 1 and 3 The highly pure pravastatin sodium praduced by the present Inventive method is preferablly useful for hypercholesteremia therapy and for this purpose it can administemd to a mammalian patient by any route of administration. A daily bral regimen is the most preferred prescribed method of administration. In human subjects with normal hepatic SUBSTITUTE SHEET (RULE 26) VPO 02/030415 PG'Y7L1S01/31230 function and moderate body weight, a reduction in serum cholesterol levels is typically observed with daily oral dosages of 10 mg or more pravastatin sodium. The quantity of the highly pure pravastatin sodium administered may be any effective amount.
Preferred oral dosages of the present invention contain from about 10 mg to about 40 mg of pravastatin sodium. Oral dosages include tablets, pills, capsules, troches, sacbets, suspensions, powdets, lozenges, elixirs and the like. The substantially pure pravastatin sodium may be administered by any route but the most preferred route of administration is oral.
The highly pure pravastatin may be administered either alone or in a composition with pharmaoeutical excipients. Whether adnaini9tered alone or in a cotnposition, the highly pure pravastatin sodium of the invention may be in the form of a solution or a solid suoh as a powder, granules, agg,regates or any other solid form.
The compositions of the present invention include compositions for tableting.
Tableting compositions may have few or many excipients depending upon the tableting method used, the release rata desired and otber factors. For example, compositioiis of the present invention may contain diluents such as cellitlose-tlerived matuials like powdared cellulose, microcrystalline cellulose, microfine cellulose, rnethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydnoxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose salts and other substituted and unsubstituted cslluloses; starch;
pregelatiniaed starch; inorganic diluents like caleium carbanate and calcium diphosphate and other diluents known to the phanmaccutical iridustry. Yet otber suitable diluents include waxes, sugars and sugar alcohols like mannitot and sorbitol, acrylate polymers and copolymers, as well as pectin, dextrin and gelatin.
Further tableting excipients include binders, such as acacia gum, pr4gelatinized star+oh, sodium alginate, glucose and other binders used in wet and dry granulation and direct compression tableting processes. Excipients that may also be present in a solid composition of the novel forms of pravastatin sodium further include disintegrants like sodium starch glycolate, crospovidone, low-substituted hydroxypropyl cellulose and others. Additional excipients include tableting lubricants like magnesium and calcium stearate and sodium stearyl fumarate; flavorings; sweeteners; preservatives;
pharmaceutically acceptable dyes and glidants sucb as silieo,t dioxide.
SUBSTITUTE SHEET (RULE 26) WO 02/030415 PCTlU501/32230 Capsule dosages wi11 contain the solid eornposition within a capsule which may be rpade of gelatin or other encapsulating material. Tablets and powders may be coated.
Tablets and powders may be coated with an enteric ooating, The enteric-coated powder forms may hava coatings comprising phthalic acid cellulose acetate, hydroxypropylmethyl cellulose phthalate, polyvinyl aleohol phthalate, carboxymethylethyloellulose, a copolymer of styrcne and maleic acid, a eopolymer of inethaorylic acid and methyl methacrylate, and like materials, and if desired, they may be employed with suitable plasticizers and/or extending agents. A coated tablet may have a coating on the surface of the tablet or may be a tablet comprising a powder or granules with an enterie-c:oating.
The higbly pure pravastatin sodium may also be admirzistered in injectable dosages as a solute or suspended solid in a sterile solution or suspension. Suitable carriers for storile injectable dosages include water and oits.
Although the following examples illustrate the practice of the present invention in some of its embodiments, the examples should not be construed as limiting the scope of the invention. Other embodiments will be apparent to one skilled in the art from consideration of the specification and examples. =It is intended that the specification, including the examples, is considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow.

SUBSTITUTE SHEET (RULE 26) WO 42/030415 PCr/[7S01/31230 E MPI,ES
EXAMPLE I
pprificationgf Pravastatin The fcrmentation broth (100 L) was acidified to from about 2.5 to about 5.0 by addition of sulfnric acid. The acidified tbrmentation broth was extraoted with i-butyl acetate (3x50 L). The yield of i-butyl acetate extraction was found to be 95%
by HPLC
analysis calibrated to the internal standard in the broth. The cornbined i-butyl acetate phases were then extracted with water (35 L) at about pH 7.5 to about pH 11.0 by addition of concentrated ammonium hydroxide. The resulting aqueous pravastatin solution was then rcacidified to a pH of about 2.0 to about 4.0 by addition of 5M sulfuric acid and back-extracted with i-butyl aoetate (8 L). The resuIting solution of pravastatin in i-butyl acetate was partially dried over perlite and NazSO4. The pravastatin solution was decanted and then filtered from the drying age,mts and decolorized over activated charaoal (1.7 g). The solution was then filtered to remove the charcoal and transferred to a flask equipped with a gas inlet.
Ammonia gas was then introduced into the beadspace above the solution with rapid stirring. The precipitated crystals of ammonium pravastatin catboxylate salt were collected by filtration and washed with i-butyl acetate and then acetone which yielded pravastatin ammonium salt in about 94 % purity as detennined by I3PLC coupled with UV
absorbance measured at X-238nm, The pravastatin ammonium salt was furtber purified by cry$tallization from a saturated amrnonium chloride solution as follows. The pravastatin salt oontaining 162 g of active substanee was dissolved in water (960 ml) and diluted with acetone (96 ml) and i-buty) acetate (96 ml) at about 35-40 C. The solution was cooled to about 30-32 C and pravastatin ammonium was induced to crystallize by addition of solid NH~C) until furtber addition resulted in no apparent increase in crystal formation. After adding ammonium chloride, the solution is cooled to about 0-26 C. The pravastatin ammonium crystals wera oollected by filtration and washed witb i-butyl acetate and acetone, as before, and then dried at about 40 C. The resulting pravastatin ammonium salt crystals (155.5 g) were obtained in about 98 % purity as determined by HPLC employing the afore-mentioned conditions.

SUBSTITLITE SHEET (RULE 26) WO 02/030419 PCT/[T501/31730 The pravastatin ammonium salt was further purified by another crystallization as follows, 7he pravastatin ammonium salt (15S.5 g of active substance) was dissolved in water (900 ml). Isobutanol (2 nril) was added and then the pff was raised to about pH 10 to about pH 13.7 by addition ofa concentrated solution of sodium hydroxide and the solution was stirred for 30 min. at ambient temperature. The solution was neutralized to 4 pH of about 7 by addition of sulforic acid and crystallization of pravastatin ammonium was induced by addition of solid NH4CI. The crystals (150 g) were collected by fiitration and washed with acetone. Pravastatin atnmonium was found to be about 99.3% pure by HPLC
detection using the above-daseribed conditions.
The pravastatin ammanium was then transpvsecl to the sodium salt as follows.
The pravastatin atnmonium salt crystals were dissolved in water (1800 ml). i-butyl acetate (10.5 L) was added. The solution was then acidified to a pH of between from about pH 2 to about pH 4, exact by addition of sulfuric acid, which converted pravastatin back to its frea acid. The i-butyl acetate phase, containing prrn=astatin, was washed with water (SxlOml). Pravastatin was then converted to its sodium salt and back-extracted into another aclueous phase by swirling the t-butyl acetate solution over water between about 900-2700 ml with intermiltent addition of 8m NaOH until a pH of between about pH 7.4 to about pH 13 was reached.
The pravastatin sodium salt solution was then treated with an ion exchange resin to scavenge excess sodium cations. After separation, the aqueous phase was stirred over IRC
50 in the H+ ion exchange resin for 30 min. at ambient temperature. Stirring was oontinued until a pH of about pH 7.4 to about pH 7.8 was reaehod.
T'he solution was then filtered to remove the resin and partially concentrated io a weight ot'S08 S. under vacuum. Acatonitrile (480 ml) was then added and the solution was stirred over activated carbon (5 g) to decolorize. Pravastatin sodium was obtainod as crystals by crystallization in 90% yield after further addition of acetone and acetonitrile to form a 1/3/12 mixture of water/acetone/aeetonitrile (5.9.L) with cooling to about -10 to about 0 C. Pravastatin sodium was obtained in an overall yield of 65 % in about 99.8 /a purity from the starting fermented active substance as measured by HPLC using the above-described conditions.

SUBSTITUTE SHEET (RULE 26) WO 07J030415 YSCTlLISO1l31z30 Following the procedure in Example 1, but omitting the recrystallization from the water/aeetonefacetonitrile mixture, pravastatin sodium was obtained by lyophilization of the eoncentrated solution of pravastatin sodium in water in about 99% purity and about 72% yield.

EXANlPLE 3 Following the procedura of Example 1, but further purifying the pravastatin ammonium salt by once repeating the crystaliizatlon of the pravastatin ammonium salt, pravastatin sodium was obtained in about 99.8% purity and 68.4~/p yield, Following the procedure of Example 1, but ftlrther purifying the pravastatin ismmonium salt by twice repeating the crystallization of the pravastatin ammonium salt, prgvastatin sodium was obtained in about 99.6% purity and 53 yb yield.

Following the procedure of Example 1, the fermentation broth (100 L) was acidified to pH from about 2.5 to about 5Ø by addition of sulfurio acid. The acidified fermentation broth was extracted with i-butyl acetate (3x50 L). The combined i-huryl acetate phases were then extracted witb water (35 L) having been basified to a pH of about pH 7.5 to about pH 11.0 by addition of concentrated amrnonium hydroxide.
Instead of reacidifying the aqueous extract and cxtxacting with i-butyl acetate to obtain a further enriched orgaeic solution as was done in Example 1, the aqueous extract was coneentrated to 140 $/I. under vacuum. The resulting concentrated solution was then acidified to a pH of about pH 4.0 to about pH 7.5 by addition of 1M HCI.
Ammonium chloride crystals (405 g.) were then added to the concentrated solution and the pravastatin ammonium salt was allowed to crystallize at ambient temperature. The crystals wera then isolated by filtration and washed with a saturated solution of ammonium SUBSTITUTE SHEET (RULE 26) WO 02/030415 PCT/tJSO1/31230 chloride. The crystals wera then added to water (IL) at 40 C. After dissolution, the temperature was reduced to 30 C and ammonium chloride (330 g.) was added to the solution. The solution was then stirred for 15 h at ambient temperature and crystals of pravastatin amnionium salt were recovered by filtration and washed with i-butyl acetate and after that with acetone and dried. 7fie resulting crystals were then further purified by recrystallization transposed to the sodium salt and isolated as described in Sxample 1.
Pravastatin sodiurn was obtained in about 99.9% purity and 67.7% yield.

Following the procedure of Bxarnple 1, but the pravastatin sodium salt was crystallized from 1/15 mixture of water/acetone in an overall yieid from the starting ferrnented active substancx of 64% and in 99.8 % purity as measured by HPLC.

Following the n,ethod ofExample 5, first two paragraphs, a coneentrated aqueous extract (140 g. L-i) was obtained. The eonoentrated aqueous extract was divided into equal parts=

Following the procedure in Example 1, pravastatin ammonium salt was isolated from a fermentation broth, but active substance was dissolved and crystallized after procipitation with ammonia gas.
Enriched pravastatin i-butyl acetate solution (6500 L) was decolorized over activated charcoal (6.5 kg). Then the solution was filtered to remove the charcoai and transferred to a vessel equipped with a gas inlet.
The solution contained 183.2 kg active substance.
Pravastatin ammonium salt was precipitated with ammonia gas following the procedure in Example 1.
Precipitated pravastatin ammonium salt was dissolved by adding water (1099 L) to the vessel in presence of i-butyl aoetate mother liquor.

SUBSTITUTE SHEET (RULE 26) WO 02/030415 PCT/t1301/31230 Pravastatln ammonium salt was crystallized by adding ammoniurn chloride (412 kg) into the vessel. Ammonium chloride was added in 31 parts at 30 - 32 C
during 5 hours. The suspension was stirred at 24 - 26 C for I hour. Crystals were filtered, suspended in i-butyl acetate and filtered then suspended in i-butyl acetate ;
acetone (2 ; 1) S and filtered, then suspended in acetone and filtered. Crystals were dried in vacuum after washing with acetotte.

The process yielded ptavastatin atnmonium salt in about 93 % purity as determined by 1=iPLC with UV detection at 1= 238 nm. Crystaltized active substance was 168.71cg, Following the proeedure in Example 1, pravastatin ammonium salt was isolated from a fermentation bmth, but crystallization was used instead ofprecipitation with 1S ammonia gas, Enriched pravastatin i-butyl acetate solution (4150 rni) was docolorized over activated charcoal (4.15 g). Then the solution was filtered to remove the charcoal and transferred into a flask.
Water (300 ml) was added to i-butyl acetate solution. pH was adjusted to 9.36 with conc. ammonia solution (27 nnl).
Pravastatin ammonium salt was crystallized by adding ammonium chloride (121.5 g) into the flask. Ammonium chloride was added in more parts at 30 - 32 C
during 5 hours. The suspension was stirred at 24 - 26 C for 15 hours. Crystals were filtered, more times suspended, washed and dried.
The proms yielded pravastatin ammonium salt in about 95 % purity as determined by HPLC. Crystallized active substance was 42.7 g.

SUBSTITUTE SHEt=T (RULE 26) Following the procedure of Example 8, pravastatin ammonium salt was produced in about 93 % purity.
Active substance (10 g) was dissolved in water (60 ml) : acetone (6 ml) :
isobutyl acetate (6 mt) mixture at 35 - 40 C. The solution was cooled to 30 - 32 C.
Arnmonium chloride (22 g) was added into the solution in more parts during 5 hours.
The suspension was cooled to 24 - 26 C and it was stirred for an hour then pravastatin ammonium salt was filtered, washed with isobutyl acetate then with ac.etone.
Pravastatin ammonium salt was dried at 40 C. The yield was 96 %. The purity was 97 %.
EXAMPY.E Il Following the procedure of Example 8, pravastaCin ammonium salt was produced in about 93 % purity.
Active substance (10 g) was dissolved in water (60 ml) : acetone (6 ml) :'ssobutyl acetate (6 ml) mixture at 35 - 40 C. The solution was cooled to 30 - 32 C.
Sodium chloride (11.4 g) was added into the solution in more parts during 3 hours.
Pravastatin sodium salt was filtered, washed with isobutyl acetate then with acetone then it was dried at 40 C.
The yield was 77 %. The purity was 97 %.

Following the procedure of Example 8, pravastatin anrmonium salt was produced in about 93 % purity.
Active substance (10 g) was dissolved in water (60 ml) : acetone (6 ml) :
isobutyl acetate (6 ml) mixture at 35 - 40 C. The solution was cooled to 30 - 32 C.
Lithium chloride (9.3 g) was used for salting out crystallization.
Filtered pravastatin lithium salt was washed with isobutyl acetate and dried Pravastatin Iithium salt was obtained in 96 % purity with 89 % yield.

SUBSTI7UTE SHEET (RULE 26)

Claims (27)

1. Substantially pure pravastatin sodium.

2. The pravastatin sodium of claim 1 containing less than 0.5% pravastatin lactone.

3. The pravastatin sodium of claim 1 containing less than 0.2% epiprava.

4. The pravastatin sodium of claim 1 containing less than 0.5% pravastatin lactone and less than 0.2% epiprava.

5. The pravastatin sodium of claim 1 containing less than 0.2% pravastatin lactone.

6. The pravastatin sodium of claim 1 containing less than 0.1% epiprava.

7. The pravastatin sodium of claim 1 containing less than 0.2% pravastatin lactone and less than 0.1% epiprava.

8. Substantially pure pravastatin sodium prepared by a process comprising the steps of:

a) forming an organic solution of pravastatin;
b) obtaining solid pravastatin as a pravastatin salt;
c) purifying the pravastatin salt by recrystallization;
d) transposing the cation of the pravastatin salt to pravastatin sodium; and, e) isolating pravastatin sodium substantially free of pravastatin lactone and epiprava.

9. The process of claim 8, wherein the organic solution of pravastatin is an enriched organic solution of pravastatin.

10. The process of claim 8, wherein the cation of the pravastatin salt is an amine.

11. The process of claim 10, wherein the amine is selected from the group consisting of ammonium, a primary amine, a secondary amine and a tertiary amine.

12. The process of claim 8, wherein the cation of the pravastatin salt is an alkali metal cation.

13. The process of claim 12, wherein the alkali metal cation is selected from the group consisting of lithium, sodium and potassium.

14. The process of claim 8, wherein the solid pravastatin is in the form of a crystal.

15. The process of claim 8, wherein the solid pravastatin is in the form of an amorphous precipitate.

16. The substantially pure pravastatin sodium of claim 8 wherein the organic solution of pravastatin is formed by extracting an aqueous fermentation broth with a first organic solvent, back-extracting pravastatin with an aqueous solution at pH of about 8.0 to about 9.5, acidifying the basic aqueous solution to a pH of about 2.0 to about 3.7 and extracting the acidified aqueous solution with a second organic solvent to form an organic solution of pravastatin.

17. The substantially pure pravastatin sodium of claim 16 wherein the first and second organic solvents are i-butyl acetate.

18. The substantially pure pravastatin sodium of claim 8 wherein the ammonium salt is purified by at least one crystallization from a mixture of water and an anti-solvent.

19. The substantially pure pravastatin sodium of claim 18 wherein the anti-solvent is selected from the group consisting of i-butyl acetate and acetone.

20. The substantially pure pravastatin sodium of claim 18 wherein ammonium chloride is added to the mixture of water and anti-solvent to induce crystallization of the ammonium salt.

21. The substantially pure pravastatin sodium of claim 8 wherein the ammonium salt is transposed using an acidic or chelating ion exchange resin.

22. The substantially pure pravastatin sodium of claim 8 wherein the pravastatin sodium is isolated by recrystallization.

23. The substantially pure pravastatin sodium of claim 8 wherein the pravastatin sodium is isolated by lyophilization.

24. A formulation comprising pravastatin sodium and less than about 0.5%
pravastatin lactone.

25. A pharmaceutical composition comprising the substantially pure pravastatin sodium of claim 1.

26. A pharmaceutical dosage form comprising the pharmaceutical composition of claim 25.

27. A pharmaceutical dosage form comprising the substantially pure pravastatin sodium of claim 1.