KR102812226B1 - Pharmaceutical composition comprising deoxycholic acid - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising deoxycholic acid (DCA) or a pharmaceutically acceptable salt thereof, comprising a pharmaceutically acceptable additive, having a pH of 7.0 to 8.0, and an electrical conductivity of 5 mS/cm or less at 25°C.

Description

{PHARMACEUTICAL COMPOSITION COMPRISING DEOXYCHOLIC ACID}

The present invention relates to a pharmaceutical composition comprising deoxycholic acid.

Lipolysis is the process by which neutral fat contained in fat cells is broken down into free fatty acids (FFA) and glycerol by the action of hormone sensitive lipase (HSL), and is regulated by hormones or the autonomic nervous system. Recently, lipolysis injections using this type of lipolysis are gaining popularity as one of the easier methods for weight loss or body management than exercise or diet.

The lipolytic use of deoxycholic acid (DCA) is already known. Recent publications have reported that aqueous solutions of deoxycholic acid have the property of removing fat when injected into fatty deposits in vivo (International Patent Applications WO 2005/117900 and WO2005/112942, U.S. Patent Applications US2005/0261258; US2005/0267080; US2006/127468; and US2006/0154906, etc.).

In addition, deoxycholic acid (DCA) has the advantage of high solubility, making it easy to prepare. However, despite these advantages, water-soluble compositions of deoxycholic acid (DCA) are known to form white precipitates when stored at low concentrations in water-soluble solutions optionally containing benzyl alcohol for a certain period of time, and surprisingly, it has been found that the rate of precipitation becomes faster as the concentration of deoxycholic acid (DCA) becomes lower. It is known that such precipitation occurs when subcutaneous injections of deoxycholic acid (DCA) are counter-indicated at low concentrations, and considering that the average shelf life of cosmetic injectables on the market is more than 24 months, the occurrence of a precipitate makes commercialization virtually impossible due to stability issues.

One way to address this has been to prepare the solution in a much higher concentration of deoxycholic acid (DCA), for example, at a concentration of 5 wt% to about 16 wt%, and then dilute the solution immediately before use by the physician. However, although this method may be effective for storage stability, it is not ideal for routine use, especially when a sterile injectable solution of at most about 0.2 mL is required. Furthermore, current clinical treatment methods involve up to 50 injections per treatment session, making it inconvenient to dilute the solution every time for each injection.

As another solution, a commercial product that does not produce precipitates by adjusting the pH to a range of up to 8.5 is being sold, but side effects such as a burning sensation and inflammation due to high pH have been reported. Therefore, there is a need to develop a formulation that satisfies precipitation stability at a lower pH while reducing side effects.

Korean Patent Publication No. 2019-0120712

The present invention provides a pharmaceutical composition containing deoxycholic acid (DCA), which has excellent storage stability because no precipitate is generated even at low pH compared to conventional pharmaceutical compositions containing deoxycholic acid (DCA), and which can minimize side effects such as a burning sensation and inflammation at the site of administration.

The present invention provides a pharmaceutical composition comprising deoxycholic acid (DCA) or a pharmaceutically acceptable salt thereof, comprising a pharmaceutically acceptable additive, having a pH of 7.0 to 8.0, and an electrical conductivity of 5 mS/cm or less at 25°C.

The pharmaceutical composition containing deoxycholic acid (DCA) according to the present invention can exhibit excellent storage stability without forming a precipitate during the storage period. In addition, since it has a lower pH than the pharmaceutical composition containing conventional deoxycholic acid (DCA), it becomes similar to the pH in the human body, and the local pH at the injection site is quickly restored, so that injection pain can be significantly alleviated and side effects such as a burning sensation and inflammation at the injection site can be minimized. Accordingly, the pharmaceutical composition containing deoxycholic acid (DCA) of the present invention can be usefully used for lipolysis, and for the prevention and treatment of obesity, especially localized obesity.

Figures 1 and 2 are graphs showing the results of a pH buffering capacity comparison experiment.

Hereinafter, the present invention will be described in more detail to help understand the present invention. At this time, the terms or words used in this specification and claims should not be interpreted as limited to their usual or dictionary meanings, and should be interpreted as meanings and concepts that conform to the technical idea of the present invention based on the principle that the inventor can appropriately define the concept of the term to explain his own invention in the best way.

The present invention provides a pharmaceutical composition comprising deoxycholic acid (DCA) or a pharmaceutically acceptable salt thereof, comprising a pharmaceutically acceptable additive, having a pH of 7.0 to 8.0, and an electrical conductivity of 5 mS/cm or less at 25°C.

The above deoxycholic acid or its pharmaceutically acceptable salt is a known substance and can be prepared by a known method. For example, the deoxycholic acid or its salt can be prepared as a high-purity deoxycholic acid or its salt by extracting, crystallizing, and purifying bile acid from an animal, for example, bovine bile acid. In addition, the deoxycholic acid or its salt can be purchased commercially (for example, can be purchased from Sigma-aldrich, PCA S.P.A, etc.).

The deoxycholic acid of the present invention may be in the form of a pharmaceutically acceptable salt. Deoxycholic acid is (4R)-4-((3R,5R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate, and the salt is a pharmaceutically acceptable salt, and includes, but is not limited to, a conventional metal salt form, for example, an alkali metal salt such as lithium, sodium, or potassium; an alkaline earth metal salt such as calcium or magnesium salt; or a chromium salt.

The pharmaceutical composition containing deoxycholic acid (DCA) according to the present invention may contain 1 to 50 mg/mL of deoxycholic acid (DCA) or a pharmaceutically acceptable salt thereof, more preferably 5 to 30 mg/mL, even more preferably 5 to 20 mg/mL, and even more preferably 5 to 15 mg/mL. By containing deoxycholic acid (DCA) or a pharmaceutically acceptable salt thereof within the above range, deoxycholic acid (DCA) exhibiting a lipolytic effect can be easily administered in an appropriate amount.

The pharmaceutical composition of the present invention can be preferably administered parenterally, and more preferably can be administered topically parenterally. The topical administration includes local application to areas such as under the eyes, under the chin, under the arms, buttocks, calves, back, thighs, ankles, and abdomen. For example, the pharmaceutical composition of the present invention can be formulated as a preparation for parenteral administration (including a preparation for topical administration), and the preparation for parenteral administration can be a preparation for transdermal administration, a preparation for subcutaneous administration, a preparation for intravenous administration, a preparation for intramuscular administration, or a preparation for intraperitoneal administration, and more preferably a preparation for subcutaneous administration. In addition, the preparation for parenteral administration can be a preparation for single administration or multiple administration. The preparation for multiple administration can be administered up to a total of 6 times at intervals of 7 days to 1 month with a volume of about 0.2 ml, and can be manufactured to be suitable for administration up to a maximum of 60 times per day. As needed, the parenteral formulation may be administered repeatedly at sites spaced 0.5 to 2.0 cm apart.

The above-mentioned parenteral administration preparation includes a form of solution, emulsion, suspension, lyophilization, etc., and preferably includes a form of solution or emulsion. The above-mentioned liquid preparation can be filled into an ampoule, syringe, or vial, and more preferably can be filled into a syringe to reduce the occurrence of precipitation or gelation. In addition, the sterility of the preparation can be secured by sterilizing it with a sterilizing filter before filling, or by post-sterilizing it after filling, if possible.

The parenteral administration preparation in the form of the above liquid preparation can be prepared according to a formulation method commonly used in the field of pharmaceutical science using pharmaceutically acceptable additives and/or carriers. Accordingly, the pharmaceutical composition of the present invention can include pharmaceutically acceptable additives selected from the group consisting of isotonic agents, preservatives (or antibacterial agents), pH regulators, stabilizers, solubilizers, solubility enhancers, surfactants, antioxidants, and analgesics; and pharmaceutically acceptable carriers selected from the group consisting of organic solvents and aqueous solvents.

The pH adjusting agent includes a pH adjusting agent commonly used in injectable preparations, and examples thereof include, but are not limited to, diethanolamine, acetic acid, meglumine, sodium citrate, sodium hydroxide, adipic acid, citric acid, hydrochloric acid, lactic acid, sulfuric acid, tartaric acid, phosphoric acid, and the like. The composition of the present invention may contain the pH adjusting agent in a concentration of 0.1-5 mg/mL, preferably 0.1-4 mg/mL, more preferably 0.1-3 mg/mL, even more preferably 0.1-2 mg/mL, and even more preferably 0.1-1.5 mg/mL. The pharmaceutical composition of the present invention may have a pH in the range of pH 7.0 to pH 8.0, for example, a range of pH 7.3 to pH 8.0, and more specifically, a range of pH 7.5 to 8.0. A pharmaceutical composition having the above pH range can minimize pain and/or inflammation when administered topically. For example, the body of a normal healthy person maintains a pH of 7.2 to 7.8, except for gastric juice and urine, and among these, body fluids such as blood usually have a pH of around 7.4 (approximately a range of pH 7.35-7.45). Even a small change in the pH value can be sensitively reacted to, for example, in the case of a composition exceeding pH 8, the pH may be too high compared to the pH of the body fluid, which may cause side effects such as pain such as a burning sensation or an inflammatory reaction at the site of administration. Therefore, it is preferable for a composition to be administered into the body to have a pH value similar to that of the body fluid. The pharmaceutical composition of the present invention can have a range of pH 7.0 to pH 8.0, for example, a range of pH 7.3 to pH 8.0, and more specifically, a range of pH 7.5 to 8.0, and thus, when administered into the body, side effects such as pain such as a burning sensation or an inflammatory reaction can be minimized.

The pharmaceutical composition containing the deoxycholic acid (DCA) according to the present invention has an electrical conductivity of 5 mS/cm or less at 25°C. More specifically, the electrical conductivity at 25°C may be 0.1 mS/cm to 5 mS/cm, more specifically 0.1 mS/cm to 4 mS/cm, and even more specifically 0.1 mS/cm to 3 mS/cm.

The pharmaceutical composition containing deoxycholic acid (DCA) according to the present invention may substantially not include a pH buffer. Since the pharmaceutical composition containing deoxycholic acid (DCA) according to the present invention does not substantially use an ionic pH buffer, it can lower the electrical conductivity at 25°C and satisfy 5 mS/cm or less. In the past, it was common to use a pH buffer to maintain a certain range of pH. Generally, those used as the pH buffer include, for example, adipic acid, boric acid, calcium carbonate, calcium hydroxide, calcium lactate, tricalcium phosphate, sodium phosphate, citric acid, maleic acid, malic acid, sodium glutamate, sodium acetate, sodium bicarbonate, boric acid, trisodium citrate, sodium lactate, triethanolamine, and anhydrous sodium biphosphate, and sodium biphosphate and/or citric acid are representatively used. However, the inventor of the present invention confirmed that the pH is maintained at a constant range in the range of pH 7.0 to 8.0 even without including a pH buffer. Furthermore, by not including a pH buffer, gelation and precipitation do not occur even in the range of pH 7.0 to 8.0, so that the storage stability is significantly improved. In addition, since a pH buffer is not used to maintain pH, the local pH of the injection site can be recovered more quickly and injection pain can be significantly alleviated because there is no pH buffering effect by the buffer.

That the composition of the present invention substantially does not use the pH buffer means that the composition of the present invention substantially does not include a pH buffer and thus satisfies the electrical conductivity of 5 mS/cm or less at 25°C. Therefore, under the premise that the composition of the present invention has an electrical conductivity of 5 mS/cm or less at 25°C, the composition of the present invention may include a small amount of pH buffer. Specifically, the content of the pH buffer may be added at a concentration of 0.1 to 30 mM, 0.1 to 25 mM, 0.1 to 20 mM, 0.1 to 17 mM, 0.1 to 15 mM, 0.1 to 13 mM, 0.1 to 10 mM, 0.1 to 7 mM, or 0.1 to 5 mM based on the composition.

The pharmaceutical composition containing deoxycholic acid (DCA) according to the present invention can exhibit excellent storage stability without generating precipitate during the storage period despite having a low pH of pH 8.0 or lower, compared to the pharmaceutical composition containing conventional deoxycholic acid (DCA). In addition, since it has a low pH, it becomes similar to the pH of the human body, pH 7.4, and the local pH of the injection site is quickly recovered, so that injection pain can be significantly alleviated, and side effects such as a burning sensation and inflammation at the injection site can be minimized.

In addition, in the pharmaceutical composition containing the deoxycholic acid (DCA) according to the present invention, the additive may include at least one selected from the group consisting of a polyol, an amino acid, and a non-ionic surfactant. In the past, ionic substances such as sodium chloride acting as an isotonic agent were mainly used as additives, but in the pharmaceutical composition according to one embodiment of the present invention, at least one selected from the group consisting of a polyol, an amino acid, and a non-ionic surfactant can be used instead of the ionic substance such as sodium chloride as an additive. Accordingly, the electrical conductivity may be lower than in the case of using an ionic substance as in the past, and the electrical conductivity may satisfy 5 mS/cm or less at 25°C. More specifically, the electrical conductivity may be from 0.1 mS/cm to 5 mS/cm at 25°C, more specifically from 0.1 mS/cm to 4 mS/cm at 25°C, and even more specifically from 0.1 mS/cm to 3 mS/cm. The pharmaceutical composition according to one embodiment of the present invention can effectively prevent precipitation and gelation despite low pH, thereby improving storage stability.

The above polyol is a substance having a plurality of hydroxyl groups and may include sugars (reduced and non-reduced) and sugar alcohols. Non-limiting examples of the polyol include at least one selected from the group consisting of sucrose, glycerol, mannitol, sorbitol, propylene glycol, trehalose, ethyleneglycol, maltose, glucose, and polyethyleneglycol, and considering the effect of preventing precipitation and gelation, at least one of sucrose, glycerol, and mannitol is more preferably, but is not limited thereto.

The above amino acid may be at least one selected from the group consisting of histidine, methionine, glycine, alanine, valine, leucine, isoleucine, tyrosine, proline, phenylalanine, asparagine, glutamine, serine, threonine, cysteine, and tryptophane, and considering the effect of preventing precipitation and gelation, histidine and/or methionine are more preferably, but are not limited thereto.

The above nonionic surfactant may be at least one selected from the group consisting of poloxamer and polysorbate, and examples thereof include, but are not limited to, poloxamer 188, polysorbate 20, and polysorbate 80.

The pharmaceutical composition containing the deoxycholic acid (DCA) according to one embodiment of the present invention may most preferably use at least one of sucrose, glycerol, and mannitol, considering the effect of preventing precipitation and gelation.

The pharmaceutical composition containing deoxycholic acid (DCA) according to one embodiment of the present invention may contain at least one additive selected from the group consisting of the polyol, the amino acid, and the non-ionic surfactant at 0.1 to 500 mM, preferably 0.1 to 400 mM, and more preferably 0.3 to 350 mM, 0.3 to 300 mM, 0.3 to 250 mM, or 0.3 to 230 mM. By containing the additive within the above range, the osmotic pressure of the composition can be configured to be similar to that of body fluid.

When the pharmaceutical composition containing deoxycholic acid (DCA) of the present invention includes the polyol, particularly when it includes at least one of sucrose, glycerol and mannitol as the polyol, it may further include a pH regulator or pH buffer within a range of 5 mS/cm or less in terms of electrical conductivity at 25°C. The above-mentioned additionally added pH adjuster or pH buffer may be at least one selected from the group consisting of sodium citrate, sodium hydroxide, adipic acid, citric acid, phosphoric acid, sodium phosphate, disodium phosphate, monosodium phosphate, anhydrous sodium biphosphate, calcium carbonate, calcium hydroxide, calcium lactate, maleic acid, malic acid, sodium glutamate, sodium acetate, sodium bicarbonate, trisodium citrate, sodium lactate, and triethanolamine, and may be sodium phosphate and/or sodium citrate when the effect of preventing precipitation and gelation is taken into consideration. The amount of the pH regulator or pH buffer further added may be added within a range where the electrical conductivity of the composition of the present invention becomes 5 mS/cm or less at 25°C, and specifically, may be added at a concentration of 0.1 to 30 mM, 0.1 to 25 mM, 0.1 to 20 mM, 0.1 to 17 mM, 0.1 to 15 mM, 0.1 to 13 mM, 0.1 to 10 mM, 0.1 to 7 mM, or 0.1 to 5 mM based on the final composition.

Meanwhile, the pharmaceutical composition containing deoxycholic acid (DCA) according to the present invention may not contain benzyl alcohol. In addition, the pharmaceutical composition containing deoxycholic acid (DCA) according to the present invention may not contain benzalkonium chloride, benzalthonium chloride, phenol, cresol, chlorocresol, and chlorobutanol. Conventionally, aqueous solutions containing deoxycholic acid (DCA) have used benzyl alcohol as a preservative. However, in aqueous solutions containing benzyl alcohol, low concentrations of deoxycholic acid often cause precipitation after storage for a certain period of time, and in particular, benzyl alcohol can cause irritation when in contact with the skin, which is problematic. In addition, due to these problems of benzyl alcohol, there are cases of conventional aqueous solutions containing deoxycholic acid (DCA) containing benzalkonium chloride, benzalthonium chloride, phenol, cresol, chlorocresol, and chlorobutanol as preservatives instead of benzyl alcohol. However, due to the nature of preservatives that kill or prevent the reproduction of microorganisms, it has been known in many experimental and clinical studies that exposure to preservatives can cause irritation and toxicity to the human body. Therefore, it may be more desirable not to use such preservatives in single-dose formulations.

Accordingly, the pharmaceutical composition containing deoxycholic acid (DCA) according to the present invention may not contain benzyl alcohol, and may not contain benzalkonium chloride, benzalthonium chloride, phenol, cresol, chlorocresol, and chlorobutanol. Accordingly, the occurrence of precipitation and gelation during storage can be effectively suppressed, skin irritation upon skin contact can be reduced, and potential toxicity risk due to the formulation can be reduced.

The pharmaceutical composition containing the deoxycholic acid (DCA) according to the present invention can be used for lipolysis and can be used for the prevention or treatment of obesity.

In addition, the present invention may include a method for inducing lipolysis or a method for treating obesity, which comprises administering to a mammal in need of inducing lipolysis or treating obesity a pharmaceutical composition comprising a therapeutically effective amount of the deoxycholic acid or a pharmaceutically acceptable salt thereof.

Furthermore, the present invention provides the use of a pharmaceutical composition comprising deoxycholic acid or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for inducing lipolysis or preventing or treating obesity.

The above "treatment" means stopping or delaying the progression of a disease when used on a subject showing symptoms of the disease, and the above "prevention" means stopping or delaying the signs of the disease when used on a subject not showing symptoms of the disease but at a high risk of such.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[Experimental Example 1: pH Buffering Capacity Comparison Experiment]

10 mM sodium phosphate, 10 mM TRIS, and 10 mg/mL deoxycholic acid (obtained from Sigma-Aldrich) were dissolved in water for injection, and then NaOH aqueous solution or HCl aqueous solution was added to adjust the pH to 7.5. (When dissolving deoxycholic acid, NaOH aqueous solution was added little by little until deoxycholic acid was completely dissolved, and then HCl aqueous solution was added to adjust the pH.) Then, to confirm the buffering capacity in the range of pH 7.5 to 7.0, 40 mL of solution was taken, 40 ㎕ of 0.5 M HCl was added at a time, and the pH was measured. In addition, to confirm the buffering capacity in the range of pH 7.5 to 8.0, 40 mL of solution was taken, 40 ㎕ of 0.5 M NaOH was added at a time, and the pH was measured. To calculate the buffering capacity of each solution, the amount of added base or acid was linearly regression analyzed against the change in pH to derive the slope between the two variables. The results are shown in Table 1, Figures 1 and 2.

Buffering capacity (mEq/L/pH) pH 7.5-7.0 pH 7.5-8.0 10mM sodium phosphate 4.9 3.1 10mM Tris 2.2 4.0 10 mg/mL deoxycholic acid 4.0 1.2

Referring to Table 1, Figures 1 and 2, it was confirmed that sodium phosphate, TRIS pH buffer, which is commonly used around pH 8.0, and 10 mg/mL deoxycholic acid (DCA) exhibited similar buffering capacity, and in particular, the pH buffering capacity of 10 mg/mL deoxycholic acid (DCA) was excellent in the pH range of 7.5-7.0.

Example 1

To 80-85 ml of water for injection at about 25-30°C, 25 mg/mL of deoxycholic acid (obtained from Sigma-Aldrich) was added, and 6N sodium hydroxide aqueous solution was added little by little while stirring at 300 rpm to dissolve the deoxycholic acid. After visually confirming that all deoxycholic acid was dissolved, additional stirring was performed at 300 rpm for about 30 minutes to ensure sufficient dissolution. The pH of the obtained solution was adjusted to pH 8.0 with 0.5 N hydrochloric acid, and then the final volume was adjusted to 100 ml with water for injection. After sterilization filtration through a membrane filter (PES 0.22 μm filter, Millipore, USA), the solution was filled into vials, and stored at 5°C. The electrical conductivity at 25°C was 4.9 mS/cm.

Comparative Example 1

It was prepared in the same manner as in Example 1, except that the pH was adjusted to 8.0 and 20 mM sodium phosphate was added. The electrical conductivity at 25°C was 9.1 mS/cm.

Comparative Example 2

It was prepared in the same manner as in Example 1, except that the pH was adjusted to 8.0 and 20 mM TRIS was added. The electrical conductivity at 25°C was 5.6 mS/cm.

Comparative Example 3

It was prepared in the same manner as in Example 1, except that the pH was adjusted to 8.0 and 20 mM sodium bicarbonate was added. The electrical conductivity at 25°C was 9.0 mS/cm.

[Experimental Example 1: Storage Stability Experiment]

The formulations manufactured in Example 1 and Comparative Examples 1 to 3 were immediately taken out of a 5°C chamber and then allowed to warm to room temperature for 1 hour at 25°C to compare whether they precipitated or gelled. The results are shown in Table 2.

Immediately after the release After 1 hour of room temperature Gelation Sedimentation Gelation Sedimentation Example 1 0 0 0 0 Comparative Example 1 2.5 2 1 2 Comparative Example 2 0 1 0 1 Comparative Example 3 0 0 0 1

* 0: No sediment or gel visible to the naked eye by a trained operator

1: Presence of sediment or gel visible to the naked eye by a trained operator

2: Presence of sediment or gel at a level that can be detected by an untrained worker with the naked eye within seconds.

3: Presence of sediment or gel at a level immediately visible to the naked eye by an untrained worker.

Referring to Table 2 above, in Comparative Examples 1 to 3 where the electrical conductivity exceeded 5 mS/cm using a pH buffer, precipitation and/or gelation occurred, whereas in Example 1 where the electrical conductivity was 5 mS/cm or less and no pH buffer was used, it could be confirmed that precipitation and/or gelation did not occur immediately after dissolution and even after warming to room temperature.

Examples 2 to 6, Comparative Example 4

To 80-85 ml of water for injection at about 25-30°C, 10 mg/mL of deoxycholic acid (obtained from Sigma-Aldrich) was added, and 6N sodium hydroxide aqueous solution was added little by little while stirring at 300 rpm to dissolve the deoxycholic acid. After visually confirming that all deoxycholic acid was dissolved, additional stirring was performed at 300 rpm for about 30 minutes to ensure sufficient dissolution. The pH of the obtained solution was adjusted to pH 7.5 with 0.5 N hydrochloric acid, and the additives shown in Table 3 below were added and dissolved, and the final volume was adjusted to 100 ml with water for injection. After sterilization filtration through a membrane filter (PES 0.22 μm filter, Millipore, USA), it was filled into a vial, and stored at 5°C.

Comparative Example 5

Sodium phosphate dibasic was added to 80-85 ml of water for injection at about 25-30℃ to 1.42 mg/mL and sodium hydroxide was added to 1.43 mg/mL, and stirred for 20 minutes. Deoxycholic acid (obtained from Sigma-Aldrich) was added to 10 mg/mL, and stirred at 300 rpm for about 60 minutes to sufficiently dissolve. The pH of the obtained solution was adjusted to pH 7.5 with 0.5 N hydrochloric acid, and then 4.38 mg/mL of sodium chloride and 9 mg/mL of benzyl alcohol as additives were added, and stirred sufficiently for about 10 minutes to dissolve. The final volume was adjusted to 100 ml with water for injection. After sterilization filtration through a membrane filter (PES 0.22 μm filter, Millipore, USA), it was filled into a vial, and stored at 5℃.

[Experimental Example 2: Storage Stability Experiment]

The preparations manufactured in Examples 2 to 6 and Comparative Examples 4 to 5 were stored at 5°C for 2 months and then compared for precipitation and gelation. The results are shown in Table 3.

Example 2 Example 3 Example 4 Example 5 Example 6 Comparative Example 4 Comparative Example 5 Additives Sucrose 100mM Glycerol 100mM Histidine 50mM Methionine 50mM Poloxamer 188** 0.6mM NaCl
50mM NaCl
74.9mM Electrical conductivity
(mS/cm) 2.3 2.4 2.7 2.5 2.3 7.4 11.6 Osmotic pressure
(mOsm/kg) 148 146 93 88 38 147 287 pH 7.49 7.50 7.52 7.53 7.52 7.45 7.48 Sedimentation occurs after 2 months at 5℃
Whether* 0 0 0 0 0 3 3

* 0: No sediment or gel visible to the naked eye by a trained operator

1: Presence of sediment or gel visible to the naked eye by a trained operator

2: Presence of sediment or gel at a level that can be detected by an untrained worker with the naked eye within seconds.

3: Presence of sediment or gel at a level immediately visible to the naked eye by an untrained worker.

** Poloxamer188 has an average molecular weight of 8400 daltons, which is converted to molar concentration of 5 mg/mL.

Referring to Table 3, it was confirmed that in Comparative Example 4, which used an ionic substance such as NaCl as an additive, precipitation occurred, whereas in Examples 2 to 6, which used polyol, amino acid, and nonionic surfactant as additives, no precipitation occurred even at pH 7.5.

Examples 7-22

It was manufactured in the same manner as Example 2, except that additives were added and pH was adjusted as shown in Table 4 below.

Comparative Example 6

It was manufactured in the same manner as in Comparative Example 5, except that the pH was adjusted to 8.3.

Comparative Example 7

It was manufactured in the same manner as in Comparative Example 5, except that benzyl alcohol was not included.

Comparative Example 8

Sodium phosphate dibasic (1.42 mg/mL) was added to 80-85 ml of water for injection at about 25-30°C and stirred for 20 minutes. Sodium hydroxide was dissolved in the obtained solution to about 1.07 mg/mL based on the final volume, and 10 mg/mL deoxycholic acid (obtained from Sigma-Aldrich) was added and stirred at 300 rpm for about 60 minutes to dissolve. The pH of the obtained solution was adjusted to pH 8.3 with hydrochloric acid, and then sodium chloride as an additive was added to a final volume of 147 mM and the final volume was adjusted to 100 ml with water for injection. After filtering through a membrane filter (PES 0.22 μm filter, Millipore, USA), it was filled into a vial. The filled vial was stored at 5°C.

Comparative Example 9

It was manufactured in the same manner as Comparative Example 8, except that the pH was adjusted to 7.5.

Sucrose (mM) Glycerol (mM) Met
(mM) P188**
(mM) PS20
(mM) ppH Electrical Conductivity (mS/cm) Osmotic pressure
(mOsm/kg) Example 7 87.6 162.9 - - - 7.5 1.7 296 Example 8 146.1 108.6 - - - 7.5 1.6 306 Example 9 204.5 54.3 - - - 7.5 1.6 314 Example 10 146.1 108.6 20.1 - - 7.5 1.6 321 Example 11 146.1 108.6 - 0.36 - 7.5 1.6 307 Example 12 146.1 108.6 - - 2.44 7.5 1.6 305 Example 13 146.1 108.6 20.1 0.36 - 7.5 1.5 325 Example 14 146.1 108.6 20.1 - 2.44 7.5 1.5 326 Example 15 87.6 162.9 - - - 8.0 1.7 292 Example 16 146.1 108.6 - - - 8.0 1.6 296 Example 17 204.5 54.3 - - - 8.0 1.6 312 Example 18 146.1 108.6 20.1 - - 8.0 1.7 319 Example 19 146.1 108.6 - 0.36 - 8.0 1.6 304 Example 20 146.1 108.6 - - 2.44 8.0 1.6 301 Example 21 146.1 108.6 20.1 0.36 8.0 1.7 331 Example 22 146.1 108.6 20.1 - 2.44 8.0 1.7 321 Comparative Example 5 - 7.5 11.6 287 Comparative Example 6 - 8.3 11.7 288 Comparative Example 7 - 7.5 11.5 204 Comparative Example 8 - 8.3 17.7 318 Comparative Example 9 - 7.5 18.0 317

* Met: Methionine

P188: Poloxamer 188

PS20: polysorbate 20

** Poloxamer188 has an average molecular weight of 8400 daltons, which is converted to molar concentration of 5 mg/mL.

[Experimental Example 3: Storage Stability Experiment]

The formulations manufactured in Examples 7 to 22 and Comparative Examples 5 to 9 were filled into vials or syringes and stored at 5°C for 11 days, 1 month, and 2 months. Precipitation and gelation were compared immediately after dispensing and after being cooled to room temperature at 25°C. The results are shown in Table 5.

11th 1 month 2 months vial
(Immediately after drying/after returning to room temperature) syringe
(Immediately after drying/after returning to room temperature) vial
(Immediately after drying/after returning to room temperature) syringe
(Immediately after drying/after returning to room temperature) vial
(Immediately after drying/after returning to room temperature) syringe
(Immediately after drying/after returning to room temperature) Example 7 - - - - - - Example 8 - - - - - - Example 9 - - - - - - Example 10 - - - - - - Example 11 - - - - - - Example 12 - - - - - - Example 13 - - - - - - Example 14 - - - - - - Example 15 - - - - - - Example 16 - - - - - - Example 17 - - - - - - Example 18 - - - - - - Example 19 - - - - - - Example 20 - - - - - - Example 21 - - - - - - Example 22 - - - - - - Comparative Example 5 Some gels/- Some gels/- Gel/- Gel/- Gel/- Gel/- Comparative Example 6 - - - - - - Comparative Example 7 Gel+precipitation/gel+precipitation Gel+precipitation/gel+precipitation Gel+precipitation/gel+precipitation Gel+precipitation/gel+precipitation Gel+precipitation/gel+precipitation Gel+precipitation/gel+precipitation Comparative Example 8 Gel/sedimentation - Gel+sediment/some gel+sediment Some gels/- Gel+sediment/some gel+sediment Some gels/- Comparative Example 9 Gel+precipitation/gel+precipitation Gel+precipitation/gel+precipitation Gel+precipitation/gel+precipitation Gel+precipitation/gel+precipitation Gel+precipitation/gel+precipitation Gel+precipitation/gel+precipitation

* - : No gel or sedimentation

Referring to Table 5, it can be confirmed that in the cases of Examples 7 to 22, no gel or precipitation occurred in the vials and syringes immediately after dispensing and after returning to room temperature after 11 days, 1 month, and 2 months of storage. In the case of Examples 7 to 22, since they do not contain benzyl alcohol and the pH is similar to that in the body, they are expected to be excellent in terms of safety and pain during administration.

On the other hand, when looking at Comparative Examples 5 to 9, it was confirmed that benzyl alcohol was effective in preventing gelation/precipitation, but benzyl alcohol has safety issues in countries including several European countries. Meanwhile, it was confirmed that the degree of precipitation was less in the syringe compared to the vial.

Example 23-34

To 80-85 ml of water for injection at about 25-30°C, sodium hydroxide was added to a final concentration of 1.07 mg/mL and deoxycholic acid (DCA, obtained from Sigma-Aldrich) was added to a final concentration of 10 mg/mL, and then stirred at 300 rpm for about 20 minutes until sufficiently dissolved. To the obtained solution, sodium phosphate monobasic, sodium citrate, glycerol, and sucrose were added in concentrations according to Table 6 below, and further stirred until all were dissolved. Subsequently, if necessary, 1 N sodium hydroxide was added in small amounts to adjust the pH as shown in Table 6 below, and then the final volume was adjusted to 100 ml with water for injection. After sterilization filtration with a membrane filter (PES 0.22 μm filter, Millipore, USA), the solution was filled into vials and stored at 55°C for 3 months, 40°C and 25°C for 4 months, and 5°C for 5 months. All vials were clean and free of foreign matter immediately after filling, and had an osmotic pressure of 284-306 mOsm/kg and an electrical conductivity of 2.79-3.87 mS/cm.

DCA (mg/mL) NaOH (mg/mL) Sodium Phosphate Monobasic (mM) Sodium citrate(mM) Sucrose(mM) Glycerol (mM) pH Electrical Conductivity (mS/cm) Osmolality (mOsm/kg) Example 23 10 1.07 5 5 220 0 7.8 2.82 297 Example 24 10 1.07 5 5 0 220 7.8 3.25 286 Example 25 10 1.07 5 5 110 110 7.8 3.02 292 Example 26 10 1.07 10 5 220 0 7.8 3.38 306 Example 27 10 1.07 10 5 0 220 7.8 3.87 294 Example 28 10 1.07 10 5 110 110 7.8 3.62 303 Example 29 10 1.07 5 5 220 0 7.6 2.79 300 Example 30 10 1.07 5 5 0 220 7.6 3.23 287 Example 31 10 1.07 5 5 110 110 7.6 2.99 291 Example 32 10 1.07 5 5 220 0 8.0 2.87 298 Example 33 10 1.07 5 5 0 220 8.0 3.29 284 Example 34 10 1.07 5 5 110 110 8.0 3.09 292

After storage for the specified storage period at each temperature, the vials were taken out and tested for foreign substances. After storage at 55°C for 3 months, 40°C for 4 months, 25°C for 4 months, and 5°C for 5 months, no foreign substances or gelation occurred in any of the vials of Examples 23-34. This confirmed that all of the compositions of Examples 23-34 were compositions that stably maintained DCA.

Example 35-38

The gelation after low-temperature exposure and the time it takes for the gel to return to a liquid state after gelation were evaluated as follows according to the liquid formulation of DCA. 1.07 mg/mL of sodium hydroxide and 10 mg/mL of deoxycholic acid (DCA, obtained from Sigma-Aldrich) were added to 70-90 mL of water for injection at about 20-30°C, and the mixture was stirred until sufficiently dissolved. Sodium phosphate monobasic and/or sodium citrate were added to the obtained solution at the concentrations shown in Table 7 below, to form four types of formulations using the basic formulations of Examples 35-38. In the composition of the basic formulation examples 35-38 above, additional polyol was added and stirred additionally so that polyol was not added, mannitol was 25 mM, 50 mM, 250 mM, and 500 mM, sucrose was 25 mM, 50 mM, 250 mM, 500 mM, glycerol was 25 mM, 50 mM, 250 mM, 500 mM, and all were dissolved. If necessary, 1 N HCl or 1 N NaOH was added in small amounts to adjust the pH to 7.5, and a final volume of 100 mL was made with water for injection, producing a total of 52 formulations. After each of these was sterilized and filtered through a membrane filter (PES 0.22 μm filter, Millipore, USA), 2 mL was filled into vials, and stored at 5°C for 23 days.

DCA (mg/mL) NaOH (mg/mL) Sodium Phosphate Monobasic (mM) Sodium citrate(mM) Polyol pH Example 35 10 1.07 0 0 Polyol-free or with 25 mM, 50 mM, 250 mM or 500 mM of one of Mannitol/Sucrose/Glycerol 7.5 Example 36 10 1.07 5 0 Example 37 10 1.07 0 5 Example 38 10 1.07 5 5

After 3 days of storage at 5℃, after 23 days of storage, the presence or absence of gelation was checked at 15 minutes, 30 minutes, 45 minutes, 60 minutes, and 90 minutes after being stored at room temperature, and the presence or absence of foreign substances was checked at 90 minutes after being stored at room temperature. When observing the presence or absence of gelation after 3 days and 23 days of storage, gelation was checked in a cold room to ensure that there was no effect due to exposure to room temperature.

All formulations of Examples 35-38 did not form gels after storage at 5°C for 3 days. Accordingly, all formulations were confirmed to be stable for up to 3 days at 5°C.

The formulations of Example 35 group, which did not contain either sodium phosphate or sodium citrate, did not form gels even when stored at 5°C for up to 23 days. The formulations of Examples 36, 37, and 38 groups did not form gels until 5°C for up to 3 days, but gels were confirmed to form after 23 days of storage. The presence or absence of gels was confirmed at 15, 30, 45, 60, and 90 minutes after exposure to room temperature for these formulations, and the times at which gels disappeared (time and minutes after exposure) are shown in Table 8 below.

Salt Polyol Time taken for the gel to change to liquid after being exposed to room temperature at 5℃ (min)* Polyol 0mM Polyol 25mM Polyol 50mM Polyol 250mM Polyol 500mM Example 35 doesn't exist Mannitol 0 0 0 0 0 Sucrose 0 0 0 0 Glycerol 0 0 0 0 Example 36 Sodium Phosphate monobasic 5mM Mannitol 30 30 30 30 30 Sucrose 30 30 15 0 Glycerol 30 30 30 15 Example 37 Sodium Citrate 5mM Mannitol 90 45 45 45 30 Sucrose 30 30 15 0 Glycerol 30 30 30 15 Example 38 Sodium Phosphate monobasic 5mM,
Sodium Citrate 5mM Mannitol 90 90 90 90 90 Sucrose 90 45 45 30 Glycerol 90 45 45 30

*: When time is 0, no gel occurs after 23 days of storage at 5℃.

In the group of Example 36, the formulation containing 500 mM sucrose did not form a gel even after storage at 5°C for 23 days. The formulation containing 250 mM sucrose disappeared after 15 minutes of being stored at room temperature, and the other formulations all disappeared after 30 minutes, confirming the absence of gel. No foreign substances were found in any vials as a result of a foreign substance test. Therefore, it was confirmed that all formulations of the group of Example 36, even if a gel is formed at a low temperature, return to a liquid state when left at room temperature for about 30 minutes and can be injected into a patient. In addition, it was confirmed that the formulation containing 500 mM sucrose has a gel prevention effect, and when containing 250 mM sucrose, the time taken for a gel formed at a low temperature to return to a liquid state is shortened, thereby increasing the convenience of use.

In the case of the Example 37 group, all compositions including polyols took a shorter time to become liquid than those without polyols. No gel was found in the formulations including polyols within 45 minutes, and no foreign substances were found in the formulations without polyols after 90 minutes. Accordingly, it was confirmed that all formulations of Example 37 became liquid after 90 minutes of being left at room temperature even if a gel was formed, and that they were injectable to patients. In addition, it was confirmed that formulations including polyols such as mannitol, sucrose, or glycerol were more convenient to use because they changed to liquid more quickly when left at room temperature even if a gel was formed during low-temperature storage.

For group Example 38, the formulations containing 50 mM, 250 mM, or 500 mM sucrose or glycerol took a shorter time to return to a liquid phase when left at room temperature than the other formulations, and for the other samples, they were all liquid after 90 minutes of storage at room temperature. Accordingly, it was confirmed that all formulations of Example 38 were all liquid after 90 minutes of storage at room temperature even if a gel was formed, and that they were injectable to patients. In addition, it was confirmed that formulations using polyols were more convenient to use because gels formed at low temperatures returned to a liquid phase more quickly at room temperature when an appropriate polyol was included.

Examples 39-44

To evaluate the effect of metal ions on a liquid formulation of deoxycholic acid (DCA), a solution containing 10 mg/mL of DCA, 1.07 mg/mL of sodium hydroxide, pH 7.8, and an appropriate amount of HCl was prepared. The preparation method was the same as the preparation method of the polyol-free formulation in Example 35, except that the final pH was adjusted to 7.8. Iron chloride was added to the prepared solution to an iron concentration of 10 ppm, and foreign substances were observed with the naked eye, confirming that metal ions could cause deoxycholic acid (DCA) precipitation. Metal ions can originate from stainless steel of equipment used in the production process, impurities of excipients, packaging containers, etc.

In order to confirm whether sodium citrate improves the stability of a liquid deoxycholic acid formulation against metal ions, the following formulation preparation and stability test were conducted. The compositions of Examples 39-44 were prepared in a similar manner to the compositions of Examples 35-38. The prepared compositions were stored at room temperature for 6 days in a container made of SUS316L, filtered using a membrane filter made of PES material, and then filled into vials at 2 mL each, stored in a 55°C stability chamber for 16 days, and then visually observed to compare the degree of foreign matter formation. It was confirmed that the degree of foreign matter formation was significantly reduced in the compositions of Examples 40 and 42 containing sodium citrate compared to Examples 39 and 41 having the same composition but not containing sodium citrate, and thus it was confirmed that sodium citrate contributes to the stability of the deoxycholic acid (DCA) formulation. In addition, when comparing the compositions of Examples 41 and 43 with the composition of Example 39, it was confirmed that phosphate and glycerol also contributed to the stability of the deoxycholic acid formulation.

DCA (mg/mL) NaOH (mg/mL) Sodium Phosphate Monobasic (mM) Glycerol (mM) Sodium citrate(mM) pH 55℃ 16d later
Degree of foreign body formation* Example 39 10 1.07 0 0 7.5 3 Example 40 10 1.07 0 5 7.5 1 Example 41 10 1.07 5 0 7.5 2 Example 42 10 1.07 5 5 7.5 0 Example 43 10 1.07 5 220 0 7.5 0 Example 44 10 1.07 5 220 5 7.5 0

* 0: No sedimentation visible to the naked eye by a trained operator

1: Presence of sediment that can be visually observed by a trained operator

2: Presence of sedimentation levels that can be detected visually by an untrained worker within seconds.

3: Sedimentation at a level that can be readily detected by the naked eye by an untrained worker.

Claims (18)

Containing deoxycholic acid (DCA) or a pharmaceutically acceptable salt thereof; a pH buffer; and a pharmaceutically acceptable nonionic additive,
The pH is 7.0 to 8.0,
The above nonionic additive is a polyol,
The above polyol is at least one selected from the group consisting of sucrose, glycerol and mannitol,
The above pH buffer is at least one selected from the group consisting of a citrate buffer, a phosphate buffer and a TRIS buffer,
A pharmaceutical composition which does not contain a preservative.
In claim 1,
A pharmaceutical composition comprising the deoxycholic acid or a pharmaceutically acceptable salt thereof in a concentration of 1 mg/mL to 50 mg/mL.
In claim 1,
A pharmaceutical composition wherein the nonionic additive is included in a concentration of 0.1 mM to 500 mM.
delete delete In claim 1,
A pharmaceutical composition wherein the pH buffer is included at a concentration of 0.1 mM to 30 mM.
In claim 1,
The above citrate buffer is at least one selected from the group consisting of citric acid, sodium citrate and trisodium citrate,
A pharmaceutical composition wherein the phosphate buffer is at least one selected from the group consisting of phosphoric acid, sodium phosphate, sodium phosphate monobasic, and sodium phosphate dibasic.
In claim 7,
A pharmaceutical composition wherein the citrate buffer is sodium citrate and the phosphate buffer is sodium phosphate.
In claim 1,
A pharmaceutical composition wherein the pharmaceutical composition does not contain sodium chloride.
In claim 1,
A pharmaceutical composition wherein the preservative is at least one selected from the group consisting of benzyl alcohol, benzalkonium chloride, benzalthonium chloride, phenol, cresol, chlorocresol and chlorobutanol.
In claim 1,
The above pharmaceutical composition is a pharmaceutical composition having an osmotic pressure similar to that of a body fluid.
In claim 11,
A pharmaceutical composition having an osmotic pressure of 250 mOsm/kg to 330 mOsm/kg.
In claim 12,
A pharmaceutical composition having an osmotic pressure of 284 mOsm/kg to 306 mOsm/kg.
In claim 1,
The above pharmaceutical composition is a pharmaceutical composition having an electrical conductivity of 5 mS/cm or less at 25°C.
In claim 1,
A pharmaceutical composition wherein the above pharmaceutical composition is formulated as an aqueous parenteral administration preparation.
In claim 1,
The above pharmaceutical composition is a pharmaceutical composition for subcutaneous injection.
In claim 1,
The above pharmaceutical composition is a pharmaceutical composition used for decomposing fat.
In claim 1,
The above pharmaceutical composition is a pharmaceutical composition for preventing or treating obesity.