Note: Descriptions are shown in the official language in which they were submitted.
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FORMULATIONS OF DEOXYCHOLIC ACID AND SALTS THEREOF
FIELD OF THE INVENTION
[0001] This invention relates to aqueous pharmaceutical formulations
containing very low
concentrations of a salt of deoxycholic acid ("DCA"), wherein the formulation
is maintained
at a pH such that precipitation of DCA is substantially inhibited. In a
preferred embodiment,
the pharmaceutical composition is buffered to maintain a physiologically
acceptable pH such
that the composition is suitable for injection.
BACKGROUND
[0002] Recently published literature reports that the aqueous solutions of DCA
has fat
removing properties when injected into fatty deposits in vivo (See, WO
2005/117900 and WO
2005/112942, US2005/0261258; US2005/0267080; US2006/127468; and
U52006/0154906).
DCA injected into fat tissue degrades fat cells via a cytolytic mechanism to
provide the
desired aesthetic results.
[0003] Notwithstanding the benefits of aqueous formulations of DCA, it has
been found
that at low concentrations of DCA (i.e., less than or about 2% w/v;) in
aqueous solutions
which optionally contain benzyl alcohol, forms a precipitate after storage
over a period of
time. Surprisingly, it has been found that the lower the concentration DCA,
the higher is the
rate of precipitation notwithstanding any significant change in the pH of the
solution. This
precipitation at very low concentrations is a problem for commercialization as
a precipitate is
counter-indicated for subcutaneous injections of DCA.
[0004] In each treatment regimen, the current clinical trials of aqueous
formulations of
DCA employ multiple injections of small amounts of the aqueous formulation
into different
sites defining the fat deposit to be treated.
[0005] As is apparent, aqueous formulations of DCA used in such treatments
overlap with
the problems arising from precipitation of the DCA. That is to say that an
initially clear
aqueous solution of DCA when stored for a period of time, will form a
precipitate at
commercially relevant concentrations of DCA notwithstanding the fact that the
pH of these
solutions are between about 7.50 and about 8.0 which are substantially above
the pKa of
deoxycholic acid.
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[0006] Thus, there is a need for stabilizing the low concentration aqueous
solutions of
deoxycholic acid or a salt thereof against precipitation during a shelf life
of at least 2 months.
SUMMARY OF THE INVENTION
[0007] It has been surprisingly found that aqueous solutions of a salt of
deoxycholic acid at
concentrations of from about 0.4% w/v to less than about 2% w/v can be
stabilized by
adjusting the pH of the solution to above about 8 and preferably from a
physiologically
acceptable pH from about 8.1 to about 8.5. The pH is preferably maintained
within this range
by using buffers.
[0008] Accordingly, in one of its composition aspects, this invention is
directed to aqueous
formulations consisting essentially of a salt of deoxycholic acid at a
concentration of from
about 0.4% w/v to less than about 2% w/v and optionally a preservative
effective amount of
benzyl alcohol which formulations are stabilized against precipitation by
adjusting the pH of
the initially formed clear solution to a pH of from about 8.1 to about 8.5. In
another
embodiment, this invention is directed to an aqueous formulation consisting
essentially of a
salt of deoxycholic acid at a concentration of from about 0.5% w/v to about 1%
w/v and
optionally a preservative effective amount of benzyl alcohol which
formulations are
stabilized against precipitation by adjusting the pH of the initially formed
clear solution to a
pH of from about 8.1 to about 8.5.
[0009] In another embodiment, this invention is directed to an aqueous
formulation
consisting essentially of:
a sterile aqueous solution buffered to a pH of about 8.3;
about 0.5% w/v or about 1% w/v of a salt of deoxycholic acid;
optionally a preservative effective amount of benzyl alcohol; and
about 1% w/v of sodium chloride,
wherein the composition is stable against precipitation.
[0010] Also disclosed herein is a method for stabilizing an aqueous
composition of a salt of
deoxycholic acid against precipitation during storage wherein the
concentration of the
deoxycholic acid salt is an amount effective to lyse fat cells provided that
the amount of the
deoxycholic acid salt ranges from about 0.4% w/v to less than 2% w/v, which
method
comprises:
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forming an aqueous solution of a deoxycholic acid salt at an initial pH above
its pKa;
adjusting the pH of the aqueous solution to a pH of from about 8.1 to about
8.5; and
optionally including a sufficient amount of buffer to maintain the pH at from
about 8.1 to about
8.5.
[0010A] Various embodiments of the claimed invention relate to an injectable
aqueous solution
comprising: from about 0.4% w/v to less than 2% w/v of a salt of deoxycholic
acid; and a pH-
adjusting agent, wherein said solution is at a pH of about 8.1 to about 8.5.
[0010B] Various embodiments of the claimed invention relate to an injectable
aqueous solution
consisting essentially of: water; from about 0.4% w/v to less than 2% w/v of a
salt of deoxycholic
acid; sodium chloride; a pH-adjusting agent; and an optional preservative,
wherein said solution is
at a pH of about 8.1 to about 8.5.
[0010C] Various embodiments of the claimed invention relate to an injectable
aqueous solution
comprising: about 0.5% w/v of sodium deoxycholate; about 0.9% w/v benzyl
alcohol; and about
1% w/v of sodium chloride, wherein the solution is buffered to a pH of about
8.3.
[0010D] Various embodiments of the claimed invention relate to an injectable
aqueous solution
comprising: about 1% w/v of sodium deoxycholate; about 0.9% w/v benzyl
alcohol; and about 1%
w/v of sodium chloride, wherein the solution is buffered to a pH of about 8.3.
[0010E] Various embodiments of the claimed invention relate to a method for
stabilizing an aqueous
solution of a salt of deoxycholic acid against precipitation during a shelf
life, comprising forming
an aqueous solution of a deoxycholic acid salt at an initial pH above the pKa
of the deoxycholic
acid salt and adjusting the pH of the aqueous solution to be 8 or above,
wherein the aqueous
solution comprises from about 0.4% w/v to less than 2% w/v deoxycholic acid
salt and a
pharmaceutically acceptable excipient.
[0010F] Various embodiments of the claimed invention relate to a method for
stabilizing an aqueous
solution of a salt of deoxycholic acid against precipitation during a shelf
life, comprising forming
an aqueous solution of a deoxycholic acid salt at an initial pH above the pKa
of the deoxycholic
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acid salt and adjusting the pH of the aqueous solution to be about 8, wherein
the aqueous solution
comprises from about 0.4% w/v to less than 2% w/v deoxycholic acid salt and a
pharmaceutically
acceptable excipient.
[0010G1 Various embodiments of the claimed invention relate to a method for
stabilizing an aqueous
solution of a salt of deoxycholic acid against precipitation during a shelf
life, comprising forming
an aqueous solution of a deoxycholic acid salt at an initial pH above the pKa
of the deoxycholic
acid salt and adjusting the pH of the aqueous solution to be 8 or above,
wherein the aqueous
solution comprises: from about 0.05% w/v to less than 2% w/v deoxycholic acid
salt; and a
pharmaceutically acceptable excipient, a pharmaceuticaly acceptable carrier,
or both.
[001011] Various embodiments of the claimed invention relate to a method for
stabilizing an aqueous
solution of a salt of deoxycholic acid against precipitation during a shelf
life, comprising forming
an aqueous solution of a deoxycholic acid salt at an initial pH above the pKa
of the deoxycholic
acid salt and adjusting the pH of the aqueous solution to be about 8, wherein
the aqueous solution
comprises: from about 0.05% w/v to less than 2% w/v deoxycholic acid salt; and
a
pharmaceutically acceptable excipient, a pharmaceuticaly acceptable carrier,
or both.
[0011] Also disclosed herein is a method to lyse a fat cell comprising
administering to said cell a
composition according to this invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Fig. 1 illustrates (67 X enhancement) that an aqueous composition of
water and 0.9 % w/v
benzyl alcohol and no sodium deoxycholate contains only a trace of
precipitate, which is presumed to be
bioprene tubing particles.
[0013] Fig. 2 illustrates (67 X enhancement) that an aqueous composition of
water, 0.5 % w/v sodium
deoxycholate and 0.9 % w/v benzyl alcohol contains significant amounts of
precipitate which is
presumed to be deoxycholate crystals.
[0014] Fig. 3 illustrates (67 X enhancement) that an aqueous composition of
water, 1 % w/v sodium
deoxycholate and 0.9 % w/v benzyl alcohol contains significant amounts of
precipitate, albeit less than
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that of 0.5 % w/v sodium deoxycholate. As before, the precipitate is presumed
to be deoxycholate
crystals.
[0015] Fig. 4 illustrates (67 X enhancement) that an aqueous composition of
water, 2 % w/v sodium
deoxycholate and 0.9 % w/v benzyl alcohol contains significant amounts of
precipitate but substantially
less than that observed in Figs. 2 and 3.
DETAILED DESCRIPTION
[0016] As used herein, certain terms have the following defined meanings.
[0017] All numerical designations, e.g., pH, temperature, time, concentration,
and molecular weight,
including ranges, are approximations which are varied ( +) or (¨) by
increments of 0.1. It is to be
understood, although not always explicitly stated that all numerical
designations are preceded by the
term "about". The term "about" also includes the exact value "X" in addition
to minor increments of
"X" such as "X + 0.1" or "X ¨ 0.1." It also is to be understood, although not
always explicitly stated,
that the reagents described herein are merely exemplary and that equivalents
of such are known in the
art.
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[0018] As used herein, the term "comprising" is intended to mean that the
compositions and
methods include the recited elements, but do not exclude others.
[0019] "Consisting essentially of" when used to define compositions and
methods, shall
mean excluding any active ingredients. An "active ingredient" is a substance
intended to
furnish pharmacological activity or other direct effect in the diagnosis,
cure, mitigation,
treatment, or prevention of disease or to affect the structure or any function
of the human
body. Thus, for example, a composition consisting essentially of the elements
as defined
herein would not exclude trace contaminants from the isolation and
purification method and
pharmaceutically acceptable carriers, such as phosphate buffered saline,
preservatives, and
the like but would exclude enzymes such as phosphatases, and proteins. Non-
limiting
examples of such proteins are heparin, albumin, and the like
[0020] "Consisting of" shall mean excluding more than trace elements of other
ingredients
and substantial method steps for administering the compositions of this
invention.
Embodiments defined by each of these transition terms are within the scope of
this invention.
[0021] As used herein, the term "salt of deoxycholic acid" or "a salt thereof"
refers to
pharmaceutically acceptable salts of (4R)-4-((3R,5R,10S,12S,13R,17R)-3,12-
dihydroxy-
10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate
having an
alkali metal or an ammonium ion as the cation. Preferred are alkali metal
salts, with sodium
salts being more preferred.
0- Na
0
:
pill
.Ow
HO
H
[0022] Sodium deoxycholate or sodium (4R)-4-((3R,5R,10S,12S,13R,17R)-3,12-
dihydroxy-
10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate can
be
prepared according to the methods disclosed in PCT/US2010/061150 titled
"Methods for the
Purification of Deoxycholic Acid," filed on December 17, 2010.
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[0023] As used herein, the term "aqueous pharmaceutical formulation" refers to
a
composition of a deoxycholic acid or a salt thereof in water suitable for
administration to a
patient preferably via subcutaneous injection from a syringe.
[0024] As used herein, the term "buffer" refers to an aqueous solution
comprising a mixture
of a weak acid and its conjugate base or a weak base and its conjugate acid. A
buffer has the
property that the pH of the solution changes very little when a small amount
of acid or base is
added to it. Buffer solutions are used as a means of keeping pH at a nearly
constant value in
a wide variety of chemical applications. Examples of suitable buffers include
phosphate
buffers and those known in the literature (see, for example, Troy, D.B., ed.
(2005)
Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams
& Wilkins).
[0025] As used herein, the term "base" refers to various typically water-
soluble
compounds, molecules or ions that in solution have a pH greater than 7. Such
compounds,
molecules or ions are able to take up a proton from an acid or are able to
give up an unshared
pair of electrons to an acid. Examples of suitable bases include metal
carbonates and
bicarbonates, for example sodium carbonate, calcium carbonate, magnesium
carbonate, zinc
carbonate, sodium bicarbonate and the like; and metal hydroxides, for example
sodium
hydroxide, potassium hydroxide, and the like, such as those known in the
literature (see, for
example, Troy, D.B., ed. (2005) Remington: The Science and Practice of
Pharmacy, 21st ed.,
Lippincott Williams & Wilkins).
[0026] As used herein, the term "metal carbonates" refers to the metal salt of
C032-. For
example, sodium carbonate, calcium carbonate, magnesium carbonate, zinc
carbonate, and
the like.
[0027] As used herein, the term "metal bicarbonates" refers to the metal salt
of HCO3. For
example, sodium bicarbonate, and the like.
[0028] As used herein, the term "metal hydroxides" refers to the metal salt of
-OH. For
example, sodium hydroxide, potassium hydroxide, and the like.
[0029] As used herein, the terms "sterile water" or "water for injection"
refer to a sterile,
nonpyrogenic preparation of water for injection which contains no
bacteriostat, antimicrobial
agent or added buffer. In general, the osmolar concentration of additives
totals at least 112
mOsm/liter (two-fifths of the normal osmolarity of the extracellular fluid -
280 mOsm/liter).
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0 OH
[0030] As used herein, the term "benzyl alcohol" refers to the compound .
[0031] As used herein, the term "precipitation" refers to the formation of a
solid in a
solution and is readily differentiated from gel formation.
[0032] As used herein, the term "solution" refers to a substantially
homogeneous mixture
comprising two or more substances dissolved in a solvent.
[0033] As used herein, the terms "substantially inhibit precipitation" and
"inhibits
precipitation" means to inhibit most or all visible precipitation so as to
maintain homogeneity
for a period of time ranging from at least 1 month to at least 1 year.
[0034] As used herein, the term "relative standard deviation for homogeneity"
or "HE"
refers to the value obtained by dividing the standard deviation of the
homogeneity by the
absolute value of the mean. An HE less than 10 indicates very good
homogeneity.
Formulations
[0035] Knowledge about the chemical and physical stability of a drug
composition in the
desired media for delivery is valuable. In the longer term, the stability of
the composition
will dictate the shelf life of the marketed product. It is preferable that the
active ingredient in
a pharmaceutical composition be at the required concentration when
administered to a
patient.
[0036] In the discussion below, sodium deoxycholate is recited for
illustrative purposes
only and it is understood that other pharmaceutically acceptable salts of
deoxycholic acid can
be used interchangeably with the sodium salt.
[0037] Current clinical methods for the administration of a sodium
deoxycholate to a
patient to dissolve fat include the administration via subcutaneous injections
of a low
concentration (i.e., <2% w/v) of an aqueous solution of the salt of
deoxycholic acid where the
amount of the salt of deoxycholic acid is sufficient to lyse fat cells (about
0.4% w/v and
higher). At such concentrations, as it has been shown that the low
concentration is beneficial
for the effective and safe removal of fat deposits in the body. However, it
has been observed
that a precipitate forms at such relatively low concentrations of sodium
deoxycholate in
aqueous media. This precipitation results in a limited shelf life of aqueous
solutions of
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sodium deoxycholate, even at cold temperatures (3-5 C). In one embodiment,
the sodium
salt can be replaced by another alkali metal salt.
[0038] This instability of aqueous solutions of sodium deoxycholate can be
circumvented
by the preparation of an aqueous solution of sodium deoxycholate at a
concentration of about
5% to about 16% w/v, and having the practitioner dilute the pharmaceutical
composition of
the sodium deoxycholate solution just prior to use. Whereas this dilution
method is effective
to allow for both storage stability and effective patient dosing, it is not
ideal as a method for
routine use especially if a sterile injectable solution of no more than about
2 mL is required.
Moreover, current clinical plans include up to 50 injections per treatment
session.
[0039] It has been found that aqueous formulations of sodium deoxycholate at
concentrations ranging from about 0.4% w/v to less than about 2% w/v can be
stabilized by
adjusting the pH of the solution. This invention is directed to an aqueous
formulation
consisting essentially of a salt of deoxycholic acid at a concentration
ranging from about
0.4% w/v to less than about 2% w/v and optionally a pharmaceutically
acceptable excipient
such as a preservative effective amount of benzyl alcohol and/or a pH
adjusting buffer,
wherein said formulation is maintained at a pH of about 8.1 to about 8.5.
[0040] In another embodiment, the aqueous formulation is lyophilized to
provide for a
stable composition which is ready to be reconstituted by addition of the
appropriate amount
of water. In this embodiment, this invention comprises lyophilized
compositions as described
above which optionally further contain a lyophilization aid.
[0041] In one embodiment, the aqueous formulation contains about 0.5% w/v of a
salt of
deoxycholic acid. In another embodiment, the aqueous formulation contains
about 1% w/v of
a salt of deoxycholic acid.
[0042] In a further embodiment, the water employed in the aqueous formulation
is sterile
water. In still a further embodiment, the preservative effective amount of
benzyl alcohol is
about 0.9% w/v benzyl alcohol and the pH of the formulation is about 8.3. In
one
embodiment, said salt is an alkali metal salt. In another embodiment, said
salt is a sodium
salt.
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[0043] In one embodiment, the pharmaceutical formulations disclosed herein are
suitable
for injection into a human. The method of injection can be any type of
injection, such as
subcutaneous injection, as well as other forms of injection.
[0044] In one preferred aspect of this invention, the precipitation of the
salt of deoxycholic
acid in the aqueous formulation is inhibited for a period of at least about
six months. In
another aspect, the precipitation is inhibited for a period of at least about
one year. In yet
another aspect, the precipitation is inhibited for a period of at least about
two years.
[0045] It is contemplated that when stored at various temperatures, for
example at ambient
or cold temperatures, the formulation can have an increased shelf life. In
certain
embodiments, the composition is stored at a temperature of from about 17 C to
about 27 C.
In some embodiments, the temperature of the formulation is increased to a
temperature of
about 25 C to about 37 C. In other embodiments, the formulation is stored at
a temperature
of from about 2 C to about 8 C.
[0046] In certain embodiments, the pH of the formulation ranges from about 8.1
to about
8.5. In one embodiment, the pH of the composition is about 8.1, or
alternatively, about 8.2,
or alternatively, about 8.3, or alternatively, about 8.4, or alternatively,
about 8.5. In a
preferred embodiment, the pH of the formulation is about 8.3.
[0047] In one embodiment, the pH is established by the use of a base. It is
contemplated
that any base can be used to increase the pH of the composition provided that
it does not react
with the sodium deoxycholate and will not cause harm to the patient. In some
embodiments,
the base is selected from the group consisting of metal carbonates, metal
bicarbonates, metal
hydroxides, or a mixture thereof Examples of bases include, but are not
limited to, a base
selected from the group consisting of sodium carbonate, calcium carbonate,
magnesium
carbonate, zinc carbonate, sodium bicarbonate, sodium hydroxide and potassium
hydroxide
or a mixture thereof In one embodiment, the base is sodium hydroxide.
[0048] In certain cases, the pH of the composition may be maintained at the
desired pH
during storage with the use of a buffer. Various buffers are known in the art
and it is
contemplated that any buffer having buffering capacity at the desired pH can
be used in the
formulations disclosed herein. In one embodiment, the buffer is a phosphate
buffer. The
amount of phosphate in the composition can be determined to provide a desired
pH and salt
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concentration. In one embodiment, the composition comprises about 10 mM
phosphate
buffer. In a preferred embodiment, the composition comprises about 10 mM
dibasic sodium
phosphate buffer.
[0049] In some embodiments, the composition comprises at least one excipient
to aid in
achieving a composition with desired properties, such as increased solubility,
preservability
or to provide an isotonic solution. Such excipients are known in the art. In
one embodiment,
the composition comprises about 1% w/v sodium chloride. In another embodiment,
the
composition comprises about 0.9% w/v benzyl alcohol. In some embodiments, the
composition comprises about 0.9% w/v benzyl alcohol and about 1% w/v sodium
chloride.
[0050] In some embodiments, the pH of the composition is established by use of
a base and
optionally maintained by use of a buffer.
[0051] In a preferred embodiment, this invention provides a stabilized
composition
comprising:
a phosphate buffer of a pH of about 8.3;
about 0.5% w/v or about 1% w/v of sodium deoxycholate;
a preservative effective amount of benzyl alcohol; and
about 1% w/v of sodium chloride,
wherein the composition is stabilized against precipitation.
[0052] In a further embodiment, the phosphate buffer is 10 mM dibasic sodium
phosphate
buffer.
[0053] In one embodiment, the preservative effective amount of benzyl alcohol
is about
0.9% w/v.
[0054] The formulations disclosed herein comprise from about 0.4% w/v to less
than about
2% w/v of a salt of deoxycholic acid in water maintained at a pH sufficient to
substantially
inhibit precipitation of the salt of deoxycholic acid. The amount of
precipitation or
homogeneity of the composition can be measured using various methods. For
example, it can
be measured quantitatively using light scattering by illuminating the
composition with a
spectrophotometer. Or alternatively, the homogeneity can be measured
qualitatively by
observing the visual clarity of the solution with the eye. In some
embodiments, the
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composition has a relative standard deviation for homogeneity of less than
about 5%.
Alternatively, the composition has a relative standard deviation for
homogeneity of less than
about 4%, or alternatively, about 3%, or alternatively, about 2%, or
alternatively, about 1%.
[0055] In another embodiment, this invention is directed to a composition
consisting
essentially of:
a sterile aqueous solution buffered to a pH of about 8.3;
about 0.5% w/v or 1% w/v of sodium deoxycholate;
about 0.9% w/v benzyl alcohol; and
about 1% w/v of sodium chloride,
wherein the composition is stable against precipitation.
[0056] In another embodiment, this invention is directed to a composition
consisting of:
an aqueous solution buffered to a pH of about 8.3;
about 0.5% w/v or about 1% w/v of sodium deoxycholate;
about 0.9% w/v benzyl alcohol; and
about 1% w/v of sodium chloride,
wherein the composition is stable against precipitation.
[0057] In some embodiments, the solutions herein do not include lipids,
phospholipids, or
phosphatidylcholine. In some embodiments, the solutions herein include up to
5% w/w, w/v,
or v/v lipids, specifically phospholipids, or more specifically
phosphatidylcholine. Preferably,
the amount of lipids used is less than that of sodium deoxycholate or another
salt of
deoxycholic acid.
[0058] In some embodiments, the aqueous pharmaceutical composition of the
invention can
further comprise a second therapeutic agent selected from the group consisting
of: anti-
microbial agents, vasoconstrictors, anti-thrombotic agents, anti-coagulation
agents, suds-
depressants, anti-inflammatory agents, analgesics, dispersion agents, anti-
dispersion agents,
penetration enhancers, steroids, tranquilizers, muscle relaxants, and anti-
diarrhea agents. In
some embodiments, a solution is in a container that contains up to 500 mL of
solution. Such
container can be a syringe or syringe-loadable container.
[0059] In some embodiments, the formulations further comprise a molecule known
to cause
fat to die by an orthogonal mechanism. Such molecules include neuropeptide Y
(NPY)
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antagonists including, but not limited to, NPY receptor antagonists, such as
BIBP-3226
(Amgen), BIBO-3304 (Boehringer Ingleheim), BMS-192548 and AR-H040922 (Bristol-
Myers Squibb), LY-357897 (Eli Lilly), 1229U91 and GW438014S (GlaxoSmithKline),
JNJ-
5207787 (Johnson & Johnson), Lu-AA-44608 (Lundbeck), MK-0557 (Merck NPY), NGD-
95-1 (Neurgogen), NLX-E201 (Neurologix), CGP-71683 (Novartis), PD-160170
(Pfizer),
SR-120819A, BIIE0246, and S.A.0204 (Sanofi Aventis), S-2367 (Shiongli),
dihydropyridine
and dihydropyridine derivatives that are NPY receptor antagonists, bicyclic
compounds that
are NPY receptor antagonists, carbazole NPY receptor antagonists, and
tricyclic compounds
that are NPY receptor antagonists (See, e.g., WO 2006/133160 and U.S.
6,313,128). Also
contemplated are fat selective pro-apoptotic peptides such as the CKGGRAKDC
peptide that
homes to white fat vasculature (See, Kolonin M.G. et al., Nat. Med., 2004,
10(6): 625-32).
[0060] Another aspect of the invention relates to mixing adipo-ablative bile
acids, such as,
deoxycholic acid (DCA) with agents that kill fat cells. In one aspect, this
invention
contemplates a means to enhance the aesthetic effects of deoxycholate
injections by mixing
into the deoxycholate injectate a molecule that causes fat to die by an
orthogonal mechanism.
Examples of such candidate molecules include, but are not limited to,
neuropeptide Y (NPY)
antagonists and fat selective pro-apoptotic peptides. Since fat cell killing
may be required to
mediate the desired effects, the effects of an agent with fat killing ability
can be enhanced via
the addition of a molecule with potent fat cell killing effects. Additionally,
molecules that
require access to the vasculature to kill (such as certain pro-apoptotic
peptides that bind to
proteins expressed on the luminal side of capillaries) can gain access to
these proteins
because deoxycholate may cause vascular leakage. Thus, such agents can be
synergistic with
deoxycholate potentially creating a more potent means to mediate body
contouring in fewer
therapeutic sessions.
[0061] Examples of NPY antagonists include, but are not limited to, NPY
receptor
antagonists, such as BIBP-3226 (Amgen), BIBO-3304 (Boehringer Ingleheim), BMS-
192548
and AR-H040922 (Bristol-Myers Squibb), LY-357897 (Eli Lilly), 1229U91 and
GW4380145
(GlaxoSmithKline), JNJ-5207787 (Johnson & Johnson), Lu-AA-44608 (Lundbeck), MK-
0557 (Merck NPY), NGD-95-1 (Neurgogen), NLX-E201 (Neurologix), CGP-71683
(Novartis), PD-160170 (Pfizer), SR-120819A, BIIE0246, and S.A.0204 (Sanofi
Aventis), S-
2367 (Shiongli), dihydropyridine and dihydropyridine derivatives that are NPY
receptor
antagonists, bicyclic compounds that are NPY receptor antagonists, carbazole
NPY receptor
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antagonists, and tricyclic compounds that are NPY receptor antagonists. See,
e.g., WO
2006/133160 and U.S. 6,313,128.
[0062] Exemplary fat selective pro-apoptotic peptides includes, but is not
limited to,
CKGGRAKDC peptide that homes to white fat vasculature. See, Kolonin M.G. et
al., Nat.
Med. June 10(6):625-32 (2004).
[0063] Sodium deoxycholate or sodium (4R)-4-((3R,5R,10S,12S,13R,17R)-3,12-
dihydroxy-
10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate can
be
prepared according to the methods disclosed in PCT/US2010/061150 titled
"Methods for the
Purification of Deoxycholic Acid," filed on December 17, 2010. Other salts of
deoxycholic
acid can be prepared likewise by the skilled artisan.
Methods
[0064] Disclosed herein are methods for stabilizing an aqueous formulation of
deoxycholic
acid salt against precipitation during storage wherein the concentration of or
a salt thereof is
an amount effective to lyse fat cells provided that the amount of salt ranges
from about 0.4%
w/v to about less than 2% w/v, which method comprises:
forming an aqueous solution of the salt of deoxycholic acid at an initial pH
above its
pKa;
adjusting the pH of the aqueous solution to a pH of from about 8.1 to about
8.5; and
optionally including a sufficient amount of buffer to maintain the pH at from
about
8.1 to about 8.5.
[0065] In one aspect of this invention, methods disclosed herein substantially
stabilize the
formulation of deoxycholic acid salt against precipitation over a period of
time preferably for
at least about six months. In another aspect, the methods stabilize the
formulation of
deoxycholic acid salt against precipitation for a period of at least about one
year. In yet
another aspect, the methods stabilize the formulation of deoxycholic acid salt
against
precipitation for a period of at least about two years.
[0066] It has been found that the pH of the solution can inhibit the
precipitation of
deoxycholic acid or a salt thereof at concentrations of from about 0.4% w/v to
less than about
2% w/v in water to allow deoxycholic acid or a salt thereof, to be maintained
in solution. In
one embodiment, the pH is established by the use of a base. It is contemplated
that any base
12
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can be used to increase the pH of the composition provided that it does not
react with
deoxycholic acid or a salt thereof In some embodiments, the base is selected
from the group
consisting of metal carbonates, metal bicarbonates, and metal hydroxides, or a
mixture
thereof Examples of bases include, but are not limited to, a base selected
from the group
consisting of sodium carbonate, calcium carbonate, magnesium carbonate, zinc
carbonate,
sodium bicarbonate, sodium hydroxide and potassium hydroxide or a mixture
thereof In one
embodiment, the base is sodium hydroxide.
[0067] In certain embodiments, the pH ranges from about 8.1 to about 8.5. In
one
embodiment, the pH of the composition is about 8.1, or alternatively, about
8.2, or
alternatively, about 8.3, or alternatively, about 8.4, or alternatively, about
8.5. In a preferred
embodiment, the pH of the aqueous solution is about 8.3.
[0068] In certain cases, the pH of the composition may need to be maintained
with the use
of a buffer. Various buffers are know in the art and it is contemplated that
any buffer having
buffering capacity at the desired pH can be used in the formulations disclosed
herein. In one
embodiment, the buffer is a phosphate buffer. The amount of phosphate required
to provide a
desired pH and salt concentration can be calculated using methods well known
in the art. In
one embodiment, the composition comprises about 10 mM phosphate buffer. In
another
embodiment, the phosphate buffer is 10 mM dibasic sodium phosphate buffer.
[0069] In certain cases, the pH is established by use of a base and optionally
maintained by
use of a buffer.
[0070] In one embodiment, the methods disclosed herein provide formulations
which are
suitable for injection into a human. The method of injection can be any type
of injection,
such as subcutaneous injection, as well as other forms of injection.
Therefore, in some
embodiments, the aqueous solution comprises sterile water or water for
injection (WFI).
[0071] In one aspect, it may be that one or more excipients are used to
maintain the
solubility, or increase the preservability of deoxycholic acid salt present in
the formulation.
In one embodiment, the method comprises adding about 1% w/v benzyl alcohol. In
some
embodiments, the formulation also comprises at least one excipient to aid in
achieving an
isotonic solution. Such excipients are known in the art. In one embodiment,
the method
comprises adding about 1% w/v sodium chloride. In some embodiments, the method
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comprises adding both 1% w/v benzyl alcohol and 1% w/v sodium chloride. In
some
embodiments, the method comprises adding both 0.9% w/v benzyl alcohol and 0.9%
w/v
sodium chloride. Using the methods disclosed herein, an aqueous solution
comprising less
than about 2% w/v of deoxycholic acid salt is maintained at a pH sufficient to
substantially
inhibit precipitation of deoxycholic acid salt. The amount of precipitation or
homogeneity of
the composition can be measured using various methods. For example, it can be
measured
quantitatively by measuring the light scattering via illumination by a
spectrophotometer. Or
alternatively, the homogeneity can be measured qualitatively by simply
observing the visual
clarity of the solution with the eye. In some embodiments, the method provides
a
pharmaceutical composition having a relative standard deviation for
homogeneity of less than
about 5%. Alternatively, the relative standard deviation for homogeneity of
less than about
4%, or alternatively, about 3%, or alternatively, about 2%, or alternatively,
about 1%.
[0072] The storage temperature can assist in maintaining the solubility of
deoxycholic acid
salt in the formulation. In certain embodiments, the storage temperature is
from about 17 C
to about 27 C. In some embodiments, the storage temperature is about 25 C to
about 37 C.
In other embodiments, the storage temperature is from about 2 C to about 8
C.
[0073] It is contemplated that the concentration of the salt of deoxycholic
acid in the
formulation is about 0.5% w/v, or alternatively about 0.7% w/v, or
alternatively about 1%
w/v, or alternatively about 1.2% w/v, or alternatively about 1.4% w/v, or
alternatively less
than about 2% w/v. In a preferred embodiment, the salt of deoxycholic acid is
sodium
deoxycholate. In another preferred embodiment, the composition comprises 0.5%
w/v of
sodium deoxycholate. In another preferred embodiment, the composition
comprises 1% w/v
of sodium deoxycholate.
[0074] In one embodiment, the aqueous formulation is split into a plurality of
individual
solutions which are separately administered to the fat cells. For example, the
aqueous
formulation is split into 5, 10, 15, 20, 25 or 30 separate solutions and, in
some cases, up to 50
separate solutions.
[0075] In a preferred embodiment, the salt of deoxycholic acid is sodium
deoxycholate. As
the methods of this invention include subcutaneous injections, there is also
provided a syringe
comprising a chamber, a plunger and an injection needle wherein the chamber
comprises a
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formulation of this invention. Preferably, the chamber is sufficient to hold
at least 2 mL and
preferably no more than 4 mL of the formulation.
[0076] In another embodiment, this invention provides a synthesis of DCA from
protected
commercially available 9-a,17-I3-dihydroxy-5-a-androstan-3-one as shown in
scheme 1
below.
Scheme 1: Synthesis of DCA
oR1 oR1 0
Oil reduce III R3 00 oxidize pe protect _..
. OW0' R30µs' OW
0
H H H
1 2 3
07) )11 O'N(1)11 0 eNt'pri
00 0 0
04,0
eliminate oxidize 1.1111
hydrogenate
_,..
_,..
.11100
R3us R30,,0100 ..111w
R30µ
H H
H
5 6
4
0 neN(7)
RV% OH 0
RV'
oilb clir::::H
- /
. RV' . c1g7j0
reduce Wittig addition
.
H 7 H
8 H
9
OH ''= OH '-= OH '-=
CO2R CO2R CO2H
R30µs
01111 reduce, hydrolyze salt
,.. sodium deoxycholate
formation
. OW
R30 . HO\sµ
H H H
10 11 12 (DCA)
[0077] The 9-a,17-I3 hydroxyl groups of commercially available 9-a,17-I3-
dihydroxy-5-a-
androstan-3-one are differentially protected with hydroxyl protecting groups
which can be
removed under conditions where one of the hydroxyl groups is regenerated while
the other
hydroxyl group remains protected. Such differential protection is referred to
as orthogonal
protection and uses well known reagents and reaction conditions. In one
example, one
hydroxy group is protected as an acetyl group, whereas the other hydroxy group
is protected
as a benzyl group. Each group can be selectively removed under reaction
conditions that
retain the other hydroxyl protecting group intact.
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[0078] It is contemplated that the relatively sterically protected 9-a-
hydroxyl group may not
need to be protected as the reactions contemplated prior to elimination of
that group are likely
inhibited at this position due to steric hindrance. Regardless, protection of
this hydroxyl
group adequately insures that the group remains intact until elimination of
the hydroxyl group
via dehydration is desired.
[0079] The 3-one group of orthogonally protected 9-a,17-13-dihydroxy-5-a-
androstan-3-
one, compound 1, is reduced with conventional reducing agent such as sodium
borohydride
to provide the 3-a-hydroxy derivative which is then protected with yet another
orthogonal
protecting group to provide compound 2.
[0080] The hydroxyl protecting group at the 17-position of compound 2 is then
selectively
removed and the hydroxyl group so regenerated is then oxidized with a suitable
oxidation
reagent such as Cr03 to provide the 17-keto derivative, compound 3. The 17-
keto group in
compound 3 is protected as a ketal under standard ketalization conditions such
as reaction
with 1,2-dihydroxyethane or 1,3-dihydroxypropane to give compound 4 (which
illustrates
ketal formation with the 1,2-dihydroxyethane for illustrative purposes only).
[0081] Deprotection of the 9-a-hydroxyl as necessary is followed by
dehydration of that
hydroxyl group under conditions such as acid-catalyzed elimination provides
the 9,10-
unsaturated derivative, compound 5. Generation of a 12-keto group is
accomplished by
allylic oxidation of compound 5 with oxidation reagents such as chromic acid
or TBHP (tert-
butyl hydroperoxide) and Na0C1 to provide compound 6. See, for example, U.S.
Patent
Application Serial No. 61/348,686. Alternatively, the allylic oxidation is
accomplished by
using about 2 to 5 equivalents of TBHP and about 0.3 to 0.5 equivalents of CuI
as a catalyst.
The reaction is carried out in a solvent such as acetonitrile at 40 C for
about 40-55 hours.
The slow portionwise addition of TBHP results in more efficient oxidation. The
product
formed contains a mixture of compound 6 and the corresponding allylic alcohol.
The product
mixture is then oxidized with PCC to give compound 6.
[0082] Hydrogenation of compound 6 under standard conditions such as 10% Pd/C
and H2
provides compound 7. Reduction of the 12-keto group in compound 7 with
reagents such as
LiA1(0But)3H provides the 12-hydroxy derivative, compound 8. Olefination of
compound 8
under standard Wittig conditions such as using ethyltriphenylphosphonium
bromide in
presence of a base such as potassium tert-butoxide provides compound 9.
Addition of an
alkyl acrylate such as methyl acrylate in presence of a Lewis acid provides
compound 10,
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wherein R is an alkyl group such as methyl. Reduction of the double bond in
compound 10
again proceeds under standard hydrogenation conditions such as Pd/C and H2 to
provide
compound 11. Deprotection of the 3-0R3 followed by hydrolysis with a base such
as LiOH
provides DCA, compound 12.
[0083] Compound 12 (crude DCA) was further purified with methanol wash and
recrystallized from ethanol. It was diluted with 2 mol% Me0H in CH2C12 (25
vol) and
heated to 35-37 C for 1 hour. The slurry was allowed to cool to 28-30 C and
filtered. The
filter cake was washed with CH2C12 (5 vol) and dried under vacuum at 40 C to
afford DCA.
[0084] DCA was dissolved in 10% DI water/ Et0H (12 vol), polish filtered over
celite and
washed with 10% DI water/ Et0H (3 vol). The resulting 15 volume filtrate was
added to DI
water (30 vol) and a thin white slurry was afforded. The slurry was held for
24 hours,
filtered, washed with DI water (20 vol) and dried under vacuum at 40 C to
afford DCA.
[0085] Conversion of DCA to a pharmaceutically acceptable salt such as sodium
deoxycholate proceeds via conventional conditions. Alternatively, conversion
of a
pharmaceutically acceptable salt of DCA such as sodium deoxycholate to DCA
also proceeds
via conventional conditions.
[0086] In another embodiment, this invention provides a stabilized formulation
comprising:
a buffered aqueous solution having a pH of about 8.1 to about 8.5 and further
comprising about 0.5% of sodium deoxycholate and about 0.9% of benzyl alcohol,
wherein the formulation is stabilized against precipitation, and the sodium
deoxycholate is
prepared according to scheme 1.
[0087] In another embodiment, this invention provides a stabilized formulation
comprising:
a buffered aqueous solution having a pH of about 8.1 to about 8.5 and further
comprising about 1% of sodium deoxycholate and about 0.9% of benzyl alcohol,
wherein the formulation is stabilized against precipitation, and the sodium
deoxycholate is
prepared according to scheme 1.
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Examples
[0088] In the examples and elsewhere in the specification, abbreviations have
the following
meanings:
Mg = Milligram
mL = Milliliter
Mm = Millimeter
mM = Millimolar
T = Time
UV = Ultraviolet
v/v = Volume /Volume
w/v = Weight/Volume (g/mL)
w/w = Weight/ Weight
WFI = Water for Injection
mOsm = Milliosmole
[0089] The invention is further understood by reference to the following
examples, which
are intended to be purely exemplary of the invention.
Example 1
Concentration dependent precipitation from a solution of sodium deoxycholate
[0090] Solutions of sodium deoxycholate at different concentration were
evaluated for
precipitate formation after 1 week of storage. The results are depicted in
Figs. 1-4, and
demonstrate that at about 0.5% and at about 1 % (w/v) concentration of sodium
deoxycholate
in an aqueous solution containing only water and 0.9 % w/v benzyl alcohol, a
significant
amount of precipitate is formed such that it would inhibit use of the solution
as a composition
for subcutaneous injections. By visual inspection of Figs. 1-4, the amount of
precipitation
can be rated as tabulated below.
Table 1
% w/v Sodium Precipitation Comment
Deoxycholate Rating
0 1 Precipitation substantially invisible to the
naked eye
0.5 10 Significant amounts of precipitate visible to the naked
eye
1.0 7 Significant amounts of precipitate visible to the naked
eye
but less than that for 0.5% w/v
2.0 2 Precipitation visible to the naked eye but present in
substantially smaller amounts compared to the 0.5 and 1%
solutions above
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[0091] The precipitation rating estimates that "0" refers a clear solution and
that "10" refers
to a mixture exhibiting substantial precipitation readily visible to the naked
eye.
[0092] Such an observation demonstrates that in the concentration ranges
tested, the
precipitation phenomena was substantially affected by deoxycholate
concentration. To
ascertain pH's effect on precipitation, the pH of the solutions were measured,
as provided in
Table 2, which demonstrates that the pH of the solutions were substantially
the same,
especially for the 1% and the 2% solutions. The inverse aqueous solubility of
sodium
deoxycholate, where a more dilute solution (0.5% or 1%) provides more
precipitation than a
more concentrated solution (2%), is a surprising observation and also
evidences that the
precipitation phenomena is not directly related to pH, because the pH of the
solutions were
substantially the same again, especially for the 1% and 2% solutions.
Table 2
% w/v Sodium Reading # Temperature/ C pH
Deoxycholate
0 1 24.0 7.75
0 2 24.1 7.58
0.5 1 24.7 7.77
0.5 2 24.5 7.71
1 1 24.6 7.93
1 2 24.5 7.97
2 1 24.9 8.07
2 2 24.7 8.06
[0093] Accordingly, this invention provides that the surprising precipitation
from dilute,
0.4% to less than 2% (w/v), salt of deoxycholic acid solutions are inhibited,
to the extent that
such solutions are useful for subcutaneous injections, by increasing the
solution pH.
Example 2
Sodium deoxycholate (API) formulations with and without benzyl alcohol
1. A composition of sodium deoxycholate (0.5% and 1% ) was prepared
comprising
sodium phosphate (10 mM), sodium chloride (75-90 mM), benzyl alcohol (0.9%),
deoxycholic acid, pH 8.3.
2. An isotonic composition of sodium deoxycholate without benzyl alcohol
was
prepared using the free acid form, namely, deoxycholic acid, as follows.
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a. Preparation of 100 mL isotonic batches at 10 mg/mL
[0094] 1.0 g of the deoxycholic acid (DCA) added to the solution only after a
basic solution
was made with 70 mL water, 142 mg anhydrous dibasic sodium phosphate and 267
iut 10M
NaOH. It took about 20 minutes for the API to go into solution. The pH of the
solution was
11.1. The rapid addition of HC1 was known to cause some precipitation, so 225
ILLL of 1M
HC1 was slowly added to bring the solution to pH 8.3. The solution was allowed
to mix for
an additional 15 minutes. After bringing the volume up to 100 mL with water,
the osmolality
was found to be 51 mOsm. Addition of 859 mg of NaCl brought the osmolality up
to 305
mOsm.
[0095] The solution so prepared could optionally be lyophilized to provide for
a lyophilized
product which could be reconstituted by addition of the appropriate amount of
sterile water.
Accordingly, this invention also provides for lyophilized products of the
solutions disclosed
herein.
b. Preparation of 1000 mL isotonic batches at 10 mg/mL
[0096] The results from section a (above) did not scale up perfectly when
multiplied ten
fold. To 900 mL of water, 1.4g anhydrous dibasic sodium phosphate, 8.6 g NaC1,
and 2.7 mL
10 M NaOH were added. 10.0 g of DCA was then added and allowed to mix to
clarity for 30
minutes. The pH of the solution was 10.4. 1.5 mL 1 M HC1 was slowly added and
allowed to
mix for 5 minutes. The final pH was 8.1. An additional 20 iut of 10M NaOH had
to be
added to bring the pH to 8.3. After bringing the volume up to 1000 mL with
water, the
osmolality was 314 mOsm.
[0097] Based on observations of the pH change during the addition of 1 M HC1,
it was
determined that for 1000 mL batches at 10 mg/mL API, just 1.0 mL of 1M HC1
should be
immediately added and then slowly titrated with small volumes of the acid. The
suggested
order of addition for 1000 mL of 10 mg/mL API is outlined in Table 3.
c. Preparation of 100 mL isotonic batches at 5 mg/mL
[0098] 0.50 g of deoxycholic acid (DCA) was added to the solution only after a
basic
solution was made with 70 mL water, 142 mg anhydrous dibasic sodium phosphate
and 134
iut 10M NaOH. It took about 20 minutes for the API to go into solution. The pH
was 10.7.
The rapid addition of HC1 was known to cause some precipitation, so 115 ILLL
of 1 M HC1 was
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slowly added to bring the solution to pH 8.3. The solution was allowed to mix
for an
additional 15 minutes. After bringing the volume up to 100 mL with water, the
osmolality
was found to be 39 mOsm. Addition of 859 mg of NaCl brought the osmolality up
to 294
mOsm.
d. Preparation of 1000 mL isotonic batches at 5 mg/mL
[0099] The results from section c (above) did not scale up perfectly when
multiplied ten
fold. To 900 mL of water, 1.4g anhydrous dibasic sodium phosphate, 8.6 g NaC1,
and 1.3 mL
10M NaOH were added. 5.0 g of DCA was then added and allowed to mix to clarity
for 30
minutes. The pH was 8.6. After adding just 350 iut 1M HC1, the pH dropped to
8Ø An
additional 25 iut of 10M NaOH had to be added to bring the pH to 8.4. After
bringing the
volume up to 1000 mL with water, the osmolality was 305 mOsm. Based on
observations of
the pH change during the addition of 1 M HC1, it was determined that for 1000
mL batches at
5 mg/mL API, that the solution should be slowly titrated with small volumes of
1M HC1.
The suggested order of addition for 1000 mL of 5 mg/mL is outlined in Table 3.
Table 3. Order of addition (left to right) for isotonic 1000 mL benzyl alcohol
free
formulation
Dibasic
API 10M
Anhydrous
DCA NaC1 1M HC1
pH
Concentration Sodium NaOH
Phosphate
10 mg/mL 1.4g 2.7 mL 10.0g 8.6g 1.0 mL +
8.3
incremental
addition to
final pH
5 mg/mL 1.4 g 1.3 mL 5.0 g 8.6 g incremental
8.3
addition to
final pH
21
