Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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PRAVASTATIN SODIUM SUBSTANTIALLY FREE OF PRAVASTATIN LACTONE
AND EPI-PRAVASTATIN, AND COMPOSITIONS CONTAINING SAME
CROSS-REFERENCE TO RELATED APPLICATION
S The present application claims priority under 35 U.S.C. 119(e) to U.S.
Provisional
Application No. 60/238,276, filed October 5, 2000, which is incorporated
herein by
reference.
FIELD OF THE INVENTION
The present invention relates to statins and more particularly to pravastatin
sodium
and processes for isolating it as a product of enzymatic hydroxylation of
compactin from a
fermentation broth.
BACKGROUND OF THE INVENTION
Statin drugs are currently the most therapeutically effective drugs available
for
reducing the level of LDL in the blood stream of a patient at risk for
cardiovascular
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 [1 S-
[la(~3',b~)2a,6a,8[i(R'),8aa]]-1,2,6,7,8,8a-hexahydro-(3,8,6-trihydroxy-2-
methyl-8-(2-
methyl-1-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 form is represented by Formula (Ib), with atoms labeled to indicate
numbering of
the atoms.
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HO COOH ~..IO~a~a~0
O ,,.OH O Y\s,,v~0
I I
/3\ /~\
(Ia) ~O H a ~ O H
CI-ig CH3 CHg /awlii\ iCFi3
7 8a
Ri6\5 41a~~ 3
Pravastatin, compactin (Formula Ia, R=H), lovastatin (Formula Ia, R=CH3),
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 (3 and 8
positions with
respect to the carboxylic acid group. The carboxylic 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 forms. Lactonization causes processing difficulties 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 isolate them in
high yield.
Presently, the most economically feasible method of making pravastatin is by
microbial hydroxylation of compactin at the C-6 position. Although enzymatic
processes
are highly stereoselective, it is common for pravastatin sodium obtained after
isolation
from a fermenation broth to be contaminated with a significant amount of the C-
6 epimer
of pravastatin ("epiprava"). The C-6 position is bis-allylic and, hence, the C-
6 atom is
prone to epimerize. Careful control of pH and other conditions during
isolation of
pravastatin is required in order to minimize epimerization. Known methods of
isolating
pravastatin from a fermentation broth 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 meets 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 chromatographic
purification.
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SUMMARY OF THE INVENTION
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
substantially pure
pravastatin sodium.
A preferred embodiment of the process involves extraction of pravastatin from
an
aqueous fermentation broth into an organic solvent, back-extraction of
pravastatin into a
basic aqueous solution and a re-extraction into an organic 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 ammonium salt and then purification by recrystallization
of the
ammonium 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
1 S pravastatin may then be isolated in a highly pure state from solution by
recrystallization,
lyophilization or other means.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides pravastatin sodium substantially free of
pravastatin
lactone and epiprava and a downstream process for isolating pravastatin sodium
from a
fermentation broth in such high purity.
Enzymatic Hydroxylation of Compactin
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. Preferrably,
the
enzymatic hydroxylation is conducted using a living culture of Steptomyces,
with a nutrient
mixture of compactin and dextrose. If the broth is neutral or basic upon
completion of the
fermentation, then an acid is added to it to bring the broth to a pH of
between about 1 and
6, preferably between 1 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,
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preferably one having a pH of less than 1 as a 1M solution in water.
Acidification of the
fermentation broth converts any pravastatin carboxylate salts in the broth to
the free acid
and/or lactone.
Isolation of Substantially Pure Pravastatin Sodium
Pravastatin is first obtained from an aqueous fermentation broth in a
relatively
highly concentrated organic solution by a sequence of extraction and back-
extraction steps.
In the first step, pravastatin is extracted from the fermentation broth. CZ-C4
alkyl
formates and C,-C4 alkyl esters of CZ-C4 carboxylic acids are capable of
efficient extraction
of pravastatin from an aqueous fermentation broth. The alkyl group may be
linear,
branched or cyclic. Preferred esters include ethyl formate, h-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, h-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 butyrates, methyl isobutyrate, 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 formate are especially well suited.
The most
preferred extraction solvent is i-butyl acetate. Other organic solvents may be
substituted
for the esters. Halogenated halocarbons, aromatic compounds, ketones and
ethers may be
used, such as dichloromethane, chloroform, carbon tetrachloride, 1,2-
dichloroethane,
benzene, butyl methyl ketone, 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 preferably NaOH, NH40H or KOH, most
preferably
NaOH. The extraction solvent is preferablly 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 including the
subjective
judgment that sufficient contacting has occurred for complete extraction.
Multiple back-
extractions 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
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concentrate the pravastatin by using a volume of aqueous base that is less
than the volume
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 preferablly 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.
Pravastatin is preferablly re-extracted into one of the organic solvents
previously
described as suitable for extracting pravastatin from the fermentation broth.
The organic
solvent may 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
about 50 % (v/v) of the aqueous extract, more preferably from about 33% (v/v)
to about
20% (v/v) and still more 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 this
preferred mode for practicing the invention. This preferred mode achieves a
balance of
solvent economy and high product yield. Deviations from this preferred mode
which
further enhance the yield by repeated extractions where only one has been
described above
do not necessarily depart from the spirit of the invention. Before proceeding
to obtain
pravastatin 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
MgS04, NazS04, CaS04, silica, perlite and the like, and optionally decolorized
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 from the enriched organic
solution
with ammonia or an amine. The amine may be a primary, secondary or tertiary
amine.
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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 amines
include,
but are not limited to, methyl, dimethyl, trimethyl, ethyl, diethyl, triethyl
and other C,-C6
primary, secondary and tertiary amines; and further include morpholine, N-
methylmorpholine, isopropyl cyclohexyl amine, piperidine and the like.
Regardless of the
absence, presence or multiplicity of substitution on nitrogen, a salt formed
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 ammonia.
Precipitation of the ammonium salt of pravastatin also may be induced by
addition
of an ammonium salt either alone or in combination with the ammonia or amine.
The
preferred ammonium salts are the following salts of ammonia: NH4Cl, NH4Br,
NH4I,
(NH4)zS04, NH4N03, (NH4)3P04, (NH4)zSzOa and NH40Ac, the most preferred being
NH4Cl. Ammonium salts and high boiling liquid and solid amines may be added by
conventional means, preferably in an area with good ventilation, either as
solids, neat
liquids or solutions in aqueous or organic solvent. Addition of gaseous
ammonia requires
special equipment for handling 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 and/or 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
temperature is not allowed to exceed 40°C. Although temperatures as
high as 80°C may
be experienced without significant 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 means, the addition should
be ceased.
When 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,
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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 precipitated crystals, the pravastatin ammonium
salt is
preferablly purified by one or more, or most preferably three,
recrystallizations. To purify
the pravastatin ammonium salt, the salt is preferablly dissolved in water. The
polarity of
the solution is preferablly decreased by addition of an anti-solvent. The anti-
solvent is
preferablly a water-soluble organic solvent or solvent mixture in which the
pravastatin salt
is poorly soluble, i-butyl acetate and acetone being preferred.
The pravastatin salt may be allowed to recrystallize spontaneously, or may be
induced to recrystallize by taking the further step of adding a common ion.
According to
the preferred process wherein pravastatin is purified as its ammonia salt,
NH4Cl is added to
induce recrystallization of the ammonium salt.
The recrystallization may be performed at between about -10 ° C and
about 40 ° C,
preferably between about 0°C and about 40°C. After the
pravastatin salt has been
substantially recrystallized 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
temperature 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 ammonium salt, the pravastatin ammonium
salt
is preferablly transposed to pravastatin sodium. Pravastatin is preferablly
liberated from
the ammonium salt by dissolving in an aqueous solvent, acidifying with any
protic acid,
but preferably 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 i-butyl acetate, is
optionally contacted
with the acidified solution until pravastatin is substantially completely
transfered to the
organic phase. The organic phase is preferablly separated from the aqueous
phase and,
after optionally washing with water to remove ammonium residues, the
pravastatin is
preferablly back-extracted with aqueous sodium hydroxide solution at a pH of
from about
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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 canons are
scavenged to attain a near 1:1 equivalence of sodium cation and pravastatin
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 exchange resins which may be used are those
having sulfonic acid (S03-H+) groups. These include the commercial products
Amberlite~
IR-118, IR-120 252H; Amberlyst~ 15, 36; Amberjet 1200(H) (Rohm and
Haas);Dowex~
SOWX series, Dowex HCR-W2, Dowex 650C, Dowex Marathon C, Dowex DR-2030, and
Dowex HCR-S, ion exchange resins (Dow Chemical Co.); DIAION SK 102 to DIAION
SK 116 resin series and Lewatit SP 120 (Bayer) . The preferred strong acid
cationic
exchange resins are Amberlite~ 120, Dowex SOWX and DIAION SK series.
Weak acid cationic exchange resins include those which have pendant carboxylic
acid groups. Weak acid cationic exchange resins include the commercial
products
Amberlite CG-50, IRP-64, IRC 50 and C67, Dowex CCR series, Lewatit CNP series
(Bayer) and DIAION WK series (Mitsubishi), of these, the most preferred are
Amberlite~
IRC50, Lewatit CNP 80 and DIAION WK 10. Less preferred are the chelate type
exchange resins. Some of the commercial varieties that are available include
Amberlite~
IRC-718, and IRC-467.
The solution containing pravastatin sodium salt and excess sodium canons 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 of the resin or by stirring a
sufficient quantity of
the resin in a flask with the solution. The mode of contact 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 pH
will vary with
dilution. Reduction in the pH of the pravastatin sodium solution from a higher
pH to a
lower pH and then leveling off of the pH at the lower level is an indication
of substantial
completion of scavenging excess Na+ ions. After scavenging is substantially
complete, the
pravastatin sodium solution is preferablly separated from the resin in a
conventional
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manner. It may either be collected as the eluent from a column or bed or may
be separated
by filtration, decantation and the like.
Pravastatin sodium may be isolated from the pravastatin sodium solution by
crystallization. Efficient crystallization may first require partial removal
of the water,
which can be conducted by vacuum distillation or nano-filtration. Preferably,
the aqueous
pravastatin sodium salt solution is concentrated from about 20 to about 50
w/v% before
crystallizing. If necessary, after concentration the aqueous pravastatin
sodium solution can
be adjusted to a pH of between about 7 and about 10 with an ion exchange resin
in H+
form.
Addition of a water-soluble organic 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 acetate
mixtures. One of the most preferred solvent system for crystallizing
pravastatin sodium is
a 1/3/12 water/acetone/acetonitrile mixture formed by concentrating the
pravastatin
sodium solution to about 30 w/v% and then adding an appropriate volume of 1/4
acetone/acetonitrile mixture. The most preferred crystallization solvent
mixture is water-
acetone (1:15).
Pravastatin sodium also may be isolated by lyophilization 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% (w/w)
contamination by epiprava. Pravastatin sodium further may be isolated with
less than
0.2% (w/w) pravastatin lactone and 0.1% epiprava by adhering to the preferred
embodiments of the invention, two of which are exemplified in Examples 1 and 3
The highly pure pravastatin sodium produced by the present inventive method is
preferablly useful for hypercholesteremia therapy and for this purpose it can
administered
to a mammalian patient by any route of administration. A daily oral regimen is
the most
preferred prescribed method of administration. In human subjects with normal
hepatic
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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, sachets,
suspensions,
powders, 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 pharmaceutical excipients. Whether administered alone or in a
composition, the
highly pure pravastatin sodium of the invention may be in the form of a
solution or a solid
such as a powder, granules, aggregates 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 rate desired and other factors. For example,
compositions of the
present invention may contain diluents such as cellulose-derived materials
like powdered
cellulose, microcrystalline cellulose, microfine cellulose, methyl cellulose,
ethyl cellulose,
hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl
cellulose,
carboxymethyl cellulose salts and other substituted and unsubstituted
celluloses; starch;
pregelatinized starch; inorganic diluents like calcium carbonate and calcium
diphosphate
and other diluents known to the pharmaceutical industry. Yet other suitable
diluents
include waxes, sugars and sugar alcohols like mannitol and sorbitol, acrylate
polymers and
copolymers, as well as pectin, dextrin and gelatin.
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 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 such as silicon dioxide.
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Capsule dosages will contain the solid composition within a capsule which may
be
made of gelatin or other encapsulating material. Tablets and powders may be
coated.
Tablets and powders may be coated with an enteric coating. The enteric-coated
powder
forms may have coatings comprising phthalic acid cellulose acetate,
hydroxypropylmethyl
cellulose phthalate, polyvinyl alcohol phthalate, carboxymethylethylcellulose,
a copolymer
of styrene and malefic acid, a copolymer of methacrylic 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 enteric-coating.
The highly pure pravastatin sodium may also be administered in injectable
dosages
as a solute or suspended solid in a sterile solution or suspension. Suitable
carriers for
sterile injectable dosages include water and oils.
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.
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EXAMPLES
EXAMPLE 1
Purification of Pravastatin
The fermentation broth (100 L) was acidified to from about 2.5 to about S.0 by
addition of sulfuric acid. The acidified fermentation broth was extracted 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 combined 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 reacidified to a pH of about 2.0 to about 4.0 by addition of SM sulfuric
acid and back-
extracted with i-butyl acetate (8 L). The resulting solution of pravastatin in
i-butyl acetate
was partially dried over Perlite and Na2S04. The pravastatin solution was
decanted and
then filtered from the drying agents and decolorized over activated charcoal
(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 headspace above the solution with
rapid
stirring. The precipitated crystals of ammonium pravastatin carboxylate salt
were collected
by filtration and washed with i-butyl acetate and then acetone which yielded
pravastatin
ammonium salt in about 94 % purity as determined by HPLC coupled with UV
absorbance
measured at ~,=238nm.
The pravastatin ammonium salt was further purified by crystallization from a
saturated ammonium chloride solution as follows. The 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
pravastatin ammonium was induced to crystallize by addition of solid NH4Cl
until further
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 were
collected by filtration and washed with 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.
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The pravastatin ammonium salt was further purified by another crystallization
as
follows. The pravastatin ammonium salt (155.5 g of active substance) was
dissolved in
water (900 ml). Isobutanol (2 ml) was added and then the pH was raised to
about pH 10 to
about pH 13.7 by addition of a concentrated solution of sodium hydroxide and
the solution
was stirred for 30 min. at ambient temperature. The solution was neutralized
to a pH of
about 7 by addition of sulfuric acid and crystallization of pravastatin
ammonium was
induced by addition of solid NH4C1. The crystals (150 g) were collected by
filtration and
washed with acetone. Pravastatin ammonium was found to be about 99.3% pure by
HPLC
detection using the above-described conditions.
The pravastatin ammonium was then transposed to the sodium salt as follows.
The
pravastatin ammonium 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
free acid. The i-butyl acetate phase, containing pravastatin, was washed with
water
(5x l Oml). Pravastatin was then converted to its sodium salt and back-
extracted into
another aqueous phase by swirling the i-butyl acetate solution over water
between about
900-2700 ml with intermittent 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 canons. After separation, the aqueous phase was stirred
over IRC
50 in the H+ ion exchange resin for 30 min. at ambient temperature. Stirring
was
continued until a pH of about pH 7.4 to about pH 7.8 was reached.
The solution was then filtered to remove the resin and partially concentrated
to a
weight of 508 g. under vacuum. Acetonitrile (480 ml) was then added and the
solution
was stirred over activated carbon (5 g) to decolorize. Pravastatin sodium was
obtained as
crystals by crystallization in 90% yield after further addition of acetone and
acetonitrile to
form a 1/3/12 mixture of water/acetone/acetonitrile (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%
purity from the starting fermented active substance as measured by HPLC using
the above-
described conditions.
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EXAMPLE 2
Following the procedure in Example 1, but omitting the recrystallization from
the
water/acetone/acetonitrile mixture, pravastatin sodium was obtained by
lyophilization of
the concentrated solution of pravastatin sodium in water in about 99% purity
and about
72% yield.
EXAMPLE 3
Following the procedure of Example 1, but further purifying the pravastatin
ammonium salt by once repeating the crystallization of the pravastatin
ammonium salt,
pravastatin sodium was obtained in about 99.8% purity and 68.4% yield.
EXAMPLE 4
Following the procedure of Example 1, but further purifying the pravastatin
ammonium salt by twice repeating the crystallization of the pravastatin
ammonium salt,
pravastatin sodium was obtained in about 99.6% purity and 53% yield.
EXAMPLE 5
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 sulfuric acid. The
acidified
fermentation broth was extracted with i-butyl acetate (3x50 L). The combined i-
butyl
acetate phases were then extracted with water (35 L) having been basified to a
pH of about
pH 7.5 to about pH 11.0 by addition of concentrated ammonium hydroxide.
Instead of reacidifying the aqueous extract and extracting with i-butyl
acetate to
obtain a further enriched organic solution as was done in Example l, the
aqueous extract
was concentrated to 140 g/L 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 were then isolated by filtration and washed with a saturated solution
of ammonium
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chloride. The crystals were then added to water (1L) 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 ammonium salt were recovered by filtration and washed with i-butyl
acetate
and after that with acetone and dried. The resulting crystals were then
further purified by
recrystallization transposed to the sodium salt and isolated as described in
Example 1.
Pravastatin sodium was obtained in about 99.9% purity and 67.7% yield.
EXAMPLE 6
Following the procedure of Example l, but the pravastatin sodium salt was
crystallized from 1/15 mixture of water/acetone in an overall yield from the
starting
fermented active substance of 64% and in 99.8 % purity as measured by HPLC.
EXAMPLE 7
1 S Following the method of Example S, first two paragraphs, a concentrated
aqueous
extract (140 g. L'') was obtained. The concentrated aqueous extract was
divided into equal
parts.
EXAMPLE 8
Following the procedure in Example 1, pravastatin ammonium salt was isolated
from a fermentation broth, but active substance was dissolved and crystallized
after
precipitation 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
charcoal 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 acetate mother liquor.
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Pravastatin ammonium salt was crystallized by adding ammonium 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 1 hour. Crystals
were filtered,
suspended in i-butyl acetate and filtered then suspended in i-butyl acetate :
acetone (2 : 1)
and filtered, then suspended in acetone and filtered. Crystals were dried in
vacuum after
washing with acetone.
The process yielded pravastatin ammonium salt in about 93 % purity as
determined
by HPLC with UV detection at 1= 238 nm. Crystallized active substance was
168.7 kg.
EXAMPLE 9
Following the procedure in Example 1, pravastatin ammonium salt was isolated
from a fermentation broth, but crystallization was used instead of
precipitation with
ammonia gas.
Enriched pravastatin i-butyl acetate solution (4150 ml) was decolorized 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 ml).
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 S
hours. The suspension was stirred at 24 - 26 °C for 15 hours. Crystals
were filtered, more
times suspended, washed and dried.
The process yielded pravastatin ammonium salt in about 95 % purity as
determined
by HPLC. Crystallized active substance was 42.7 g.
EXAMPLE 10
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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 ml) mixture at 35 - 40 °C. The solution was cooled to 30 -
32 °C. Ammonium
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
acetone.
Pravastatin ammonium salt was dried at 40 °C. The yield was 96 %. The
purity was
97 %.
EXAMPLE 11
Following the procedure of Example 8, pravastatin ammonium salt was produced
in about 93 % purity.
1 S 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. 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 %.
EXAMPLE 12
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 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 lithium salt was obtained in 96 % purity with 89 % yield.
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