U.S. patent application number 12/771726 was filed with the patent office on 2010-09-02 for oxidative degeneration products of atorvastatin calcium.
This patent application is currently assigned to Lek Pharmaceuticals D.D.. Invention is credited to Andrej Bastarda, Rok GRAHEK, Andrej Kocijan, Darko Kocjan, Matjaz Kracun.
Application Number | 20100219063 12/771726 |
Document ID | / |
Family ID | 35457712 |
Filed Date | 2010-09-02 |
United States Patent
Application |
20100219063 |
Kind Code |
A1 |
GRAHEK; Rok ; et
al. |
September 2, 2010 |
OXIDATIVE DEGENERATION PRODUCTS OF ATORVASTATIN CALCIUM
Abstract
The present invention relates to oxidative degradation products
of atorvastatin calcium and the process of the preparation thereof.
The present invention also relates to atorvastatin calcium
substantially free of oxidative degradation products and the
pharmaceutical compositions containing such atorvastatin
calcium.
Inventors: |
GRAHEK; Rok; (Kranj, SI)
; Kocjan; Darko; (Ljubljana, SI) ; Bastarda;
Andrej; (Vrhnika, SI) ; Kocijan; Andrej;
(Ljubljana, SI) ; Kracun; Matjaz; (Ljubljana,
SI) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
Lek Pharmaceuticals D.D.
Ljubljana
SI
|
Family ID: |
35457712 |
Appl. No.: |
12/771726 |
Filed: |
April 30, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11632608 |
Mar 9, 2007 |
|
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PCT/EP2005/007739 |
Jul 15, 2005 |
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12771726 |
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Current U.S.
Class: |
204/157.72 ;
204/157.69 |
Current CPC
Class: |
A61P 9/00 20180101; C07D
493/04 20130101; C07D 303/48 20130101; C07D 301/00 20130101; A61P
43/00 20180101; C07D 207/416 20130101; A61P 3/06 20180101; A61P
9/10 20180101; C07D 498/14 20130101 |
Class at
Publication: |
204/157.72 ;
204/157.69 |
International
Class: |
B01J 19/12 20060101
B01J019/12; C07D 498/14 20060101 C07D498/14; C07D 493/04 20060101
C07D493/04; C07D 207/00 20060101 C07D207/00; C07D 303/48 20060101
C07D303/48 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 16, 2004 |
SI |
P200400209 |
Jul 16, 2004 |
SI |
P200400348 |
Claims
1. A process for the preparation of a compound selected from the
group consisting of
4-[6-(4-fluoro-phenyl)-6-hydroxy-1b-isopropyl-6a-phenyl-1a-phenylcarbamoy-
l-hexahydro-1,2-dioxa-5a-aza-cyclopropa[a]inden-3-yl]-3-(R)hydroxy-butyric
acid,
4-(4-Fluoro-phenyl)-2,4-dihydroxy-2-isopropyl-5-phenyl-3,6dioxa-bic-
yclo[3.1.0]hexane-1-carboxylic acid phenylamide,
4-[1b-(4-Fluoro-phenyl)-6-hydroxy-6-isopropyl-1a-phenyl-6a-phenylcarbamoy-
l-hexahydro-1,2-dioxa-5a-azacyclopropa[a]inden-3-yl]-3-(R)-hydroxy-butyric
acid and 3-(4-Fluoro-benzoyl)-2
isobutyryl-3-phenyl-oxirane-2-carboxylic acid phenylamide
comprising exposing a solution of an atorvastatin salt to sunlight
or artificial sunlight.
2. The process according to claim 1 wherein the atorvastatin salt
is selected from the group consisting of atorvastatin calcium,
atorvastatin sodium, atorvastatin potassium, atorvastatin magnesium
and atorvastatin ammonium.
3. The process according to claim 1 further comprising one or more
isolation steps.
4. The process according to claim 3 wherein the isolation step is
selected from the group consisting of preparative normal phase
chromatography and reverse phase chromatography.
5. The process according to claim 4 wherein the preparative normal
phase chromatography comprises using silica gel or silica-based
bonded phases functionalized with a group selected from
aminopropyl, cyanopropyl, diol and nitrophenyl.
6. The process according to claim 5 wherein the preparative normal
phase chromatography comprises using a mobile phase comprising a
mixture of an alcohol selected from the group consisting of
methanol, ethanol, propanol and acetonitrile and a non-polar
solvent selected from the group consisting of hexane,
dichloromethane, methylcyclohexane, and any combination
thereof.
7. The process according to claim 4 wherein in the preparative
reverse phase chromatography comprises using octadecylsylan or
octylsilan bonded on silica gel.
8. The process according to claim 7 wherein the preparative reverse
phase chromatography comprises using a mobile phase comprising a
mixture of water with an organic or inorganic buffer and one or
more organic modifiers selected from the group consisting of
alcohols and acetonitrile.
Description
[0001] The present invention belongs to the field of organic
chemistry and relates to oxidative degradation products of
atorvastatin calcium and the processes for the preparation thereof.
The present invention also relates to atorvastatin calcium
substantially free of oxidative degradation products and
pharmaceutical compositions containing such atorvastatin
calcium.
[0002] The purity of the pharmaceutical active substances has
always been considered as an essential factor in ensuring drug
safety and quality. As it is well known in the art, the result of
the many different complex steps in the production of a
pharmaceutical active substance is not only the desired product but
also impurities which are structurally closely related compounds.
Additionally, many pharmaceutical active substances are sensitive
to environmental influences such as for example temperature, pH,
humidity, light, gases, oxygen, carbon dioxide, reactivity of the
ambient medium during handling or storage. Such environmental
influences may cause transformation of the pharmaceutically active
compound into degradation products which are often less effective
than the active compound. Apart from the lower efficacy,
degradation products may also cause undesirable side effects thus
negatively affecting the safe use of a medicament. Even a very low
percent of impurities or degradation products present in the active
substance may significantly impair drug safety. Therefore, it is
very important that a pharmaceutical active substance is as pure as
possible when administered; this means that the percentage of
degradation products and impurities present in the pharmaceutical
active substance should be minimal.
[0003] Moreover, the pharmaceutical excipients used in the
pharmaceutical dosage form may also have an influence on the amount
of degradation products and impurities present in the
pharmaceutical active substances. Degradation products of the
pharmaceutical excipients themselves may act as reactive sites
triggering degradation reactions of the pharmaceutical active
substances in a pharmaceutical dosage form.
[0004] The sensitivity of various pharmaceutical active substances
to oxidative degradation is described by Waterman, KC., et al, in
"Stabilization of Pharmaceuticals to Oxidative Degradation",
Pharmaceutical Development and Technology, 7(1), 2002, 1-32, and
possible approaches to the stabilization of pharmaceutical active
substances against oxidative degradation are also presented. The
above-mentioned article suggests that the study of oxidative
mechanism in solid pharmaceutical dosage forms is difficult and
demanding, as it is indicated by few reports in said area, but
however teaches that an active substance per se and more frequently
an active substance in a pharmaceutical dosage form may oxidize.
Byrn, S. R., et al. (Solid-State Chemistry of Drugs, 2.sup.nd Ed.,
SSCI, West Lafayette, 1999) disclose that molecular oxygen from the
atmosphere reacts with organic crystals and that said reactivity
depends on the crystal form and morphology of the active substance,
which determine permeability to oxygen and its solubility in the
crystal lattice, respectively. In some examples the reactivity is
shown to decrease with increased melting point indicating that
higher crystalline lattice energy inhibits diffusion of oxygen.
[0005] For the prevention or reduction of the oxidation of an
active substance in a pharmaceutical formulation different
approaches have been used until now, such as, for example: [0006]
1. increasing the concentration of the active substance in a
pharmaceutical formulation, in the case that oxidation is caused by
the presence of peroxide and metallic impurities in excipients;
[0007] 2. addition of chelating agents (e.g. citric acid, EDTA,
fumaric acid and maleic acid) for removal of metallic impurities
present in excipients; [0008] 3. use of high-purity pharmaceutical
excipients; [0009] 4. use of alternative pharmaceutical excipients,
or decrease in the amount of excipients in the pharmaceutical
composition, particularly where the excipients are the cause of
oxidation due to a peroxide impurity; [0010] 5. use of antioxidants
which are capable of preventing or reducing the formation of
peroxides in a pharmaceutical composition. However, such
antioxidants do not reduce the level of the already present
peroxides at the same time. Some suitable antioxidants have been
described previously, including: [0011] chain terminators (as e.g.
thiols and phenols); [0012] reducing agents which are more readily
oxidized than an active substance and thus remove present oxygen
(e.g. sulfites and ascorbic acid) wherein their combination may act
synergistically (e.g. a combination of ascorbic palmitate and
tocopherol); [0013] peroxide scavengers which degrade peroxides
(e.g. Fe.sup.2+) on the principle of Fenton's procedure. However,
their use is limited because by this procedure a free hydroxyl
radical may be formed which may further induce reactions of free
radicals and thus degradation of an active substance; [0014]
cyclodextrins which cover the site of an active substance,
subjected to oxidation (Waterman, K. C., et al, Stabilization of
Pharmaceuticals to Oxidative Degradation, Pharmaceutical
Development and Technology, 7(1), 2002, 1-32).
[0015] However, for individual active substances it is impossible
to envisage optimal modes and few publications are available in the
field (Waterman, K. C., et al, Stabilization of Pharmaceuticals to
Oxidative Degradation, Pharmaceutical Development and Technology,
7(1), 2002, 1-32).
[0016] Atorvastatin calcium, which has the chemical name
hemicalcium salt of
(R--(R*,R*))-2-(4-fluorophenyl)-.beta.,.delta.-dihydroxy-5-(1-methylet-
hyl)-3-phenyl-4((phenylamino) carbonyl)-1H-pyrol-1-heptanoic acid,
is known as an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A
(HMG-CoA) reductase. It was described first time in U.S. Pat. No.
5,273,995. Processes for the preparation of atorvastatin calcium
and key intermediates thereof are described in U.S. Pat. Nos.
5,003,080; 5,097,045; 5,103,024; 5,124,482; 5,149,837; 5,155,251;
5,216,174; 5,245,047; 5,248,793; 5,280,126; 5,342,952; and
5,397,792.
[0017] HMG-CoA reductase inhibitors are known to be
pharmaceutically active substances which are sensitive to the pH of
the environment, humidity, light, temperature, carbon dioxide and
oxygen. They are known as effective therapeutically active
substances for the treatment of dyslipidemias and cardiovascular
diseases, selected from the group consisting of dyslipidemia,
hyperlipidemia, hypercholesterolemia, atherosclerosis,
arteriosclerosis, coronary artery diseases, coronary heart disease
and the like, associated with the metabolism of lipids and
cholesterol. The mechanism of action of statin compounds is by the
inhibition of the biosynthesis of cholesterol and other sterols in
the liver of humans or animals. They are competitive inhibitors of
HMG-CoA reductase or 3-hydroxy-3-methyl-glutaryl-coenzyme A
reductase, an enzyme which catalyses the conversion of HMG-CoA to
mevalonate in the liver of humans or animals, which is an important
step in the biosynthesis of cholesterol in the liver. Recent
studies indicate that, in addition to the above-mentioned
therapeutic effects, statins also have other therapeutic effects
and, accordingly, they are useful in the treatment of diseases,
abnormal conditions and disorders which are selected from the group
consisting of vascular disorders, inflammatory disease, allergic
disease, neurodegenerative disease, malignant disease, viral
disease (WO 0158443), abnormal bone states, (WO 0137876),
amyloid-.beta. precursor protein processing disorders such as
Alzheimer's disease or Down's Syndrome (WO 0132161).
[0018] To date next to nothing has been published on how to avoid
the presence of oxidation products in atorvastatin substance, and
the degradation products of atorvastatin calcium have not
previously been identified. The prevention of oxidation of
atorvastatin calcium by means of carrying out the preparation
process in an inert atmosphere, and by packaging in suitable
packaging under an inert atmosphere was described in the Slovenian
patent application SI P-200200244. The structure of one degradation
product of atorvastatin calcium having the chemical name:
3-(4-Fluoro-benzoyl)-2-isobutyryl-3-phenyl-oxirane-2-carboxylic
acid phenylamide, and its preparation by photodecomposition was
described in the article Hurley, T. R. et al, Tetrahedron 49, 1993,
1979-1984.
[0019] In view of the importance of obtaining an active substance
with a high level of purity there exists a need for the
characterization of each impurity or degradation product present in
the active substance and/or pharmaceutical composition. The
characterization of each impurity or degradation product present in
an active substance or pharmaceutical composition is particularly
important in respect of those active substances for which the
response factor of an impurity and/or degradation product for a
specified analytical method (e.g. HPLC) varies from the response
factor of the active substance. Namely, in such cases it can happen
that the active substance is declared as pharmaceutically
acceptable according to regulatory requirements although the real
level of impurities or degradation products is in fact outside the
permitted values.
[0020] The present invention meets a need in the art for obtaining
atorvastatin calcium product in a highly pure form, having a low
content of oxidative degradation products, by a technologically
simple manner and at a high yield, by characterization of three
oxidative degradation products present in atorvastatin calcium
and/or pharmaceutical compositions thereof.
[0021] One object of the present invention relates to novel
compounds, which are oxidative degradation products of atorvastatin
calcium, having the following chemical formulae and chemical
names:
a) compound with formula I
##STR00001##
and chemical name
4-[6-(4-Fluoro-phenyl)-6-hydroxy-1b-isopropyl-6a-phenyl-1a-phenylcarbamoy-
l-hexahydro-1,2-dioxa-5a-aza-cyclopropa[a]inden-3-yl]-3-(R)-hydroxy-butyri-
c acid (from hereon referred to as ATV-cycloIP); b) compound with
formula II
##STR00002##
and chemical name
4-(4-Fluoro-phenyl)-2,4-dihydroxy-2-isopropyl-5-phenyl-3,6-dioxa-bicyclo[-
3.1.0]hexane-1-carboxylic acid phenylamide (from hereon referred to
as ATV-epoxy furan); c) compound with formula III
##STR00003##
and chemical name
4-[1b-(4-Fluoro-phenyl)-6-hydroxy-6-isopropyl-1a-phenyl-6a-phenylcarbamoy-
l-hexahydro-1,2-dioxa-5a-aza-cyclopropa[a]inden-3-yl]-3-(R)-hydroxy-butyri-
c acid (from hereon referred to as ATV-cycloFP).
[0022] The oxidative degradation product of atorvastatin calcium
described in the article of Hurley, T. R. et al, Tetrahedron 49,
1993, 1979-1984 has the following chemical formula IV
##STR00004##
and chemical name:
3-(4-Fluoro-benzoyl)-2-isobutyryl-3-phenyl-oxirane-2-carboxylic
acid phenylamide (from hereon referred to as ATV-epoxy dion).
[0023] The oxidative degradation of atorvastatin calcium may be
represented by the following scheme:
TABLE-US-00001 ##STR00005## R.sub.2 R.sub.3 R.sub.4 R.sub.5
##STR00006## ##STR00007## ##STR00008## ##STR00009## ##STR00010##
##STR00011## ##STR00012## ##STR00013##
[0024] The compounds with formula I, II, III and IV according to
the present invention are present in atorvastatin calcium substance
as oxidative degradation products, accordingly, it is very
important that their amount should be minimized and that they
should be detectable only in a very small amount. As impurities
they present a risk of being toxic or otherwise harmful to the
patient. For these reasons it is essential that their presence in
the substance should be minimized. On the other hand it is very
important that the level of these impurities is monitored
accurately, accordingly their content should be determined
correctly and exactly, e.g. by using standards (compound with known
chemical structure and known assay) for quantitative
determination.
[0025] The present invention also relates to novel processes for
the preparation of compounds having the formula I, II, III and IV
according to the present invention.
[0026] The novel compounds according to the present invention may
be prepared by oxidation of solid atorvastatin in the form of a
salt (for example as a calcium, sodium, potassium, magnesium, or
ammonium salt) in an air or oxygen atmosphere at an elevated
temperature, e.g. from 40 to 90.degree. C. The reaction may last
from 1 to several days. The oxidation may be performed in a
solution of the atorvastatin salt in water and/or an organic
solvent and/or mixtures of solvents, such as for example
acetonitrile, methanol, ethanol, propanol, dichloromethane or
methylene chloride; with the addition of hydrogen peroxide or by
blowing air or oxygen through the solution at the temperature of
about 40 to 90.degree. C. The solid atorvastatin salt may be
prepared by any known process.
[0027] The novel compounds according to the present invention may
also be prepared by photo-oxidation of atorvastatin in the form of
a salt (e.g. as a calcium, sodium, potassium, magnesium, ammonium
salt) by exposing a solution of the atorvastatin salt to sunlight
or artificial sunlight. The atorvastatin salt may be prepared by
any known process.
[0028] The novel compounds according to the present invention
prepared by the processes as described above may be isolated by
preparative normal phase or reverse phase chromatography.
[0029] In preparative normal phase chromatography silica gel or
silica-based bonded phases for example aminopropyl, cyanopropyl,
diol, or nitrophenyl bonded stationary phases may be used. The
mobile phase comprises a mixture of a polar modifier alcohol, for
example methanol, ethanol, propanol or acetonitrile, and of a
non-polar solvent as for example hexane, dichloromethane,
methylcyclohexane, or a combination of more than two solvents
listed above.
[0030] In preparative reverse phase chromatography octadecylsylan
or octylsilan bonded on silica gel may be used. The mobile phase
comprises a mixture of water with an organic or inorganic buffer in
the concentration range from 5 mM to 100 mM and the pH range from 2
to 8, together with one or more organic modifiers selected from
alcohols, such as for example methanol, ethanol and propanol, or
acetonitrile.
[0031] One or more chromatographic steps may be used during the
isolation of novel compounds according to the present invention.
The solvents used in chromatographic steps may be removed by
evaporation and/or by freeze drying.
[0032] The novel compounds according to the present invention
prepared and isolated by the methods as described above were
structurally characterized by Mass Spectrometry and Nuclear
Magnetic Resonance spectroscopy in order to determine the chemical
structure of the said novel compounds. The methods of
characterization and their results are presented in the examples
described below.
[0033] During the development work on preparing stable atorvastatin
calcium compositions it has been found that atorvastatin calcium
degrades when it comes into contact with air or more precisely with
oxygen. Surprisingly the use of different antioxidants such as for
example buthylated hydroxyanisole, buthylated hydroxytoluene,
fumaric acid, propyl galate, sodium sulfite, sodium meta bisulfite,
sodium ascorbate did not prevent or diminish the formation of the
oxidative degradation products. Surprisingly, it has been found
that by lowering the content of oxygen in the surrounding
atmosphere of atorvastatin calcium or a pharmaceutical composition
comprising atorvastatin calcium the formation of the oxidative
degradation products in atorvastatin calcium may be significantly
reduced. The observed reduction in oxidative degradation products
was linearly proportional to the lowering of the oxygen content in
the surrounding atmosphere. This lowering of the oxygen content may
be performed by substitution of oxygen with an inert gas, for
example nitrogen or argon, or by lowering the atmospheric pressure
surrounding the atorvastatin calcium.
[0034] Besides carrying out the process of the preparation of
atorvastatin calcium in an inert atmosphere and storing it in an
inert atmosphere it is very important to monitor the amount of
oxidative degradation products present in atorvastatin calcium
substance and in the pharmaceutical composition comprising thereof.
For determining the amount of undesired compounds it is necessary
to provide standards of these compounds (this means the compounds
with known chemical structure and assay) to be able to perform the
exact quantitative analysis. This is of specific importance in
cases where the response factor in e.g. HPLC analysis of impurity
and/or degradation product is different in comparison to the
response factor of the active substance. HPLC analysis is normally
used for impurity determination in pharmaceutically active
substances and pharmaceutical compositions.
[0035] Surprisingly, it has been found that some of the compounds
present in atorvastatin calcium substance exhibit different
response factor in comparison to atorvastatin calcium itself when
HPLC analysis at 250 nm is performed. Namely, the novel compound
according to the present invention with formula I exhibits 0.41
response factor, the novel compound according to the present
invention with formula II exhibits 0.72 response factor, the novel
compound according to the present invention with formula III
exhibits 0.48 response factor and the compound with formula IV
exhibits 1.20 response factor in comparison to atorvastatin calcium
itself.
[0036] The content of impurities in an active substance and/or
pharmaceutical composition is an important factor for the safety of
the drug, therefore the content of impurities should be minimized.
This is especially crucial for degradation products because their
content in the drug rises during the shelf life of the drug.
[0037] A further object of the present invention is the provision
of atorvastatin calcium substantially free of oxidative degradation
products and pharmaceutical compositions containing said
atorvastatin calcium and at least one pharmaceutically acceptable
excipient.
[0038] The present invention provides substantially pure
atorvastatin calcium which comprises less than about 0.29 weight %
oxidative degradation products.
[0039] The present invention provides substantially pure
atorvastatin calcium which comprises less than about 0.09 weight %
ATV-cycloIP.
[0040] The present invention provides substantially pure
atorvastatin calcium which comprises less than about 0.05 weight %
ATV-epoxy furan.
[0041] The present invention provides substantially pure
atorvastatin calcium which comprises less than about 0.09 weight %
ATV-cycloFP.
[0042] The present invention provides substantially pure
atorvastatin calcium which comprises less than about 0.06 weight %
ATV-epoxy dione.
[0043] The Table 1 below shows the amount of each oxidative
degradation product present in atorvastatin calcium exposed to
various atmospheric conditions under which the process for the
preparation was carried out.
[0044] When atorvastatin calcium is prepared or stored in an air
atmosphere at the room temperature oxidative degradation products
are formed. This can be avoided when atorvastatin calcium is stored
in a nitrogen atmosphere.
TABLE-US-00002 TABLE 1 The content of oxidative degradation
products in atorvastatin calcium when atorvastatin calcium is
prepared in air and in a nitrogen atmosphere. Produc- Content of
Content of Content of Content of tion atmo- ATV-cycloIP ATV-epoxy
ATV-cycloFP ATV-epoxy sphere in % furane in % in % dion in % air
0.088 0.066 0.093 0.069 nitrogen 0.013 0.011 0.018 0.016 The
analysis was performed using the response factor 1.00 for all
substances.
TABLE-US-00003 TABLE 2 The content of oxidative degradation
products in atorvastatin calcium prepared in air or in a nitrogen
atmosphere. Produc- Content of Content of Content of Content of
tion atmo- ATV-cycloIP ATV-epoxy ATV-cycloFP ATV-epoxy sphere in %
furane in % in % dion in % air 0.215 0.093 0.193 0.058 nitrogen
0.032 0.016 0.038 0.013 The analysis was performed using the
response factors 0.41 for ATV-cycloIP, 0.72 for ATV-epoxy furan,
0.48 for ATV-cycloFP and 1.20 for ATV - epoxy dion.
[0045] The comparison between the Table 1 considering response
factor for oxidative degradation products as 1.00 and the Table 2
considering the determined response factors show the essential
difference in content values. When standards of the impurities are
not used and the response factor 1.00 is applied, the determined
values of oxidative degradation products in atorvastatin calcium
prepared in air or in a nitrogen atmosphere are below the values
determined by using the exact response factor. Also the content of
the oxidative degradation products determined using the response
factor 1.00 may be below the threshold of 0.10% above which the
impurities should be identified according to the generally accepted
pharmaceutical regulations. Further, due to the fact having a
response factor greater than 1.00, the content of ATV-epoxy dion as
determined using the response factor 1.00 is above the value
determined by using the exact response factor.
[0046] All further analyses were performed using the response
factors 0.41 for ATV-cycloIP, 0.72 for ATV-epoxy furan, 0.48 for
ATV-cycloFP and 1.20 for ATV-epoxy dion.
TABLE-US-00004 TABLE 3 The content of oxidative degradation
products in atorvastatin calcium stored in air or in a nitrogen
atmosphere for 24 months at room temperature (for example
25.degree. C.). Storing Content of Content of Content of Content of
atmo- ATV-cycloIP ATV-epoxy ATV-cycloFP ATV-epoxy sphere in %
furane in % in % dion in % air 0.856 0.636 0.905 0.741 nitrogen
0.094 0.052 0.088 0.063
[0047] The present invention provides a pharmaceutical composition
comprising substantially pure atorvastatin calcium which comprises
less than about 0.6 weight % oxidative degradation products and at
least one pharmaceutically acceptable excipient.
[0048] The present invention provides a pharmaceutical composition
comprising substantially pure atorvastatin calcium which comprises
less than about 0.2 weight % ATV-cycloIP and at least one
pharmaceutically acceptable excipient.
[0049] The present invention provides a pharmaceutical composition
comprising substantially pure atorvastatin calcium which comprises
less than about 0.1 weight % ATV-epoxy furan and at least one
pharmaceutically acceptable excipient.
[0050] The present invention provides a pharmaceutical composition
comprising substantially pure atorvastatin calcium which comprises
less than about 0.2 weight % ATV-cyclo FP and at least one
pharmaceutically acceptable excipient.
[0051] The present invention provides a pharmaceutical composition
comprising substantially pure atorvastatin calcium which comprises
less than about 0.1 weight % ATV-epoxy dion and at least one
pharmaceutically acceptable excipient.
TABLE-US-00005 TABLE 4 The content of oxidative degradation
products in atorvastatin calcium formulated into pharmaceutical
formulation in the form of tablets stored in air or in a nitrogen
atmosphere in Al/Al blisters for 24 months at room temperature (for
example 25.degree. C.). Storing Content of Content of Content of
Content of atmo- ATV-cycloIP ATV-epoxy ATV-cycloFP ATV-epoxy sphere
in % furane in % in % dion in % air 1.75 0.61 1.23 0.65 nitrogen
0.18 0.08 0.17 0.09
[0052] The results shown in Tables 3 and 4 show that when
atorvastatin calcium or a pharmaceutical formulation containing
atorvastatin calcium in the form of tablets are stored in an air
atmosphere at the room temperature for 24 months oxidative
degradation products dramatically increase. This can be avoided
when atorvastatin calcium is stored in a nitrogen atmosphere.
[0053] The pharmaceutical composition according to the present
invention may be administered to a mammal in a dosage form. The
dosage form contains substantially pure atorvastatin calcium
according to the present invention and at least one
pharmaceutically acceptable excipient selected from the group
consisting of diluents, binders, disintegrants, lubricants,
glidants, flavorings, sweeteners, preservatives, dyes and other
excipients used in preparing pharmaceutical composition. The
pharmaceutical composition according to the present invention can
be any dosage form that is used in pharmaceutical industry such as,
for example, tablets, orally dispersible formulations, capsules,
pellets, granulate, etc. Nitrogen or argon can be used as the inert
gas for maintenance of an inert atmosphere. The pharmaceutical
composition can be stored in an inert atmosphere in an Al/Al
blister, Al-polychloro-3-fluoroethylene homopolymer/PVC laminate
blister or bottles.
[0054] The pharmaceutical composition according to the present
invention are useful in the treatment of hypercholesterolemia and
hyperlipidemia.
[0055] The present invention is illustrated but in no way limited
by the following examples:
EXAMPLE 1
Preparation and Isolation of ATV-cycloIP, ATV-Epoxy Furan,
ATV-cycloFP and ATV-Epoxy Dion Compounds
[0056] 5 grams of atorvastatin calcium was stored in a 200 ml
airtight container in oxygen atmosphere for 30 days at 80.degree.
C. The thus prepared sample was dissolved in 50% acetonitrile/water
(vol/vol) and subjected to preparative chromatography.
Preparative chromatography: Isolation of oxidative degradation
products was carried out on a reverse-phase chromatography. Two
chromatographic separations with different mobile phases were
necessary to obtain pure compounds.
[0057] The first separation was carried out on a preparative HPLC
chromatograph equipped with Luna prep C18(2) 10 .mu.m column (200
mm.times.50 mm) and UV detector set at 250 nm. The two mobile
phases solvents, A and B, were 10 mM ammonium acetate pH 4.5 and
95% acetonitrile/5% tetrahydrofuran (vol/vol), respectively. The
flow rate was 140 ml/min. The following gradient profile was
used:
TABLE-US-00006 Time (min:sec) % B 0 5 0:22 5 0:25 30 14 75 14:10 90
15 90 15:10 5
[0058] Four fractions were collected; the pH of fractions one and
two was adjusted to 8-9 with 1M potassium hydroxide, the pH of
fraction three and four was adjusted to 2-3 with 1M hydrochloric
acid. The fractions were evaporated under reduced pressure. The
water bath temperature was kept under 30.degree. C. and condenser
was cooled with water at 0.degree. C.
[0059] All four fractions were additionally purified to obtain pure
substances.
Purification of fraction one: The conditions for the purification
of the fraction one were the same as those at the first separation,
except the mobile phase A was 10 mM ammonium hydrogen carbonate.
The following gradient profile was used:
TABLE-US-00007 Time (min:sec) % B 0 10 0:17 10 0:22 36 7 36 7:10 90
8 90 8:10 10
[0060] One fraction was collected; the pH was adjusted to 8-9 with
1M potassium hydroxide. The fraction was evaporated under reduced
pressure in the same manner as after the first chromatographic
separation.
[0061] 170 mg of pure ATV-cycloIP
(4-[6-(4-Fluoro-phenyl)-6-hydroxy-1b-isopropyl-6a-phenyl-1a-phenylcarbamo-
yl-hexahydro-1,2-dioxa-5a-aza-cyclopropa[a]inden-3-yl]-3-(R)-hydroxy-butyr-
ic acid) compound was isolated from the concentrated fraction by
freeze drying. The chromatographic purity was 97.2%.
Purification of Fraction Two:
[0062] Chromatographic conditions for the purification of the
fraction two were the same as those for the first separation with
the exception that the mobile phase A solvent was 70% 10 mM
phosphate buffer pH7.0/25% acetonitrile/5% tetrahydrofuran
(vol/vol/vol) and the following gradient profile was used:
TABLE-US-00008 Time (min:sec) % B 0 0 1:55 0 2 15 11:30 15 14:20 35
14:45 85 17:30 85
[0063] One fraction was collected and evaporated under reduced
pressure. The concentrated fraction was loaded on a reverse phase
column, buffer salts were washed out with water and the ATV-cycloFP
compound was eluted from the column with 80% acetonitrile, 20%
water (vol/vol). 185 mg of pure ATV-cycloFP
(4-[1b-(4-Fluoro-phenyl)-6-hydroxy-6-isopropyl-1a-phenyl-6a-phenylcarbamo-
yl-hexahydro-1,2-dioxa-5a-aza-cyclopropa[a]inden-3-yl]-3-(R)-hydroxy-butyr-
ic acid) compound was isolated from the concentrated fraction by
freeze-drying. The chromatographic purity was 97.5%.
Purification of Fraction Three:
[0064] Chromatographic conditions for the purification of the
fraction three were the same as those for the first separation,
except that the mobile phase A solvent was 5 mM hydrochloric acid
and the following gradient profile was used:
TABLE-US-00009 Time (min:sec) % B 0 20 0:17 20 0:22 72 7 72 7:10
20
[0065] One fraction was collected and evaporated under reduced
pressure in the same manner as the fractions from the first
separation.
[0066] 205 mg of pure ATV-epoxy furan
(4-(4-Fluoro-phenyl)-2,4-dihydroxy-2-isopropyl-5-phenyl-3,6-dioxa-bicyclo-
[3.1.0]hexane-1-carboxylic acid phenylamide) compound was isolated
from the concentrated fraction by freeze-drying. The
chromatographic purity was 93.6%.
Purification of Fraction Four:
[0067] Chromatographic conditions for the purification of the
fraction four were the same as those for the purification of the
fraction three, except that the following gradient profile was
used:
TABLE-US-00010 Time (min:sec) % B 0 20 0:17 20 0:22 75 7 75 7:10
20
[0068] One fraction was collected and evaporated under reduced
pressure in the same manner as the fractions from the first
separation.
[0069] 50 mg of pure ATV-epoxy dion
(3-(4-Fluoro-benzoyl)-2-isobutyryl-3-phenyl-oxirane-2-carboxylic
acid phenylamide) compound was isolated from the concentrated
fraction by freeze-drying. The chromatographic purity was
96.2%.
Structural Elucidation of Compound ATV-cycloIP:
Mass Spectrometry:
Conditions:
[0070] High-resolution mass spectra were obtained using a
quadrupole time-of-flight mass spectrometer Micromass Q TOF Ultima
Global. Electrospray ionisation was used. The source temperature
was set to 100.degree. C., desolvation temperature to 200.degree.
C., cone gas 0 L/h and desolvation gas 200 L/h. W geometry of TOF
analyser was employed. The instrument was calibrated using
Na-formate clusters. The sample was dissolved in 50% solution of 5
mM ammonium acetate/acetonitrile (vol/vol), and infused to mass
spectrometer with constant flow of 10 .mu.l/min. The concentration
of the sample solution was 0.05 mg/ml.
[0071] Atorvastatin calcium salt was used as the internal standard
for high-resolution measurements. A concentration of 0.01 mg/ml of
the internal standard was added to a sample solution.
[0072] Protonated molecular ion 591.2507 m/z was observed. The
calculated elemental composition was
C.sub.33H.sub.36N.sub.2O.sub.7F. The deviation between the
calculated and the measured mass was 0.5 mDa. In comparison with
atorvastatin calcium the compound ATV-cycloIP shows two additional
oxygen atoms in its chemical structure.
Nuclear Magnetic Resonance Spectroscopy:
Conditions:
[0073] .sup.1H and .sup.13C measurements were performed on a 300
MHz Varian instrument INOVA or UNITY 300. The INOVA instrument was
equipped with a 5 mm inverse detect pulsed field gradient probe.
.sup.1H and .sup.13C spectra were obtained by the measurements at
the room temperature.
[0074] Samples were dissolved in methanol, chloroform or mixture of
methanol and chloroform 2:1.
[0075] Chemical shifts in ppm are assigned with reference to the
residual signal of the solvent.
TABLE-US-00011 Solvent: CD.sub.3OD (.sup.1H and .sup.13C
measurements) Structure: ##STR00014## .sup.1H NMR spectrum
6.90-7.40 ppm aromatic protons 14H, m 4.88 ppm CD.sub.3OD s 4.39
ppm 5,3 2H, m 3.31 ppm CD.sub.3OD m 3.25 ppm 7a 1H, m 3.17 ppm
(CH.sub.3).sub.2CH 1H, m 2.87 ppm 7b 1H, m 2.54 ppm 2 2H, d
1.80-2.10 ppm 6a, 4 3H, m 1.40 ppm 6b 1H, m 1.33 and 1.32 ppm
(CH.sub.3).sub.2CH 6H, 2 .times. d .sup.13C NMR spectrum: 180.5 ppm
1 165.5 ppm CO--NH 164.4 ppm C--F d 140.0-115.0 aromatic carbons
97.2 ppm 2' 94.9 ppm 5' 74.4 ppm 4' 70.6 ppm 3' 70.4 ppm 5 69.3 ppm
3 49.1 ppm CD.sub.3OD m 46.4 ppm 2 45.0 ppm 4 37.4 ppm 7 30.5 ppm 6
29.5 ppm (CH.sub.3).sub.2CH 19.5 and 18.3 ppm
(CH.sub.3).sub.2CH
Structural Elucidation of Compound ATV-Epoxy Furan:
Mass Spectrometry:
Conditions:
[0076] High-resolution mass spectra were obtained at the same
conditions as those for compound ATV-cycloIP.
[0077] Adducts of molecular ion with sodium 472.1536 m/z and
potassium 488.1270 are observed in mass spectrum. Calculated
elemental composition in first case was C.sub.26H.sub.24NO.sub.5FNa
(deviation between calculated and measured mass was 0.0 mDa), and
in the second case C.sub.26H.sub.24NO.sub.5FK (deviation between
calculated and measured mass was 0.5 mDa).
[0078] Protonated molecular ion was not observed due to fast
elimination of water from molecule (M+H--H.sub.2O).sup.+=432.1606
m/z. Proposed elemental composition was C.sub.26H.sub.23NO.sub.4F.
The deviation between calculated and measured mass was 0.5 mDa.
That fragment ion also makes adducts with sodium and potassium.
[0079] An adduct of two molecules of compound ATV-epoxy furan and
sodium is observed at 921.3131 m/z. Proposed elemental composition
was C.sub.52H.sub.48N.sub.2O.sub.10F.sub.2Na. Deviation between
calculated and measured mass was 4.4 mDa.
Nuclear Magnetic Resonance Spectroscopy:
Conditions:
[0080] .sup.1H and .sup.13C measurements were obtained in the same
manner as for compound ATV-cycloIP.
TABLE-US-00012 Solvent: CDCl.sub.3 (.sup.1H measurements) mixture
of CD.sub.3OD:CDCl.sub.3 = 2:1 (.sup.13C measurements) Structure:
##STR00015## .sup.1H NMR spectrum 7.57 ppm --NH 1H, br 6.90-7.50
ppm aromatic protons 14H, m 6.05 ppm --OH 1H, br 4.31 ppm --OH 1H,
s 2.38 ppm (CH.sub.3).sub.2CH 1H, m 1.22 and 1.21 ppm
(CH.sub.3).sub.2CH 6H, 2 .times. d .sup.13C NMR spectrum: 215.9 ppm
imp. 165.0 ppm CO--NH 164.0 ppm C--F d 140.0-115.0 ppm aromatic
carbons 107.1 ppm 2 104.1 ppm 5 77.0 ppm CDCl.sub.3 t 75.9 ppm 4
70.4 ppm 3 49.1 ppm CD.sub.3OD m 35.6 ppm (CH.sub.3).sub.2CH 17.9
and 17.0 ppm (CH.sub.3).sub.2CH
Structural Elucidation of Compound ATV-cycloFP:
Mass Spectrometry:
Conditions:
[0081] High-resolution mass spectra were obtained in the same
manner as for the compound ATV-cycloIP.
[0082] Protonated molecular ion 591.2507 m/z was observed. The
molecular ion is much less intensive in comparison to ATV-cycloIP.
The most intensive ion in MS spectrum is 573.2406 m/z and it is
formed with the elimination of water molecule.
[0083] The calculated elemental composition for 591.2507 m/z was
C.sub.33H.sub.36N.sub.2O.sub.7F. The deviation between calculated
and measured mass was 1.4 mDa. In comparison with atorvastatin the
compound has two additional oxygen atoms in the structure.
Nuclear Magnetic Resonance Spectroscopy:
Conditions:
[0084] .sup.1H and .sup.13C measurements were obtained in the same
manner as for compound. ATV-cycloIP.
TABLE-US-00013 Solvent: CD.sub.3OD (.sup.1H and .sup.13C
measurements) Structure: ##STR00016## .sup.1H NMR spectrum
6.90-7.40 ppm aromatic protons 14H, m 4.88 ppm CD.sub.3OD s 4.13
ppm 5 1H, m 3.74 ppm 3 1H, m 3.36 ppm 7a 1H, m 3.31 ppm CD.sub.3OD
m 2.97 ppm 7b 1H, m 2.50 ppm (CH.sub.3).sub.2CH 1H, m 2.37 and 2.27
ppm 2 2H, 2 .times. m 2.07 ppm 6a 1H, m 1.85 and 1.64 ppm 4 2H, 2
.times. m 1.26 and 1.28 ppm (CH.sub.3).sub.2CH 6H, 2 .times. d 1.25
ppm 6b 1H, m .sup.13C NMR spectrum 180.2 ppm 1 167.1 ppm CO--NH
164.2 ppm C--F d 140.0-115.0 ppm aromatic carbons 96.9 ppm 2' 95.1
ppm 5' 74.5 ppm 4' 70.4 ppm 5 70.0 ppm 3' 67.5 ppm 3 49.1 ppm
CD.sub.3OD m 45.2 ppm 2 44.7 ppm 4 38.3 ppm 7 36.6 ppm
(CH.sub.3).sub.2CH 31.0 ppm 6 19.7 and 19.0 ppm
(CH.sub.3).sub.2CH
Structural Elucidation of Compound ATV-Epoxy Dion:
Mass Spectrometry:
Conditions:
[0085] High-resolution mass spectra were obtained in the same
manner as for the compound ATV-cycloIP.
[0086] Protonated molecular ion 432.1612 m/z was observed. The
calculated elemental composition was C.sub.26H.sub.23NO.sub.4F. The
deviation between calculated and measured mass was 0.1 mDa.
[0087] MS/MS spectrum of protonated molecular ion is presented in
Table 5.
TABLE-US-00014 TABLE 5 MS/MS spectra of protonated molecular ion
432 m/z -five most intensive peaks Mass RA % Calc. Mass Error mDa
Error mDa Formula 226.0664 10.97 226.0668 -0.4 -1.8
C.sub.14H.sub.9NOF 241.0659 11.86 241.0665 -0.6 -2.4
C.sub.15H.sub.10O.sub.2F 269.0603 10.67 269.0614 -1.1 -4.1
C.sub.15H.sub.10O.sub.3F 304.1138 100.00 304.1126 -0.9 -2.9
C.sub.20H.sub.15NOF 344.1082 25.07 344.1087 -0.5 -1.4
C.sub.22H.sub.15NO.sub.2F 432.1611 16.08 432.1611 0 0
C.sub.26H.sub.23NO.sub.4F
Nuclear Magnetic Resonance Spectroscopy:
Conditions:
[0088] .sup.1H and .sup.13C measurements were obtained in the same
manner as for the compound ATV-cycloIP.
TABLE-US-00015 Solvent: mixture of CD.sub.3OD: CDCl.sub.3 = 2:1
(.sup.1H and .sup.13C measurements) Structure: ##STR00017## .sup.1H
NMR spectrum 8.20-6.80 ppm aromatic protons 14H, m 4.79 ppm
CD.sub.3OD s 3.31 ppm CD.sub.3OD m 3.20 ppm (CH.sub.3).sub.2CH 1H,
m 1.20 and 1.02 ppm (CH.sub.3).sub.2CH 6H, 2 .times. d .sup.13C NMR
spectrum: 206.0 ppm (CH.sub.3).sub.2CH--CO-- 191.4 ppm phenyl-CO--
167.3 ppm F--C d 162.8 ppm CO--NH 140.0-115.0 aromatic carbons 77.0
ppm CDCl.sub.3 t 74.6 and 72.0 ppm epoxy carbon atoms 49.0 ppm
CD.sub.3OD m 38.3 ppm (CH.sub.3).sub.2CH 18.5 and 17.7 ppm CH
EXAMPLE 2
Preparation and Isolation of ATV-cycloIP Compound
[0089] 2 liters of atorvastatin solution was prepared in 80%
acetonitrile and 20% water (vol/vol), containing 1 mg of
atorvastatin per ml. The solution was put in a shallow crystallizer
dish and was exposed to solar radiation for five hours. Immediately
after that, the solution was alkalized with 1M solution of
potassium hydroxide to pH 8 to 9 and evaporated under reduced
pressure until the first occurrence of turbidity. The water bath
temperature was kept under 30.degree. C., and the condenser was
cooled with water at 0.degree. C.
[0090] The solution was then clarified with addition of a minimal
amount of acetonitrile.
[0091] Preparative chromatography and structure elucidation was
carried out in the same manner as in the example 1.
[0092] 210 mg of pure ATV-cycloIP compound was isolated from the
concentrated fraction by freeze drying. The chromatographic purity
was 96.6%.
EXAMPLE 3
Preparation and Isolation of ATV-Epoxy Furan and ATV-Epoxy Dion
Compounds
[0093] 1 liter of atorvastatin solution was prepared in 80%
acetonitrile/20% water (vol/vol)-containing 1 mg of atorvastatin
per ml. The solution was put in a shallow crystallizing dish and
was exposed to solar radiation for five hours. Immediately after
the solution was acidified with 0.5M phosphoric acid to pH 3.0. The
mixture was left at a room temperature for two hours and evaporated
under reduced pressure to cca 1/3 of the original mixture
volume.
[0094] Preparative chromatography and structure elucidations were
carried out in the same manner as in the example 1.
[0095] 120 mg of ATV-epoxy furan compound was isolated from the
concentrated fraction by freeze-drying. The chromatographic purity
was 92.6%.
[0096] 21 mg of ATV-epoxy dion compound was isolated from the
concentrated fraction by freeze-drying. The chromatographic purity
was 95.1%.
EXAMPLE 4
Preparation and Isolation of ATV-cycloFP Compound
[0097] 800 ml of solution of atorvastatin was prepared in
acetonitrile; containing 10 mg of atorvastatin per ml. 4 ml of 12M
sodium hydroxide and 40 ml of 30% hydrogen peroxide was added. The
solution was stirred at 55.degree. C. for five hours. The reaction
mixture was allowed to cool and decanted. The supernatant was
evaporated under reduced pressure to cca 50 ml. The remaining water
was discarded and the solid residue was washed with fresh water.
The solid residue was then dissolved in acetonitrile.
[0098] Preparative chromatography and structure elucidation was
carried out in the same manner as in example 1
[0099] 230 mg of pure ATV-cycloIP compound was isolated from the
concentrated fraction by freeze-drying. The chromatographic purity
was 98.3%.
EXAMPLE 5
[0100] Atorvastatin calcium can be prepared by any way described in
the literature. The only requirement during the whole process of
the preparation of atorvastatin calcium was that the inert
atmosphere must be maintained. The content of the each oxidative
degradation product ATV-cycloIP, ATV-epoxy furan, ATV-cycloFP and
ATV-epoxy dion compounds using HPLC method and detection at 250 nm
in so prepared atorvastatin calcium were below 0.04%.
EXAMPLE 6
[0101] The atorvastatin calcium obtained by procedure described in
example 1 was stored under nitrogen atmosphere at the room
temperature for 2 years. The content of each oxidative degradation
product ATV-cycloIP, ATV-epoxy furan, ATV-cycloFP and ATV-epoxy
dion compounds using HPLC method and detection at 250 nm in so
prepared atorvastatin calcium were below 0.1%.
EXAMPLE 7
[0102] The atorvastatin calcium obtained by procedure described in
example 1 was stored under air at room temperature for 2 years. The
content of the oxidative degradation product ATV-cycloIP, ATV-epoxy
furan, ATV-cycloFP and ATV-epoxy dion compounds using HPLC method
and detection at 250 nm in so prepared atorvastatin calcium were
0.856%, 0.636%, 0.905% and 0.741%, respectively.
EXAMPLE 8
[0103] Tablets were prepared using atorvastatin calcium obtained by
procedure described in example 1 and at least one pharmaceutically
acceptable excipient. In the so prepared tablets the content of the
oxidative degradation product ATV-cycloIP, ATV-epoxy furan,
ATV-cycloFP and ATV-epoxy dion compounds using HPLC method and
detection at 250 nm were 0.11%, 0.07%, 0.07% and 0.08%,
respectively.
[0104] Tablets were packed in alu/alu blisters in the nitrogen
atmosphere. Blisters were stored at the room temperature for two
years. The content of oxidative degradation product ATV-cycloIP,
ATV-epoxy furan, ATV-cycloFP and ATV-epoxy dion compounds using
HPLC method and detection at 250 nm were 0.18%, 0.08%, 0.17% and
0.09%, respectively.
EXAMPLE 9
[0105] Tablets as described in Example 8 were packed in alu/alu
blisters in air. In the so prepared tablets the content of the
oxidative degradation product ATV-cycloIP, ATV-epoxy furan,
ATV-cycloFP and ATV-epoxy dion compounds, using HPLC method and
detection at 250 nm were 0.13%, 0.09%, 0.08% and 0.08%,
respectively.
[0106] Blisters were stored at the room temperature for two years.
The content of oxidative degradation product ATV-cycloIP, ATV-epoxy
furan, ATV-cycloFP and ATV-epoxy dion compounds using HPLC method
and detection at 250 nm in so prepared atorvastatin calcium were
1.75%, 0.61%, 1.23% and 0.65% respectively.
* * * * *