BR112014000547B1 - STABLE AQUEOUS COMPOSITION IN PRECIPITATION - Google Patents

Abstract

formulations of deoxycholic acid and salts thereof. The present invention relates to an aqueous pharmaceutical composition consisting of about 0.4% w/v to less than about 2% w/v of a deoxycholic acid salt, wherein the composition is maintained at a pH of about 8.1 to about 8.5 such that the composition is stabilized against precipitation. Also described herein are methods for stabilizing an aqueous pharmaceutical composition consisting of about 0.4% w/v to less than about 2% w/v of a deoxycholic acid salt against precipitation, such method consisting of maintaining the pH of the deoxycholic acid solution. about 8.1 to about 8.5.

Description

FIELD OF INVENTION

[001] The present invention relates to aqueous pharmaceutical formulations containing very low concentrations of a salt of deoxycholic acid ("DCA"), in which 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 so that the composition is suitable for injection.

BACKGROUND OF THE INVENTION

[002] Literature reports have recently published that aqueous solutions of DCA have grease removal properties when injected into grease deposits in vivo (see, WO 2005/117900 and WO 2005/112942, US2005/0261258; US2005/0267080; US2006 /127468 and US2006/0154906). DCA injected into adipose tissue degrades fat cells through a cytolytic mechanism to provide the desired aesthetic results.

[003] Notwithstanding the benefits of aqueous formulations of DCA, it has been found that at low concentrations of DCA (ie, less than or about 2% w/v;) in aqueous solutions that optionally contain benzyl alcohol is formed a precipitate after storage for a period of time. Surprisingly, it was observed that the lower the concentration of DCA, the higher the precipitation rate despite any significant change in the pH of the solution. This precipitation at very low concentrations is a commercial problem as a precipitate is contraindicated for subcutaneous injections of DCA.

[004] In each treatment regimen, current clinical trials of aqueous formulations of ACD employ multiple injections of small amounts of the aqueous formulation in different locations, defining the fat deposit to be treated.

[005] As is apparent, aqueous formulations of DCA used in such treatments overcome the problems arising from precipitation of DCA. This means 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, despite the fact that the pH of these solutions is between about 7.50 and about 8, 0, which are substantially above the pKa of deoxycholic acid.

Thus, there is a need to stabilize low concentration aqueous solutions of deoxycholic acid or a salt thereof against precipitation for a shelf life of at least 2 months.

SUMMARY OF THE INVENTION

It has surprisingly been found that aqueous solutions of a salt of deoxycholic acid at concentrations of 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 about 8 and preferably a physiologically acceptable pH of from about 8.1 to about 8.5. The pH is preferably kept within this range using buffers.

[008] Likewise, in one of its composition aspects, this invention is directed to aqueous formulations, essentially consisting of a salt of deoxycholic acid in a concentration of about 0.4% w/v and less than about 2% w/ve, optionally, a preservative effective amount of benzyl alcohol in 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 essentially comprising a salt of deoxycholic acid at a concentration of about 0.5% w/v to about 1% w/v, and optionally a preservative effective amount of benzyl alcohol in 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.

[009] In another embodiment, this invention is directed to an aqueous formulation consisting essentially of: a sterile aqueous solution buffered at a pH of about 8.3; about 0.5% w/v or about 1% w/v of a deoxycholic acid salt; optionally, a preservative effective amount of benzyl alcohol; and about 1% w/v sodium chloride, wherein the composition is stable against precipitation.

[010] Also described in this document is a method for stabilizing an aqueous composition of a salt of deoxycholic acid against precipitation during storage, wherein the concentration of deoxycholic acid salt is an effective amount to lyse fat cells, provided that the The amount of deoxycholic acid salt ranges from about 0.4% w/v to less than 2% w/v, such method comprises: 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 about 8.1 to about 8.5.

[011] Also described in this document is a method for lysing a fat cell, ompost by administering to said cell a composition according to this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[012] Fig. 1 illustrates (67X 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 particles of bioprene tubing.

[013] Fig. 2 illustrates (67X 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 presumed to be deoxycholate crystals.

[014] Fig. 3 illustrates (67X enhancement) that an aqueous composition of water, 1% w/v sodium deoxycholate and 0.9% w/v benzyl alcohol contains significant amounts of precipitate, although less of 0.5% w/v sodium deoxycholate. As before, the precipitate is presumed to be deoxycholate crystals.

[015] Fig. 4 illustrates (67X 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 smaller than that seen in Figs. 2 and 3.

DETAILED DESCRIPTION

[016] As used in this document, certain terms have the meanings defined below.

[017] All numerical designations, eg pH, temperature, time, concentration and molecular weight, including ranges, are approximations that are varied (+) or (-) in increments of 0.1. It should be understood, though not always explicitly stated, that all numerical designations are preceded by the term "about". The term "about" also includes the exact value "X", plus small increments of "X" such as "X + 0.1" or "X - 0.1." It is also 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.

[018] In this document, the term "comprising" is intended to mean that compositions and methods include the elements recited but do not exclude others.

[019] "Consists essentially" when used to define methods and compositions, is understood to exclude any active ingredients. An "active ingredient" is a substance that is intended to have pharmacological activity or other direct effect in diagnosing, curing, mitigating, treating or preventing disease or affecting the structure or any function of the human body. Thus, for example, a composition consisting essentially of elements as defined in this term does not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers such as phosphate buffer saline, preservatives and the like, but would exclude enzymes such as phosphatases and proteins. Examples of non-imitation of such proteins are heparin, albumin and the like.

[020] "Consisting of" is meant excluding further trace elements of other ingredients and substantial method steps for administering the compositions of this invention. The modalities defined by each of these transition terms are within the scope of the present invention.

[021] In this document, the term "deoxycholic acid salt" or "a salt thereof" refers to the pharmaceutically acceptable salts of (4R)-4-((3R,5R,10S,12S,13R,17R)- 3,12-dihydroxy-10,13-dimethylhexa decahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate with an alkali metal or an ammonium ion as the cation. Alkali metal salts are preferred, with sodium salts being more preferred.

[022] Sodium or sodium deoxycholate (4R)-4-((3R,5R,10S, 12S,13R, 17R)-3,12-dihydroxy-10,13-dimethyl-hexadecahydro-1H-cyclopenta [a]fenantren-17-yl)pentanoate can be prepared according to the methods described in PCT/US2010/061150 entitled "Methods for Purification of Deoxycholic Acid" published December 17, 2010.

[023] 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.

[024] As used herein, the term "buffer" refers to an aqueous solution composed of 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 the pH at an almost 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).

[025] As used herein, the term "base" refers to various compounds, molecules or ions generally soluble in water that in solution have a pH greater than 7. Such compounds, molecules or ions are capable of taking on a proton from a acid or are capable of giving an unshared pair of electrons to an acid. Examples of suitable bases include metal bicarbonates and carbonates, 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, DB, ed. (2005) Remington: The Science and Practice of Pharmacy, 21st ed. ., Lippincott Williams & Wilkins).

[026] As used herein, the term "carbonate metals" refers to the metal salt of CO32-. For example, sodium carbonate, calcium carbonate, magnesium carbonate, zinc carbonate and the like.

[027] As used herein, "metal bicarbonates" refers to the metal salt of HCO3-. For example baking soda and the like.

[028] As used herein, the term "metal hydroxides" refers to the metal salt of -OH. For example, sodium hydroxide, potassium hydroxide and the like.

[029] As used herein, the terms "sterile water" or "water for injection" refer to a preparation of non-pyrogenic sterile water for injection that contains no antimicrobial, bacteriostatic, 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).

[030] As used herein, the term "benzyl alcohol" refers to the compound.

[031] As used herein, the term "precipitation" refers to the formation of a solid in a solution and is easily differentiated from gel formation.

[032] As used herein, the term "solution" refers to a substantially homogeneous mixture composed of two or more substances dissolved in a solvent.

[033] As used in this document, the terms "substantially inhibit precipitation" and "inhibit precipitation" mean to inhibit most or all visible precipitation in order to maintain homogeneity for a period of time ranging from at least 1 month to at least minus 1 year.

[034] As used in this document, the term "standard deviation relative to homogeneity" or "HE" refers to the value obtained by dividing the standard deviation of homogeneity by the absolute value of the mean. An HE less than 10 indicates very good homogeneity.

FORMULATIONS

[035] Knowledge about the chemical and physical stability of a drug composition in the desired medium for delivery is valuable. In the long 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 is at the required concentration when administered to a patient.

[036] 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.

[037] Current clinical methods for administering a sodium deoxycholate to a patient to dissolve fat include administration via subcutaneous injections of a low concentration (ie, < 2% w/v) of an aqueous solution of deoxycholic acid salt where the amount of deoxycholic acid salt is sufficient to lyse fat cells (about 0.4% w/v and higher). At such concentrations, as shown, the low concentration is beneficial for the safe and effective removal of fat deposits from 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 sodium deoxycholate solutions, even at low temperatures (3-5°C). In one embodiment, the sodium salt can be replaced with another alkali metal salt.

[038] This instability of aqueous solutions of sodium deoxycholate can be circumvented by preparing an aqueous solution of sodium deoxycholate in a concentration of about 5% to about 16% w/v and having the practitioner diluted the pharmaceutical composition of the solution. sodium deoxycholate before use. Since this method of dilution is effective in allowing 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. In addition, current clinical plans include up to 50 injections per treatment session.

[039] It has been observed 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 in concentrations ranging from about 0.4% w/v to less than about 2% w/v and optionally a pharmaceutically acceptable excipient as an effective amount. benzyl alcohol preservative and/or a pH adjustment buffer, wherein the formulation is maintained at a pH of from about 8.1 to about 8.5.

[040] In another embodiment, the aqueous formulation is lyophilized to provide a stable composition that is ready to be reconstituted by adding a suitable amount of water. In this embodiment, this invention comprises lyophilized compositions as described above which, optionally, further contain a lyophilization aid.

[041] In one embodiment, the aqueous formulation contains about 0.5% w/v of a deoxycholic acid salt. In another embodiment, the aqueous formulation contains about 1% w/v of a deoxycholic acid salt.

[042] In another modality, the water used in the aqueous formulation is sterile water. In yet another embodiment, the effective amount of benzyl alcohol preservative 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.

[043] In one embodiment, the pharmaceutical formulations described in this document are suitable for injection into a human. The injection method can be any type of injection, such as subcutaneous injection, as well as other forms of injection.

[044] In a preferred aspect of the present invention, the precipitation of the deoxycholic acid salt in the aqueous formulation is inhibited for a period of at least six months. In another aspect, precipitation is inhibited for a period of at least about a year. In another aspect, precipitation is inhibited for a period of at least about two years.

[045] It is observed that when stored at different temperatures, for example, at room temperature or cold, the formulation can have a longer 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 from 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.

[046] 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.

[047] In one embodiment, the pH is established by using a base. It is noted that any base can be used to increase the pH of the composition as long as it does not react with sodium deoxycholate and will not harm 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, among others, 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.

[048] In certain cases, the pH of the composition can be maintained at the desired pH during storage with the use of a buffer. Various buffers are known in the art and it is envisioned that any buffer with buffering capacity at the desired pH can be used in the formulations described herein. In one embodiment, the buffer is a phosphate buffer. The amount of phosphate in the composition can be determined to provide a desired salt concentration and pH. In one embodiment, the composition comprises a phosphate buffer of about 10 mM. In a preferred embodiment, the composition comprises a dibasic sodium phosphate buffer of about 10 mM.

[049] In some embodiments, the composition is composed of at least one excipient to help achieve a composition with desired properties, such as greater solubility, conservation or to provide an isotonic solution. Such excipients are known in the art. In one embodiment, the composition is 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.

[050] In some embodiments, the pH of the composition is established by the use of a base and, optionally, maintained by the use of a buffer.

[051] In a preferred embodiment, this invention provides a stabilized composition that includes: a phosphate buffer of a pH of about 8.3; about 0.5% w/v or about 1% w/v deoxycholate sodium; an effective preservative amount of benzyl alcohol; and about 1% w/v sodium chloride, wherein the composition is stabilized against precipitation.

[052] In another embodiment, the phosphate buffer is a 10 mM dibasic sodium phosphate buffer.

[053] In one embodiment, the effective amount of benzyl alcohol preservative is about 0.9% w/v.

[054] The formulations described 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 acid salt deoxycholic. The amount of precipitation or homogeneity of the composition can be measured using various methods. For example, this can be measured quantitatively using light scattering by illuminating the composition with a spectrophotometer. Or, alternatively, homogeneity can be qualitatively measured by looking at the visual clarity of the solution with the eye. In some embodiments, the composition has a relative standard deviation for homogeneity of less than 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%.

[055] 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 sodium deoxycholate; about 0.9% w/v benzyl alcohol; and about 1% w/v sodium chloride, wherein the composition is stable against precipitation.

[056] In another embodiment, this invention is directed to a composition consisting of: an aqueous solution buffered at a pH of about 8.3; about 0.5% w/v or about 1% w/v deoxycholate sodium; about 0.9% w/v benzyl alcohol; and about 1% w/v sodium chloride, wherein the composition is stable against precipitation.

[057] In some embodiments, solutions here do not include lipids, phospholipids, or phosphatidylcholine. In some embodiments, solutions herein include up to 5% w/w, w/v or v/v lipids, specifically phospholipids, or more specifically phosphatidylcholine. Preferably, the amount of lipid used is less than that of sodium deoxycholate or another salt of deoxycholic acid.

[058] In some embodiments, the aqueous pharmaceutical composition of the invention may further include a second therapeutic agent selected from the group consisting of: antimicrobial agents, vasoconstrictors, antithrombotic agents, anticoagulant agents, antidepressants, anti-inflammatory agents, analgesics, agents dispersal agents, anti-dispersion agents, penetration enhancers, steroids, tranquilizers, muscle relaxants and anti-diarrhoea agents. In some embodiments, a solution is in a container that holds up to 500 mL of solution. Such container may be a syringe or refillable syringe container.

[059] In some modalities, the formulations even comprise a molecule known to cause fat to die by an orthogonal mechanism. Such molecules include neuropeptide Y (NPY) 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 SA0204 (Sanofi Aventis), S-2367 (Shiongli), dihydropyridine and di derivatives -hydropyridine which are NPY receptor antagonists NGD-95-1 (Neurgogen), NLX-E201 (Neurologix), bicyclic compounds which are NPY receptor antagonists, NPY receptor antagonists carbazole, and tricyclic compounds which are NPY receptor antagonists (see, for example WO 2006/133160 and US 6,313,128). Pro-apoptotic selective fat peptides such as the CKGGRAKDC peptide that return to the white fat vasculature are also contemplated (see, Kolonin M.G. et al., Nat. Med., 2004, 10 (6): 625-32).

[060] Another aspect of the invention relates to the mixture of adipo-ablative bile acids, such as deoxycholic acid (DCA) with agents that kill adipocytes. In one aspect, this invention contemplates a means of enhancing the aesthetic effects of deoxycholate injections by mixing, into the injected deoxycholate, a molecule that causes fat to die through an orthogonal mechanism. Examples of such candidate molecules include, among others, neuropeptide Y (NPY) antagonists and fat selective pro-apoptotic peptides. Since fat cell death may be necessary to mediate the desired effects, the effects of an agent with the ability to kill fat can be potentiated by the addition of a molecule with potent fat cell killing effects. In addition, 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 can cause vascular leakage. Thus, these agents can be synergistic with deoxycholate potentially creating more potent means to mediate body contour in fewer therapeutic sessions.

[061] Examples of NPY antagonists include, among others, 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 SA0204 (Sanofi Aventis), S-2367 (Shiongli), dihydropyridine and dihydropyridine derivatives which are NPY receptor antagonists, bicyclic compounds which are NPY receptor antagonists, carbazole NPY receptor antagonists and tricyclic compounds which are NPY receptor antagonists. See, for example, WO 2006/133160 and US 6,313,128.

[062] Exemplary fat-selective pro-apoptotic peptides include, among others, CKGGRAKDC peptide that targets the white fat vasculature. See, Kolonin M.G. et al., Nat. Med. Jun 10(6): 625-32 (2004).

[063] Sodium or sodium deoxycholate (4R)-4-((3R,5R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a []phenanthrene-17-yl)pentanoate can be prepared according to the methods described in PCT/US2010/061150 entitled "Methods for the Purification of Deoxycholic Acid", filed December 17, 2010. Other salts of deoxycholic acid may be prepared in the same way by the person skilled in the art.

METHODS

[064] Described herein are methods for stabilizing an aqueous formulation of the salt of deoxycholic acid against precipitation during storage, wherein the concentration of or a salt thereof is an effective amount to lyse fat cells, provided that the amount of salt ranges from about 0.4% m/v to about less than 2% m/v, which method comprises: forming an aqueous solution of the 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

[065] Optionally including a sufficient amount of buffer to maintain the pH at from about 8.1 to about 8.5.

[066] In one aspect of this invention, methods described herein substantially stabilize the deoxycholic acid salt formulation against precipitation over a period of time, preferably at least six months. In another aspect, the methods stabilize the deoxycholic acid salt formulation against precipitation for a period of at least about one year. In yet another aspect, the methods stabilize the deoxycholic acid salt formulation against precipitation for a period of at least about two years.

[067] It has been found that the pH of the solution can inhibit the precipitation of deoxycholic acid or a salt thereof at concentrations of about 0.4% m/v to less than about 2% m/v in water to allow the deoxycholic acid or a salt thereof is kept in solution. 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, as long as 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, among others, 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.

[068] 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.

[069] In certain cases, the pH of the composition may need to be maintained with the use of a buffer. Various buffers are known in the art and it is contemplated that any buffer with buffering capacity at the desired pH can be used in the formulations described 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 known in the art. In one embodiment, the composition comprises about 10mM phosphate buffer. In another embodiment, the phosphate buffer is a 10 mM dibasic sodium phosphate buffer.

[070] In certain cases, the pH is established by the use of a base and, optionally, maintained by the use of a buffer.

[071] In one embodiment, the methods described in this document provide formulations that are suitable for injection into a human. The injection method 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).

[072] In one aspect, it may be that one or more excipients are used to maintain solubility, or enhance conservation of the 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 assist in obtaining 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 comprises adding 1% m/v benzyl alcohol and 1% m/v sodium chloride. In some embodiments, the method comprises the addition of 0.9% m/v benzyl alcohol and 0.9% m/v sodium chloride. Using the methods described herein, an aqueous solution comprising less than about 2% w/v of the deoxycholic acid salt is maintained at a pH sufficient to inhibit the precipitation of the deoxycholic acid salt substantially. 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 scattering of light through illumination by a spectrophotometer. Or, alternatively, homogeneity can be qualitatively measured simply by looking at the visual clarity of the solution with the eye. In some embodiments, the method provides a pharmaceutical composition with a relative standard deviation for homogeneity of less than 5%. Alternatively, the relative standard deviation for homogeneity is less than about 4% or alternatively about 3% or alternatively about 2% or alternatively about 1%.

[073] Storage temperature can help maintain the solubility of the 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 from about 25°C to about 37°C. In other embodiments, the storage temperature is from about 2°C to about 8°C.

[074] It is contemplated that the concentration of deoxycholic acid salt in the formulation is about 0.5% m/v or alternatively about 0.7% m/v or alternatively about 1% m /v or alternatively about 1.2% m/v or alternatively about 1.4% m/v or alternatively less than about 2% m/v. In a preferred embodiment, the deoxycholic acid salt is sodium deoxycholate. In another preferred embodiment, the composition comprises 0.5% w/v sodium deoxycholate. In another preferred embodiment, the composition comprises 1% w/v sodium deoxycholate.

[075] In one embodiment, the aqueous formulation is divided into a plurality of individual solutions, which are administered separately into the fat cells. For example, the aqueous formulation is divided into 5, 10, 15, 20, 25 or 30 separate solutions and, in some cases, up to 50 separate solutions.

[076] In a preferred embodiment, the deoxycholic acid salt 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 formulation of this invention. Preferably, the chamber is sufficient to hold at least 2 ml and preferably not more than 4 ml of the formulation.

[077] In another embodiment, this invention provides a commercially available DCA synthesis of protected 9-α,17-β-dihydroxy-5-α-androstan-3-one as shown in scheme 1 below. Scheme 1: DCA Synthesis

[078] The commercially available 9-α,17-β-hydroxyl groups of 9-α,17-β-dihydroxy-5-α-androstan-3-one are differentially protected with hydroxyl protecting groups that can be removed at 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 known reagents and reaction conditions. In one example, one hydroxyl group is protected as an acetyl group, while the other hydroxyl group is protected as a benzyl group. Each group can be selectively removed under reaction conditions that retain another hydroxyl protecting group intact.

[079] It is contemplated that the relatively sterically protected 9-α-hydroxyl group may not need to be protected, as reactions contemplated prior to elimination of the group are likely inhibited at this point due to steric blockade. Regardless, protection of this hydroxyl group adequately ensures that the group remains intact until elimination of the hydroxyl group through dehydration is desired.

[080] The 3-one group of orthogonally protected 9-α,17-β-dihydroxy-5-α-androstan-3-one, compound 1, is reduced with conventional reducing agent such as sodium borohydride to provide the derivative d 3- a-hydroxy which is then protected with yet another orthogonal protecting group to provide compound 2.

[081] The hydroxyl protecting group at the 17 position of compound 2 is then selectively removed and the regenerated hydroxyl group is then oxidized with an appropriate oxidizing reagent such as CrO3 to provide the 17-keto derivative, compound 3 17-keto group in compound 3 is protected as an acetal under standard ketalization conditions, such as reaction with 1,2-dihydroxyethane or 1,3-dihydroxypropane to give compound 4 (which illustrates acetal formation with 1,2-dihydroxyethane for illustrative purposes only).

[082] Deprotection of 9-α-hydroxyl as needed is followed by dehydration of such a hydroxyl group under conditions such as acid catalyzed elimination provides the unsaturated derivative 9,10, compound 5. The generation of a 12 group -keto is accomplished by the allylic oxidation of compound 5 with oxidizing reagents such as chromic acid or TBHP (tert-butyl hydroperoxide) and NaOCl to provide compound 6. See, for example, Patent Application Serial No. US 61 /348, 686. Alternatively, allylic oxidation is performed 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. Slow addition according to the TBHP portion 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.

[083] Hydrogenation of compound 6 under normal conditions such as 10% Pd/C and H2 provides compound 7. Reduction of the 12-keto group in compound 7 with reagents such as LiAl(OBut)3H provides 12-derivative hydroxy, compound 8. Olefination of compound 8 under normal Wittig conditions such as the use of ethyltriphenylphosphonium bromide in the presence of a base such as potassium tert-butoxide provides compound 9. Addition of an alkyl acrylate such as acrylate of methyl, in the presence of a Lewis acid provides compound 10, where R is an alkyl group, such as methyl. Reduction of the double bond in compound 10 is performed again under standard hydrogenation conditions such as Pd/C and H2 to provide compound 11. Deprotection of 3-OR3 followed by hydrolysis with a base such as LiOH provides DCA, compound 12.

[084] Compound 12 (crude DCA) was further purified by washing with methanol and recrystallized from ethanol. It was diluted with 2 mol% MeOH in CH 2 Cl 2 (25 vol.) and heated to 35-37°C for 1 hour. The slurry was cooled to 28-30°C and filtered. The filter cake was washed with CH 2 Cl 2 (5 vol.) and dried under vacuum at 40°C to obtain DCA.

[085] DCA was dissolved in 10% DI water/EtOH (12 vol.), filtered by polishing on celite and washed with 10% DI water/EtOH (3 vol.). The resulting 15 volume filtrate was added to DI water (30 vol.) and a thin white slurry provided. The slurry was kept for 24 hours, filtered, washed with DI water (20 vol.) and dried under vacuum at 40°C to obtain DCA.

[086] The conversion of DCA to a pharmaceutically acceptable salt, such as sodium deoxycholate, proceeds under conventional conditions. Alternatively, the conversion of a pharmaceutically acceptable salt of DCA, such as sodium deoxycholate, to DCA also proceeds via conventional conditions.

[087] In another embodiment, this invention provides a stabilized formulation comprising: aqueous buffer solution having a pH of about 8.1 to about 8.5 and further comprises about 0.5% sodium deoxycholate and about of 0.9% benzyl alcohol, wherein the formulation is stabilized against precipitation, and sodium deoxycholate is prepared according to scheme 1.

[088] In another embodiment, this invention provides a stabilized formulation comprising: aqueous buffer solution having a pH of from about 8.1 to about 8.5 and further comprises about 1% sodium deoxycholate and about 0 .9% benzyl alcohol, where the formulation is stabilized against precipitation, and sodium deoxycholate is prepared according to scheme 1.

EXAMPLES

[089] In the examples and elsewhere in the specification, abbreviations have the following meanings:Mg = MilligrammL = MilliliterMm = MillimetermM = MillimolarT = TimeUV = Ultravioletv/v = Volume /Volumem/v = Mass/Volume (g/mL)m /m = Mass/ Mass WFI = Water for Injection mOsm = Miliosmol

[090] 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 of a sodium deoxycholate solution

[091] Sodium deoxycholate solutions at different concentrations were evaluated for precipitate formation after 1 week of storage. The results are represented in Figures 1-4 and demonstrate that at a concentration of about 0.5% and about 1% (m/v) of sodium deoxycholate in an aqueous solution containing only water and 0.9% m/v of benzyl alcohol, a significant amount of precipitate is formed such that it would inhibit the use of the solution as a composition for subcutaneous injections. By visual inspection of Figures 1-4, the amount of precipitation can be classified as tabulated below. TABLE 1

[092] The precipitation rate estimates that "0" refers to a crystalline solution and that "10" refers to a mixture exhibiting substantial precipitation easily visible to the naked eye.

[093] Such observation demonstrates that in the concentration ranges tested, precipitation phenomena were substantially affected by the concentration of deoxycholate. To verify the effect of pH on precipitation, the pH of the solutions was measured, as shown in Table 2, which demonstrates that the pH of the solutions was substantially the same, especially for the 1% and 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 precipitation phenomena do not are directly related to pH, as the pH of the solutions was substantially the same again, especially for the 1% and 2% solutions. TABLE 2

[094] In this way, this invention provides that the surprising precipitation of the diluted, 0.4% to less than 2% (m/v), salt from deoxycholic acid solutions is inhibited, as such solutions are useful for injections. subcutaneously, increasing the pH of the solution. EXAMPLE 2 Sodium deoxycholate (API) formulations with and without benzyl alcohol1. A sodium deoxycholate composition (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, i.e., deoxycholic acid, as follows.

The. Preparation of 100 mL isotonic batches at 10 mg/mL

[095] 1.0 g of deoxycholic acid (DCA) added to the solution only after a basic solution has been made with 70 mL of water, 142 mg of anhydrous dibasic sodium phosphate and 267 μL of 10M NaOH. It took about 20 minutes for API to go into solution. The pH of the solution was 11.1. The rapid addition of HCl was known to cause some precipitation, so 225 µL of 1M HCl was slowly added to bring the solution to pH 8.3. The solution was left to mix for another 15 minutes. After bringing the volume up to 100 mL with water, the osmolality was determined to be 51 mOsm. Addition of 859 mg of NaCl brought the osmolality up to 305 mOsm.

[096] The solution thus prepared may optionally be lyophilized to provide a lyophilized product, which could be reconstituted by adding the appropriate amount of sterile water. Accordingly, this invention also provides lyophilized products from the solutions described in this document.

B. Preparation of 1000 mL isotonic batches at 10 mg/mL

[097] The results of section a (above) did not increase perfectly when multiplied ten times. To 900 ML of water, 1.4 g of anhydrous dibasic sodium phosphate, 8.6 g of NaCl and 2.7 ml of 10M NaOH were added. 10.0 g of DCA was then added and mixed until sharpened for 30 minutes. The pH of the solution was 10.4. 1.5 mL of 1M HCl was slowly added and mixed for 5 minutes. The final pH was 8.1. An additional 20 µL 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 determined to be 314 mOsm.

[098] Based on observations of the pH change during the addition of 1M HCl, it was determined that for batches of 1000 mL to 10 mg/mL API, only 1.0 mL of 1M HCl should be added immediately and then slowly titrated with small volumes of acid. The suggested order of addition for 1000 mL of 10 mg/mL API is described in Table 3.

ç. Preparation of 100 mL isotonic batches at 5 mg/mL

[099] 0.50 g of deoxycholic acid (DCA) was added to the solution only after a basic solution was made with 70 mL of water, 142 mg of anhydrous dibasic sodium phosphate and 134 μL of 10M NaOH. It took about 20 minutes for API to go into solution. The pH was 10.7. The rapid addition of HCl was known to cause some precipitation, so 115 µL of 1M HCl was slowly added to bring the solution to pH 8.3. The solution was left to mix for another 15 minutes. After bringing the volume up to 100 mL with water, the osmolality was determined 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

Tabela 3. -continuação-

[0100] The results of section c (above) did not increase perfectly when multiplied ten times. To 900 ML of water, 1.4 g of anhydrous dibasic sodium phosphate, 8.6 g of NaCl and 1.3 mL of 10M NaOH were added. 5.0 g of DCA were then added and mixed until sharpened for 30 minutes. The pH was 8.6. After adding only 350 µL of 1M HCl, the pH dropped to 8.0. An additional 25 µL 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 determined to be 305 mOsm. Based on observations of the pH change during addition of 1M HCl, it was determined that for batches of 1000 mL to 5 mg/mL API, the solution should be slowly titrated with small volumes of 1M HCl. The suggested order of addition for 1000ml of 5mg/ml API is described in Table 3. Table 3. Order of addition (left to right) for 1000ml isotonic benzyl alcohol free formulation

Table 3. -continuation-

Claims (14)

1. Precipitation-stable aqueous composition, characterized in that it consists of 0.4% m/v and less than 2% m/v of a deoxycholic acid salt and a pharmaceutically acceptable excipient, wherein said composition is maintained at a pH of 8.1 to 8.5. 2. Composition according to claim 1, characterized in that said deoxycholic acid salt is present in an amount from 0.5% m/v to 1% m/v. 3. Composition, according to claim 1 or 2, characterized in that said deoxycholic acid salt is present in an amount of 0.5% m/v. 4. Composition, according to claim 1 or 2, characterized in that said deoxycholic acid salt is present in an amount of 1% m/v. 5. Composition, according to any one of claims 1 to 4, characterized in that said excipient is a solvent, a buffer, a preservative, a lyophilization aid or any combination thereof. 6. Composition according to claim 5, characterized in that said excipient is a solvent, such as sterile water. 7. Composition, according to claim 5, characterized by the fact that said excipient is a preservative, such as benzyl alcohol. 8. Composition, according to claim 7, characterized by the fact that said preservative is 0.9% benzyl alcohol. 9. Composition, according to any one of claims 1 to 8, characterized in that said composition has a pH of 8.3. 10. Composition according to any one of claims 1 to 9, characterized in that salt is an alkali metal salt. 11. Composition according to claim 10, characterized in that said alkali metal salt is sodium. 12. Aqueous composition stable in precipitation, characterized in that it consists of: a sterile aqueous solution buffered at a pH of 8.3; 0.5% m/v sodium deoxycholate; 0.9% m/v alcohol benzylic; e1% m/v sodium chloride. 13. Precipitation-stable aqueous composition, characterized by the fact that it consists of: a sterile aqueous solution buffered to a pH of 8.3; 1% m/v sodium deoxycholate; 0.9% m/v benzyl alcohol; e1% m/v sodium chloride. 14. Composition, according to any one of claims 1 to 13, characterized in that deoxycholic acid is synthesized according to the following scheme:

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