JP6594486B2 - Formulation of deoxycholic acid and its salts - Google Patents

Description

  The present invention relates to an aqueous pharmaceutical formulation containing a very low concentration of salt of deoxycholic acid ("DCA"), which is maintained at a pH such that DCA precipitation 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.

  Recently published literature reports that aqueous solutions of DCA have the property of removing fat when injected into fatty deposits in vivo (WO 2005/117900 and WO 2005). No. 1112942, US 2005/0261258; US 2005/0267080; US 2006/127468; and US 2006/0154906). DCA injected into adipose tissue degrades adipocytes via a cell lysis mechanism, resulting in the desired aesthetic effect.

  Despite this advantage of an aqueous formulation of DCA, it precipitates after storage over a period of time at a low concentration (eg, less than 2 w / v% or about 2 w / v%) of DCA in an aqueous solution optionally containing benzyl alcohol. Things have been found to form. Surprisingly, it has been found that the rate of precipitation increases as the concentration of DCA decreases despite the fact that the pH of the aqueous solution has not changed significantly. This precipitation at very low concentrations is problematic when commercialized, as the precipitate indicates that it is not suitable for subcutaneous injection of DCA.

  In each treatment regime, current clinical trials for aqueous formulations of DCA use multiple injections of small amounts of the aqueous formulation at different sites indicating the fat deposit to be treated.

  As is apparent, the aqueous formulation of DCA used in such treatments overlaps the problems due to DCA precipitation. That is, despite the fact that an initially clear aqueous solution of DCA is stored for a period of time, the pH of this solution is about 7.50 to about 8.0, which is substantially above the pKa of deoxycholic acid. A precipitate will form at the relevant concentration of DCA available commercially.

  Therefore, a low concentration aqueous solution of deoxycholic acid or a salt thereof needs to be stabilized against precipitation for a shelf life of at least 2 months.

  Surprisingly, an aqueous solution of a salt of deoxycholic acid at a concentration of about 0.4 w / v% to less than about 2 w / v% has its pH above about 8, preferably from about 8.1 to about 8 It has been found that it can be stabilized by adjusting to a physiologically acceptable pH of .5. The pH is preferably maintained within this range using a buffer.

  Accordingly, in one aspect of the composition, the present invention provides a salt of deoxycholic acid at a concentration substantially between about 0.4 w / v% and less than about 2 w / v%, and optionally preservative. With respect to an aqueous formulation comprising an effective effective amount of benzyl alcohol, the formulation is adapted to precipitation by adjusting the pH of the initially formed clear solution to a pH of about 8.1 to about 8.5. Stabilized. In another embodiment, the present invention provides a salt of deoxycholic acid substantially at a concentration of about 0.5 w / v% to about 1 w / v% and optionally an antiseptic effective amount of benzyl alcohol. And the formulation is stabilized against precipitation by adjusting the pH of the initially formed clear solution to a pH of about 8.1 to about 8.5.

In another embodiment, the present invention substantially comprises
A sterile aqueous solution buffered to a pH of about 8.3;
About 0.5 w / v% or about 1 w / v% deoxycholic acid salt;
Optionally, an antiseptic effective amount of benzyl alcohol, and
Consisting of about 1 w / v% sodium chloride,
The composition relates to an aqueous formulation that is stable to precipitation.

Also disclosed herein is a method of stabilizing an aqueous composition of a salt of deoxycholic acid during storage against precipitation, wherein the amount of deoxycholate ranges from about 0.4 w / v% to less than 2 w / v% Where the concentration of deoxycholate is an amount effective to lyse adipocytes, the method comprising:
Making an aqueous solution of deoxycholate at an initial pH above its pKa;
Adjusting the pH of the aqueous solution to a pH of about 8.1 to about 8.5;
Optionally including a sufficient amount of buffer to maintain the pH from about 8.1 to about 8.5.

  Also disclosed herein is a method for lysing adipocytes comprising administering a composition to adipocytes according to the present invention.

It is an image which shows that the aqueous composition of water and 0.9 w / v% benzyl alcohol which does not contain sodium deoxycholate contains only a trace amount of precipitation (enlarged 67 times). Precipitates are presumed to be bioprene tube particles. Image showing that an aqueous composition of water, 0.5 w / v% sodium deoxycholate, and 0.9 w / v% benzyl alcohol contains a significant amount of precipitate (67x) (Enlarge). The precipitate is presumed to be crystals of deoxycholic acid. An aqueous composition of water, 1 w / v% sodium deoxycholate, and 0.9 w / v% benzyl alcohol contains a significant amount of precipitate, but 0.5 w / v% deoxycholic acid. It is an image showing that it is less than that of sodium (enlarged 67 times). As described above, this precipitate is presumed to be crystals of deoxycholic acid. An aqueous composition of water, 2 w / v% sodium deoxycholate, and 0.9 w / v% benzyl alcohol contained a significant amount of precipitate, but was observed in FIGS. It is an image which shows that there is substantially less than (it expands 67 times).

  As used herein, certain terms have the following defined meanings.

  All numerical representations such as pH, temperature, time, concentration, and molecular weight including ranges are approximate values that change to (+) or (−) in 0.1 units. Although not necessarily explicitly stated, all numerical designations should be understood to be preceded by the term “about”. The term “about” includes its exact value “X” in addition to a slight increase or decrease of “X” such as “X + 0.1” or “X−0.1”. Although not necessarily explicitly stated, it should be understood that the reagents described herein are exemplary only and are equivalent to those known in the art.

  As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements.

  When used to define compositions and methods, “consisting essentially of” shall mean excluding any active ingredient. An “active ingredient” is a substance that is intended to provide pharmacological activity or other direct effects or to affect the structure or any function of the human body in the diagnosis, cure, alleviation, treatment or prevention of disease. . Thus, for example, a composition consisting essentially of elements as defined herein may contain trace contaminants derived from isolation and purification methods, and pharmaceutical agents such as phosphate buffered saline, preservatives, and the like. Does not exclude acceptable carriers, but excludes enzymes and proteins such as phosphatases. Non-limiting examples of such proteins are heparin, albumin, etc.

  “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.

As used herein, the term “deoxycholic acid salt” or “salt thereof” refers to (4R) -4-((3R, 5R, 10S, 12S, 13R) having an alkali metal or ammonium ion as the cation. , 17R) -3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta [a] phenanthren-17-yl) pentanoate. Alkali metal salts are preferred, and sodium salts are more preferred.

  Sodium or sodium (4R) -4-((3R) deoxycholate by the method disclosed in International Application No. PCT / US2010 / 061150, filed Dec. 17, 2010, entitled “Methods for the Purification of Deoxycholic Acid”. , 5R, 10S, 12S, 13R, 17R) -3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta [a] phenanthren-17-yl) pentanoate can be prepared.

  As used herein, the term “aqueous pharmaceutical formulation” refers to a composition of deoxycholic acid or a salt thereof in water that is suitable for administration to a patient, preferably via subcutaneous injection from a syringe.

As used herein, the term “buffer” refers to an aqueous solution containing a mixture of a weak acid and its conjugate base or a mixture of a weak base and its conjugate acid. The buffer has the property that the pH of the solution hardly changes when a small amount of acid or base is added to it. Buffers are used as a means of maintaining a pH at a nearly constant value in a wide variety of chemical applications. Examples of suitable buffers are phosphate buffers and those known in the literature (see, eg, Troy, DB (2005) Remington: The Science and Practice of Pharmacy, 21 ^st , Lippincott Williams & Wilkins Want to be).

As used herein, the term “base” refers to various, usually water-soluble compounds, molecules or ions that have a pH of greater than 7 in solution. Such compounds, molecules or ions can pick up protons from the acid or can present a non-covalent pair of electrons to the acid. Examples of suitable bases include, for example, metal carbonates and bicarbonates such as sodium carbonate, calcium carbonate, magnesium carbonate, zinc carbonate, sodium bicarbonate; for example, literature (eg Troy, DB Ed (2005) Remington : See Science and Practice of Pharmacy, 21 ^st edition, Lippincott Williams & Wilkins)) and metal hydroxides such as sodium hydroxide and potassium hydroxide.

As used herein, the term “metal carbonate” refers to a metal salt of CO ₃ ²⁻ . Examples include sodium carbonate, calcium carbonate, magnesium carbonate, zinc carbonate and the like.

As used herein, the term “metal bicarbonate” refers to the metal salt of HCO ₃ ^— . Examples include sodium bicarbonate.

As used herein, the term "metal hydroxide" includes ^- refers to OH of metal salt. Examples include sodium hydroxide and potassium hydroxide.

  As used herein, the term “sterile water” or “water for injection” refers to a sterile, non-pyrogenic formulation of water for injection that does not contain bacteriostatic agents, antibacterial agents or additional buffers. Usually, the osmolarity of the additive will add up to at least 112 mOsm / liter (-2/5 of the normal osmotic thickness of the -280 mOsm / liter extracellular fluid).

As used herein, the term “benzyl alcohol” refers to the following compounds:

  As used herein, the term “precipitation” refers to the formation of a solid in solution and is easily distinguished from gel formation.

  As used herein, the term “solution” refers to a substantially homogeneous mixture comprising two or more substances dissolved in a solvent.

  As used herein, the term “substantially inhibit precipitation” inhibits most or all visible precipitate to maintain uniformity over a period ranging from at least one month to at least one year. It means to do.

As used herein, the term “relative standard deviation for uniformity” or “H _E ” refers to the value obtained by dividing the standard deviation of uniformity by the absolute value of the mean. Less than 10 H _E indicates that uniformity is very good.

Formulation Knowledge about the chemical and physical stability of the drug composition in the desired vehicle for delivery is valuable. During longer periods, the stability of the composition affects the shelf life of commercial products. When administered to a patient, the active ingredient of the pharmaceutical composition is preferably at the required concentration.

  In the discussion below, it is understood that sodium deoxycholate is described for illustrative purposes only, and other pharmaceutically acceptable salts of deoxycholic acid can be used interchangeably with sodium salt. The

  Current clinical procedures for administering sodium deoxycholate to patients to dissolve fat include administering a low concentration (eg, <2 w / v%) aqueous solution of a salt of deoxycholic acid by subcutaneous injection. This amount of deoxycholic acid salt is sufficient to lyse adipocytes (about 0.4 w / v% or more). At such concentrations, this low concentration has been shown to be effective for effectively and safely removing fat deposits in the body. However, it has been observed that such relatively low concentrations of sodium deoxycholate in aqueous media form precipitates. This precipitate will limit the shelf life of an aqueous solution of sodium deoxycholate, even at low temperatures (3-5 ° C.). In one embodiment, deoxycholic acid sodium salt can be replaced with another alkali metal salt.

  This instability of an aqueous solution of sodium deoxycholate is due to the fact that an aqueous solution of sodium deoxycholate is prepared at a concentration of about 5 w / v% to about 16 w / v%, and a doctor can It can be avoided by diluting the composition. This dilution method is effective to allow both storage stability and effective patient dosing, but is not ideal as a routine method, especially when only about 2 mL of sterile injection is required. Absent. In addition, current clinical plans include up to 50 injections per treatment session.

  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. The present invention includes a salt of deoxycholic acid at a concentration substantially in the range of about 0.4 w / v% to less than about 2 w / v%, and optionally an antiseptic effective amount of benzyl alcohol and / or Or a pharmaceutically acceptable excipient, such as a pH adjusting buffer, wherein the formulation is maintained at a pH of about 8.1 to about 8.5.

  In another embodiment, the aqueous formulation is lyophilized to provide a stable composition that can be easily reversed by adding an appropriate amount of water. In this embodiment, the present invention optionally comprises a lyophilized composition as described above further comprising a lyophilization aid.

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

  In a further embodiment, the water used in the aqueous formulation is sterile water. In a still 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, the aforementioned salt is an alkali metal salt. In another embodiment, the aforementioned salt is a sodium salt.

  In one embodiment, the pharmaceutical formulations disclosed herein are suitable for human injection. The method of injection can be any injection type, such as subcutaneous injection, as well as other injection forms.

  In a preferred embodiment of the invention, precipitation of the salt of deoxycholic acid in the aqueous formulation is inhibited for a period of at least about 6 months. In another embodiment, precipitation is inhibited for a period of at least about 1 year. In yet another embodiment, precipitation is inhibited for a period of at least about 2 years.

  It is believed that the formulation can extend the shelf life when stored at various temperatures, eg, ambient or cold. In certain embodiments, the composition is stored at a temperature of about 17 ° C to about 27 ° C. In some embodiments, the temperature of the formulation is raised to a temperature of about 25 ° C to about 37 ° C. In other embodiments, the formulation is stored at a temperature of about 2 ° C to about 8 ° C.

  In certain embodiments, the pH of the formulation is in the range of 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.

  In one embodiment, the pH is determined using a base. It is believed that any base can be used to raise the pH of the composition, provided that the base does not react with sodium deoxycholate and does not harm the patient. In some embodiments, the base is selected from the group consisting of metal carbonates, metal bicarbonates, metal hydroxides or mixtures 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 mixtures thereof. It is not a thing. In one embodiment, the base is sodium hydroxide.

  In some cases, the pH of the composition can be maintained at the desired pH during storage with a buffer. A variety of buffers are well known in the art, and it is contemplated that any buffer that has a 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 the desired pH and salt concentration. In one embodiment, the composition comprises about 10 mM phosphate buffer. In a preferred embodiment, the composition comprises about 10 mM sodium hydrogen phosphate buffer.

  In some embodiments, the composition comprises at least one excipient to help achieve a composition having desirable properties such as solubility, increased shelf life, or to provide an isotonic solution. Including. Such excipients are well 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.

  In some embodiments, the pH of the composition is determined using a base, and optionally maintained using a buffer.

In a preferred embodiment, the present invention provides:
A phosphate buffer having a pH of about 8.3;
About 0.5 w / v% or about 1 w / v% sodium deoxycholate;
An effective amount of antiseptic benzyl alcohol,
Providing a stabilized composition comprising about 1 w / v% sodium chloride;
This composition is stabilized against precipitation.

  In a further embodiment, the phosphate buffer is 10 mM sodium hydrogen phosphate buffer.

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

  The formulations disclosed herein comprise from about 0.4 w / v% to less than about 2 w / v% deoxy, which is maintained at a pH sufficient to substantially inhibit precipitation of the salt of deoxycholic acid. Contains salt of cholic acid in water. The amount of precipitate or composition uniformity can be measured using various methods. For example, the amount of precipitate or composition uniformity can be quantitatively measured using light scattering by illuminating the composition with a spectrophotometer. Alternatively, the uniformity can be qualitatively measured by visually observing the transparency of the solution. In some embodiments, the composition has a relative standard deviation for uniformity of less than about 5%. Alternatively, the composition has a relative standard deviation for uniformity of less than about 4%, alternatively about 3%, alternatively about 2%, alternatively about 1%.

In another embodiment, the present invention substantially comprises
A sterile aqueous solution buffered to a pH of about 8.3;
About 0.5 w / v% or about 1 w / v% sodium deoxycholate;
About 0.9 w / v% benzyl alcohol;
About a composition consisting of about 1 w / v sodium chloride,
This composition is stable to precipitation.

In another embodiment, the present invention provides:
An aqueous solution buffered to a pH of about 8.3;
About 0.5 w / v% or about 1 w / v% sodium deoxycholate;
About 0.9 w / v% benzyl alcohol;
About a composition consisting of about 1 w / v sodium chloride,
This composition is stable to precipitation.

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

  In some embodiments, the aqueous pharmaceutical composition of the present invention comprises an antibacterial agent, a vasoconstrictor, an antithrombotic agent, an anticoagulant, a foam suppressant, an anti-inflammatory agent, an analgesic, a dispersant, an antidispersant, an osmotic agent. A second therapeutic agent selected from the group consisting of a promoter, steroid, tranquilizer, muscle relaxant and antipruritic agent can further be included. In some embodiments, the solution is in a container that contains up to 500 mL of solution. Such a container can be a syringe or a container into which a syringe can be loaded.

  In some embodiments, the formulation further comprises a molecule known to cause fat loss by an orthogonal mechanism. Such molecules include neuropeptide Y (NPY) antagonists, which include BIBP-3226 (Amgen), BIBO-3304 (Boehringer Ingleheim), BMS-192522 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), NG-Ng95, E (Neurologic), CGP-71683 (Novartis), PD-160 70 (Pfizer), SR-120819A, BIIE0246 and S. A. NPY receptor antagonists such as 0204 (Sanofi Aventis), S-2367 (Shiongli); dihydropyridines and dihydropyridine derivatives that are NPY receptor antagonists; NPY receptor antagonists, bicyclic compounds that are carbazole NPY receptor antagonists; and NPY receptors These include, but are not limited to, tricyclic compounds that are body antagonists (see, eg, WO 2006/133160 and US Pat. No. 6,313,128). Fat-selective pro-apoptotic peptides such as CKGGRAKDC peptide present in white fat vasculature are also contemplated (see Kolonin M.G. et al., Nat. Med., 2004, 10 (6): 625-32).

  Another aspect of the invention relates to mixing a bile acid that can ablate fat, such as deoxycholic acid (DCA), and an agent that kills fat cells. In one aspect, the present invention contemplates a means to enhance the aesthetic effect of deoxycholic acid injection by mixing a molecule that causes fat loss through an orthogonal mechanism into the deoxycholic acid injection. 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 effect, the effect of drugs with fat killing ability is improved by adding molecules with a powerful fat cell killing effect Can be made. In addition, molecules that need access to the vasculature to kill them (such as certain pro-apoptotic peptides that bind to proteins expressed on the luminal side of capillaries) can cause deoxycholic acid to cause vascular leakage. It is possible that these proteins can be reached. Therefore, such agents are synergistic with deoxycholic acid and may create a more powerful means of mediating body correction with fewer treatment sessions.

  Examples of NPY antagonists include BIBP-3226 (Amgen), BIBO-3304 (Boehringer Ingleheim), BMS-192548 and AR-H040922 (Bristol-Myers Squibb), LY-357897 (Eli Lilly14G1L29S14) , JNJ-5207787 (Johnson & Johnson), Lu-AA-44608 (Lundbeck), MK-0557 (Merck NPY), NGD-95-1 (Neurogengen), NLX-E201 (Neurologix), CGP-716t (No.) 160170 (Pfizer), SR-120819A, BIIE0246 Preliminary S. A. NPY receptor antagonists such as 0204 (Sanofi Aventis), S-2367 (Shiongli); dihydropyridines and dihydropyridine derivatives that are NPY receptor antagonists; NPY receptor antagonists, bicyclic compounds that are carbazole NPY receptor antagonists; and NPY receptors Examples include, but are not limited to, tricyclic compounds that are body antagonists. See, for example, WO 2006/133160 and US Pat. No. 6,313,128.

  Representative examples of fat-selective pro-apoptotic peptides include, but are not limited to, CKGGRAKDC present in white fat vasculature. See Kolonin M.G. et al., Nat. Med. June 10 (6): 625-32 (2004).

  Sodium or sodium (4R) -4-((3R, deoxycholate) by the method disclosed in International Application PCT / US2010 / 061150, filed Dec. 17, 2010, entitled “Methods for the Purification of Deoxycholic Acid” 5R, 10S, 12S, 13R, 17R) -3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta [a] phenanthren-17-yl) pentanoate can be prepared. Other salts of deoxycholic acid can be similarly prepared by those skilled in the art.

Methods Disclosed herein are methods for stabilizing aqueous formulations of deoxycholate against precipitation during storage. The salt concentration is sufficient to lyse adipocytes, provided that the amount of salt ranges from about 0.4 w / v% to less than about 2 w / v%, the method comprising:
Making an aqueous solution of a salt of deoxycholic acid at an initial pH above its pKa;
Adjusting the pH of the aqueous solution to a pH of about 8.1 to about 8.5;
Optionally including a sufficient amount of buffer to maintain the pH from about 8.1 to about 8.5.

  In one aspect of the invention, the methods disclosed herein substantially stabilize the deoxycholate formulation against precipitation, preferably for a period of at least about 6 months. In another embodiment, the method stabilizes the deoxycholate formulation against precipitation for a period of at least about 1 year. In yet another embodiment, the method stabilizes the deoxycholate formulation against precipitation for a period of at least about 2 years.

  The pH of the solution can inhibit precipitation of deoxycholic acid or a salt thereof in water at a concentration of about 0.4 w / v% to less than about 2 w / v%, wherein deoxycholic acid or a salt thereof is in solution. It has been found to be possible to be maintained. In one embodiment, the pH is determined using a base. It is believed that any base can be used to raise the pH of the composition, provided that the base does not react with deoxycholic acid or its salt. In some embodiments, the base is selected from the group consisting of metal carbonates, metal bicarbonates and metal hydroxides or mixtures 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 mixtures thereof. It is not a thing. In one embodiment, the base is sodium hydroxide.

  In certain embodiments, the pH is in the range of about 8.1 to about 8.5. In one embodiment, the pH of the composition is about 8.1, alternatively about 8.2, alternatively about 8.3, alternatively about 8.4, alternatively about 8.5. In a preferred embodiment, the pH of the aqueous solution is about 8.3.

  In some cases, the pH of the composition may need to be maintained using a buffer. A variety of buffers are well known in the art, and it is contemplated that any buffer that has a 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 give the 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 sodium hydrogen phosphate buffer.

  In some cases, the pH is established using a base and optionally maintained using a buffer.

  In one embodiment, the methods disclosed herein provide a formulation suitable for human injection. The method of injection can be any type of injection, such as subcutaneous injection, as well as other forms of injection. Thus, in some embodiments, the aqueous solution comprises sterile water or water for injection (WFI).

  In one embodiment, one or more excipients may be used to maintain solubility or increase the preservability of deoxycholate present in the formulation. In one embodiment, the method comprises adding about 1 w / v% benzyl alcohol. In some embodiments, the formulation also includes at least one excipient to help achieve an isotonic solution. Such excipients are well known in the art. In one embodiment, the method comprises adding about 1 w / v% sodium chloride. In some embodiments, the method 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% deoxycholate is maintained at a pH sufficient to substantially inhibit deoxycholate precipitation. The amount of precipitate or composition uniformity can be measured using various methods. For example, by measuring light scattering via illumination with a spectrophotometer, the amount of precipitate or composition uniformity can be measured quantitatively. Alternatively, the uniformity can be measured by simply observing the clarity of the solution visually. In some embodiments, the method provides a pharmaceutical composition wherein the relative standard deviation for homogeneity is less than about 5%. Alternatively, the relative standard deviation for uniformity is less than about 4%, alternatively less than about 3%, alternatively less than about 2%, alternatively less than about 1%.

  The storage temperature can help maintain the solubility of deoxycholate 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.

  The concentration of deoxycholic acid salt in the formulation is about 0.5 w / v%, alternatively about 0.7 w / v%, alternatively about 1 w / v%, alternatively about 1.2 w / v%, alternatively about 1.4 w. / V%, or 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% sodium deoxycholate. In another preferred embodiment, the composition comprises 1 w / v% sodium deoxycholate.

  In one embodiment, the aqueous formulation is divided into multiple individual solutions that are administered separately to the adipocytes. For example, the aqueous formulation is divided into 5, 10, 15, 20, 25 or 30 separate solutions. In some cases, it is divided into up to 50 separate solutions.

  In a preferred embodiment, the salt of deoxycholic acid is sodium deoxycholate. Since the method of the present invention involves subcutaneous injection, a syringe with a chamber, plunger and needle is also provided. The chamber contains the formulation of the present invention. Preferably, the chamber is sufficient to hold at least 2 mL, preferably only 4 mL of formulation.

  In another embodiment, the present invention provides the synthesis of DCA from protected commercial 9-α, 17-β-dihydroxy-5-α-androstan-3-one as shown in the following scheme.

Scheme 1: Synthesis of DCA

  The 9-α-, 17 hydroxy group of commercially available 9-α, 17-β-dihydroxy-5-α-androstan-3-one is differentially protected with a hydroxy protecting group. Hydroxy protecting groups can be removed under the condition that one of them is regenerated while the other hydroxy group remains protected. Such differential protection is called orthogonal protection and uses well-known reagents and reaction conditions. In one example, one hydroxy group is protected as an acetyl group, while the other hydroxy group is protected as a benzyl group. Each group can be selectively removed under reaction conditions in which other hydroxy protecting groups remain intact.

  Since the possible reaction prior to 9-α-hydroxy group elimination is probably inhibited at this position due to steric hindrance, the relatively sterically protected 9-α-hydroxy group probably needs to be protected. There seems to be no. Nevertheless, protecting the hydroxy group ensures that it remains sufficiently intact until removal of the hydroxy group via dehydration is desired.

  The 3-one group (compound 1) of ortho-protected 9-α, 17-β-dihydroxy-5-α-androstan-3-one is sodium borohydride or the like so as to obtain a 3-α-hydroxy derivative. Reduction with a conventionally used reducing agent. This is then protected with another orthogonal protecting group to give compound 2.

The hydroxy protecting group at position 17 of compound 2 is then selectively removed and the so deprotected hydroxy group is then converted to a suitable such as CrO ₃ to give the 17-keto derivative (compound 3). Oxidized with oxidizing reagent. The 17-keto group in compound 3 can be combined with 1,2-dihydroxyethane or 1,3-dihydroxypropane to obtain compound 4 (illustrated with 1,2-dihydroxyethane for illustration purposes only). It is protected as a ketal under standard ketalization conditions such as

  If necessary, the deprotection of 9-α-hydroxyl is followed by dehydration of the hydroxy group under conditions such as acid catalyzed elimination to give the 9,10-unsaturated derivative (compound 5). Formation of the 12-keto group is achieved by allylating compound 5 with an oxidizing reagent such as chromic acid or TBHP (tert-butyl hydroperoxide) and NaOCl to yield compound 6. See, for example, U.S. Patent Application No. 61 / 348,686. Alternatively, allylic oxidation is achieved using about 2-5 equivalents of TBHP and about 0.3-0.5 equivalents of Cul as the catalyst. This reaction is carried out in a solvent such as acetonitrile at 40 ° C. for about 40 to 55 hours. By adding TBHP slowly little by little, it is oxidized more efficiently. The product formed contains a mixture of compound 6 and the corresponding allyl alcohol. The product mixture is then oxidized with PCC to obtain compound 6.

Compound 6 is hydrogenated to give compound 7 under standard conditions such as 10% Pd / C and H ₂ . The 12-keto group in compound 7 is reduced with a reagent such as LiAl (OBu ^t ) ₃ H to obtain a 12-hydroxy derivative (compound 8). Compound 9 is obtained by olefination of compound 8 under standard Wittig conditions such as using ethyltriphenylphosphonium bromide in the presence of a base such as potassium tert-butoxide. Compound 10 is obtained by adding alkyl acrylic acid such as methyl acrylate in the presence of Lewis acid. In the formula, R is an alkyl group such as methyl. Again, reduction of the double bond in compound 10 proceeds under standard hydrogenation conditions such as Pd / C and H ₂ to give compound 11. Subsequent to hydrolysis with a base such as LiOH, 3-OR ₃ is deprotected to yield DCA (compound 12).

Compound 12 (crude DCA) was further purified with a methanol wash 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 at 40 ° C. under reduced pressure to obtain DCA.

  DCA was dissolved in 10% DI water / EtOH (12 vol), final filtered over celite, then washed with 10% DI water / EtOH (3 vol). The resulting 15 vol filtrate was added to DI water (30 vol) to give a thin white slurry. This slurry was held for 24 hours, then washed with DI water (20 vol) and dried under reduced pressure at 40 ° C. to obtain DCA.

  Conversion of DCA to pharmaceutically acceptable salts 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.

In another embodiment, the present 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% sodium oxycholate and about 0.9% benzyl alcohol And
The formulation is stabilized against precipitation and sodium oxycholate is prepared according to Scheme 1.

In another embodiment, the present invention provides:
Providing a stabilized formulation comprising a buffered aqueous solution having a pH of about 8.1 to about 8.5 and further comprising about 1% sodium oxycholate and about 0.9% benzyl alcohol;
The formulation is stabilized against precipitation and sodium oxycholate is prepared according to Scheme 1.

In this example, and elsewhere in this specification, the abbreviations have the following meanings:

  The invention will be further understood by reference to the following examples, which are intended only to illustrate the invention.

Example 1 Concentration Dependent Precipitation from Sodium Deoxycholate Solution Solutions of different concentrations of sodium deoxycholate were evaluated for precipitate formation after storage for 1 week. The results are shown in FIGS. 1-4, and sodium deoxycholate in an aqueous solution containing only water and 0.9 w / v% benzyl alcohol has concentrations of about 0.5% and about 1% (w / v). Clearly shows that a considerable amount of precipitate is formed, thus preventing the use of this solution as a composition for subcutaneous injection. By visual inspection of FIGS. 1-4, the amount of precipitate can be assessed as expressed below.

  The score of the precipitate is evaluated as “0” representing a clear solution and “10” representing a mixture showing significant precipitate that is readily visible to the naked eye.

  Such observations indicate that within the range of concentrations tested, the precipitation phenomenon is significantly affected by the concentration of deoxycholic acid. In order to confirm the effect of pH on precipitation, the pH of each solution was measured as shown in Table 2. Table 2 shows that the pH of each solution, in particular the pH of the 1% and 2% solutions, was substantially the same. The reverse water solubility of sodium deoxycholate is a surprising observation when more dilutions (0.5% or 1%) provide more precipitate than more concentrated solutions (2%) And that the precipitation phenomenon is not directly related to pH. This is because the pH of each solution was again substantially the same, especially for the 1% and 2% solutions.

  Thus, the present invention provides surprisingly to the extent that less than 0.4% to less than 2% (w / v) diluted deoxycholic acid salt is useful for subcutaneous injection by raising the pH of the solution. It is to suppress precipitation.

Example 2 Sodium Deoxycholate (API) Formulation With and Without Benzyl Alcohol A composition of sodium deoxycholate (0.5% and 1%) containing sodium phosphate (10 mM), sodium chloride (75-90 mM), benzyl alcohol (0.9%), deoxycholic acid, pH 8.3. Prepared.
2. An isotonic composition of sodium deoxycholate without benzyl alcohol was prepared using the free acid form, deoxycholic acid, as follows.

a. Preparation of 100 mL isotonic batch at 10 mg / mL After making a base solution with 70 mL water, 142 mg dibasic sodium phosphate anhydrous, and 267 μL 10 M NaOH, add 1.0 g deoxycholic acid (DCA) It was. It took about 20 minutes for the API to dissolve in the solution. The pH of the solution was 11.1. Since adding HCl quickly is known to produce some precipitate, 225 μL of 1M HCl was slowly added to bring the solution to pH 8.3. The solution was allowed to mix for an additional 15 minutes. When the amount was made up to 100 mL with water, the osmolality was 51 mOsm. When 859 mg of NaCl was added, the osmolality rose to 305 mOsm.

  The solution so prepared can optionally be lyophilized to provide a lyophilized formulation that can be reconstituted by adding an appropriate amount of sterile water. Accordingly, the present invention also provides lyophilized formulations of the solutions disclosed herein.

b. Preparation of 1000 mL isotonic batch at 10 mg / mL The results in section a (above) did not scale up completely when increased 10-fold. To 900 mL of water, 1.4 g of dibasic sodium phosphate anhydrous, 8.6 g of NaCl, and 2.7 mL of 10M NaOH were added. Next, 10.0 g of DCA was added and mixed for 30 minutes until clear. The pH of this solution was 10.4. Slowly add 1.5 mL of 1M HCl and mix 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. When the amount was made up to 1000 mL with water, the osmolality was 314 mOsm.

  Based on observations of pH changes during the addition of 1M HCl, a batch of 1000 mL of API at 10 mg / mL should immediately add 1.0 mL of 1M HCl and then slowly titrate with a small amount of acid. Were determined. Table 3 outlines the proposed order of addition for API 1000 mL at 10 mg / mL.

c. Preparation of 100 mL isotonic batch at 5 mg / mL After making a base solution with 70 mL water, 142 mg dibasic sodium phosphate anhydrous, and 134 μL 10 M NaOH, add 0.50 g deoxycholic acid (DCA) It was. It took about 20 minutes for the API to dissolve in the solution. The pH was 10.7. Since adding HCl quickly is known to cause some precipitation, 115 μL of 1M HCl was slowly added to bring the solution to pH 8.3. The solution was mixed for an additional 15 minutes. When the amount was adjusted to 100 mL with water, the osmolality was 39 mOsm. When 859 mg of NaCl was added, the osmolality rose to 294 mOsm.

d. Preparation of 1000 mL Isotonic Batch at 5 mg / mL The results in section c (above) did not scale up completely when increased 10-fold. To 900 mL of water, 1.4 g of dibasic sodium phosphate anhydride, 8.6 g of NaCl, and 1.3 mL of 10M NaOH were added. Next, 5.0 g of DCA was added and mixed for 30 minutes until clear. The pH was 8.6. When 350 μL of 1M HCl was added, the pH dropped to 8.0. An additional 25 μL of 10M NaOH had to be added to bring the pH to 8.4. When the volume was made up to 1000 mL with water, the osmolality was 305 mOsm. Based on observations of pH changes during the addition of 1M HCl, it was determined that a batch of 1000 mL API at 5 mg / mL needed to be slowly titrated with a small amount of 1M HCl. Table 3 outlines the proposed order of addition for 1000 mL at 5 mg / mL.

Claims (10)

A method of inhibiting the precipitation of deoxycholate in injectable aqueous pharmaceutical composition, include maintaining the pH of the aqueous pharmaceutical composition to a value of 8.1 to 8.5, the aqueous pharmaceutical compositions Comprising an aqueous solution comprising from 0.4% w / v to less than 2% w / v deoxycholate and at least one pharmaceutically acceptable excipient.   2. The method of claim 1 wherein deoxycholate precipitation is inhibited for at least 6 months.   The method according to claim 1 or 2, wherein the precipitation of deoxycholate is inhibited for at least 6 months at a temperature of 17 ° C to 27 ° C, 25 ° C to 37 ° C or 2 ° C to 8 ° C.   The method according to claim 1, wherein the pH is maintained at 8.3.   The method according to any one of claims 1 to 4, wherein deoxycholate is present in an amount of 0.5% w / v to 1% w / v.   6. The at least one pharmaceutically acceptable excipient comprises a solvent, a preservative, a lyophilization aid, an isotonic agent or a combination thereof. Method.   The method according to any one of claims 1 to 6, wherein the solution is for subcutaneous injection. In injectable aqueous pharmaceutical composition to a method for inhibiting the precipitation of deoxycholate, it includes maintaining the pH of the aqueous pharmaceutical composition to a value of 8.1 to 8.5, the aqueous pharmaceutical A method wherein the composition comprises an aqueous solution comprising 0.5% w / v deoxycholate, 0.9% w / v benzyl alcohol and 1% w / v sodium chloride. In injectable aqueous pharmaceutical composition to a method for inhibiting the precipitation of deoxycholate, it includes maintaining the pH of the aqueous pharmaceutical composition to a value of 8.1 to 8.5, the aqueous pharmaceutical A method wherein the composition comprises an aqueous solution comprising 1% w / v deoxycholate, 0.9% w / v benzyl alcohol and 1% w / v sodium chloride. The method according to claim 1, wherein the salt is a sodium salt.

Images