Molecule conjugate of bile acid or bile salt and pharmaceutical composition for local fat removal

Abstract

The present invention relates to a molecular assembly of a bile acid or a bile acid salt and a composition containing the same. Specifically, it relates to a skin-penetrating deoxycholic acid formulation that dramatically improves patient compliance by developing deoxycholic acid, a lipolytic pharmacological substance, as a topical agent rather than a conventional injectable.

Description

Technology Field

[0001] The present invention relates to a molecular aggregate of a bile acid or bile acid salt and a pharmaceutical composition for local fat removal containing the same. Specifically, the invention relates to a molecular aggregate of a bile acid or bile acid salt, which is a lipolytic pharmacological substance, developed as a topical skin preparation rather than an existing injectable, thereby dramatically improving patient compliance, and a pharmaceutical composition for local fat removal containing the same. Background Technology

[0002] Bile acids and bile salts can emulsify fats and promote the action of lipase, a digestive enzyme, thereby dissolving fatty acids. Since these components are also present in the human digestive system, they can play a role in breaking down ingested fats to aid in digestion and absorption. Utilizing this pharmacological mechanism, the global company Allergan developed a fat removal injection using deoxycholic acid, a type of bile acid, and subsequently developed Belkyra™ (in Canada). Although Belkyra™ (in Canada) is an injectable medication that improves double chin by delivering it subcutaneously to induce irreversible fat cell destruction and then promoting the production of new collagen in the treatment area—a mechanism that carries less risk than liposuction surgery—it has been inconvenient. This is because it requires the use of a medical professional and necessitates multiple follow-up visits due to side effects such as pain, inflammation, bruising, swelling, contusions, and, in severe cases, facial muscle weakness and jaw nerve damage. Prior art literature

[0003] (Patent Document 0001) KR 10-2061001 B1 The problem to be solved

[0004] The present invention aims to provide a substance that ensures patient convenience and eliminates the side effects of injections, by applying a molecular aggregate of bile acid or bile acid salt capable of skin penetration to the skin, rather than a surgical method of delivering drugs by direct injection using a needle and syringe.

[0005] In addition, the present invention aims to provide a skin-permeable bile acid molecular aggregate in which storage stability is confirmed by mass-producing a skin-permeable bile acid or bile acid salt molecular aggregate and efficacy is confirmed as a result of application to animals.

[0006] In addition, the present invention aims to provide a pharmaceutical composition for localized fat removal capable of penetrating the skin, comprising the above molecular assembly. means of solving the problem

[0007] The present invention provides a molecular aggregate in which molecules of bile acid or bile acid salt are physically bonded, wherein the molecular aggregate is formed by a composition containing water, and the molecular aggregate in the composition has an aggregated structure.

[0008] In addition, according to one embodiment of the present invention, a molecular assembly can be provided in which the pH of the molecular assembly is greater than 8.5 and less than 10.

[0009] In addition, according to one embodiment of the present invention, a molecular aggregate can be provided in which the average particle size of the molecular aggregate is 1.0 to 10 nm or less.

[0010] In addition, according to one embodiment of the present invention, the molecular aggregate may provide an amorphous molecular aggregate.

[0011] In addition, according to one embodiment of the present invention, a molecular aggregate can be provided in which the rate of change in concentration over 12 months under accelerated conditions of 40±2℃ / 75±5% of the molecular aggregate is greater than 1 and less than 10%.

[0012] In addition, according to one embodiment of the present invention, a molecular aggregate can be provided in which the rate of change in particle size over 12 months under 40±2℃ / 75±5% acceleration conditions of the molecular aggregate is greater than 1 and less than 10%.

[0013] In addition, according to one embodiment of the present invention, a molecular aggregate can be provided in which the rate of change of pH over 12 months under 40±2℃ / 75±5% accelerated conditions of the molecular aggregate is greater than 0 and less than 5%.

[0014] In addition, according to one embodiment of the present invention, the bile acid may be any one of the group consisting of cholic acid, kenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid, and the bile acid salt may be any one of the salts of the group consisting of cholic acid, kenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid, thereby providing a molecular assembly.

[0015] In addition, according to one embodiment of the present invention, a pharmaceutical composition for local fat removal comprising the molecular aggregate can be provided.

[0016] In addition, according to one embodiment of the present invention, the composition can provide a pharmaceutical composition for local fat removal that is applied as a topical agent to the skin in a non-surgical, non-invasive method.

[0017] In addition, according to one embodiment of the present invention, the composition may further provide a pharmaceutical composition for local fat removal comprising one or more selected from the group consisting of glycerin, chia seed oil, glucan, hyaluronic acid, honeysuckle extract, collagen, ceramide, lecithin, betaine, trehalose, panthenol, squalane, caprylic / capric triglyceride, butylene glycol, propanediol, pentylene glycol, sodium levulinate, hydrogenated lecithin, and sodium hyaluronate.

[0018] In addition, according to one embodiment of the present invention, the composition may provide a pharmaceutical composition for local fat removal comprising 0.05 to 10 weight% of the molecular aggregate.

[0019] In addition, according to one embodiment of the present invention, a pharmaceutical composition for localized fat removal can be provided for treating a disease selected from the group consisting of obesity, fat redistribution syndrome, submental fat (a double chin formed by localized fat accumulation), lower eyelid fat herniation, lipomas, Dercum's disease, lipodystrophy, buffalo hump dystrophy, and combinations thereof.

[0020] In addition, according to one embodiment of the present invention, a pharmaceutical composition for localized fat removal can be provided, which is localized to a region selected from the group consisting of abdominal-neck fat, thigh fat, upper arm fat, visceral fat accumulation, fat generated after breast augmentation surgery, chest fat, fat spread around the arm, under-eye fat, under-chin fat, buttock fat, calf fat, back fat, thigh fat, ankle fat, cellulite, and combinations thereof.

[0021] In addition, according to one embodiment of the present invention, a pharmaceutical composition for local fat removal can be provided, which is used as any one of the formulations in the group consisting of gel, cream, ointment, unguent, spray, thickened formulation, and poultice.

[0022] In addition, according to one embodiment of the present invention, a pharmaceutical composition for local fat removal can be provided, wherein the concentration of bile acid measured in plasma 3 hours after application to the skin is 0.001 to 0.2 μg / ml. Effects of the invention

[0023] The present invention has the advantage of reducing the inconvenience of administration methods by allowing direct application to the skin, rather than a surgical method of delivering drugs by injecting them directly using needles and syringes. In addition, unlike existing product forms, it has the advantage of possessing excellent effects in skin permeability, fat breakdown, and reduction of fat accumulation, despite not being an injectable formulation.

[0024] In addition, the skin gap is 80 nm, so conventional pharmaceutical products had difficulty penetrating into the skin, but in the case of the present invention, it has a small size of 0.5 to 5 nm, so it has the effect of increasing skin permeability.

[0025] In addition, nano-sized dispersed particles have low storage stability because they are easily exposed to aggregation or Ostwald ripening phenomena, whereas the molecular aggregates and compositions of the present invention have excellent stability, with minimal changes in appearance, pH, and particle size under accelerated conditions of 40 ± 2℃ / 75 ± 5%.

[0026] In addition, the present invention has the advantage of not necessarily requiring the use of third substances, such as surfactants, micelles, cyclodextrin, lipids, albumin, water-soluble polymers, stabilizers / dispersants, nanoparticles, porous particles, etc., which were additionally used in conventional technology in addition to API and water to improve solubility. Brief explanation of the drawing

[0027] Figure 1 shows the results of Zetasizer particle size analysis of molecular aggregates of deoxycholic acid. Figure 2 is a TEM image of a molecular aggregate of deoxycholic acid. Figure 3 is a graph regarding the calibration curve of the molecular aggregate of deoxycholic acid. Figure 4 is a figure regarding the ability of a molecular aggregate of deoxycholic acid to destroy adipocytes. Figure 5 shows the differentiation process of preadipocytes into adipocytes. Figure 6 is a figure regarding the fat cell destruction ability of a molecular aggregate of deoxycholic acid. Figure 7 is a figure showing the skin penetration amount measured by collecting subcutaneous tissue after injecting a deoxycholic acid precursor once into the subcutaneous tissue of a living mouse and subsequently applying equal amounts of the deoxycholic acid precursor and the deoxycholic acid molecular aggregate of the present invention onto the skin. FIG. 8 is a figure comparing the amount of skin penetration and the amount of distribution within the skin tissue after collecting subcutaneous tissue, following a single injection of a deoxycholic acid precursor into the subcutaneous tissue of a living mouse and the subsequent application of equal amounts of the deoxycholic acid precursor and the deoxycholic acid molecular aggregate of the present invention onto the skin. Figure 9 is a figure comparing the plasma distribution amounts of a deoxycholic acid precursor and a molecular aggregate over time when the same amount was applied to the skin of a living mouse after a single subcutaneous injection of the deoxycholic acid precursor and a molecular aggregate after a single application to the skin. Figure 10 shows the results of H&E staining to evaluate the histological effects on subcutaneous tissue according to application time of a deoxycholic acid precursor and molecular aggregates applied to the skin in the same amount as a single subcutaneous injection of the deoxycholic acid precursor into the skin of a living mouse. Figure 11 is a figure showing the increase in the area of ​​the blue stained region due to the destruction of fat cells or an increase in collagen, as a result of Masson's Trichrome staining to evaluate the histological effect on subcutaneous tissue according to application time of the deoxycholic acid precursor and molecular aggregate applied to the skin of a living mouse in the same amount as when the deoxycholic acid precursor was injected subcutaneously once. Figure 12 compares the change in body weight with a negative control group when 1.0% and 2.5% of deoxycholic acid molecular aggregates were applied to the skin of obese mice twice a day for 4 weeks (N=5). Figure 13 compares the change in waist circumference with a negative control group when 1.0% and 2.5% of molecular aggregates of deoxycholic acid were applied to the skin of obese mice twice a day for 4 weeks (N=5). Figure 14 is a comparison of the change in waist circumference with a negative control group when 1.0% and 2.5% of molecular aggregates of deoxycholic acid were applied to the skin of obese mice twice a day for 4 weeks. Specific details for implementing the invention

[0028] Poorly soluble drugs have extremely low saturation solubility in aqueous solutions and extremely high interfacial tension / energy with water, making them thermodynamically unstable; consequently, they undergo sedimentation or phase separation. Therefore, to increase the drug content (i.e., solubility) to a pharmacologically meaningful level, a third substance was required, such as a surface active agent that lowers the interfacial energy or a carrier capable of holding a high amount of the drug.

[0029] These existing solubility enhancement technologies commonly require the use of a third substance other than API and water, or act as a key factor in improving solubility. For example, surfactants, micelles, cyclodextrin, lipids, albumin, water-soluble polymers, stabilizers / dispersants, nanoparticles, and porous particles are examples of such third substances.

[0030] However, the inventors of the present invention have completed the present invention by confirming that, even without using the third material mentioned above, a molecular aggregate utilizing polar interactions or hydrogen bonds is manufactured to make the surface of the structure hydrophobic, thereby increasing permeability to the phospholipid membrane, and by controlling the particle size of the aggregate to a level of 1.0 to 10 nm, the permeability to skin pores having a size of about 80 nm is increased, and through this, pharmacological substances are delivered to fat cells within the skin, thereby exhibiting excellent effects in fat breakdown and reduction of fat accumulation.

[0031] The following explains this in more detail.

[0033] terminology

[0034] In the present invention, the terms "bile acid" and "bile salt" refer to a steroid acid (and / or its carboxylate anion) and its salt, which are found in the bile of animals (e.g., humans). Non-limiting examples include a bile acid selected from the group consisting of cholic acid, kenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and litocholic acid, or a bile salt which is a salt thereof.

[0035] In the present invention, the term “molecular aggregate of bile acid or bile acid salt” refers to a molecular aggregate prepared by applying shear stress to a solution containing bile acid so that bile acid molecules or bile acid salt molecules are physically bonded, and means a structure in which bile acid molecules are clustered together.

[0036] In the present invention, “bile acid molecules or bile acid salt molecules” refers to the molecules of the bile acid or bile acid salt themselves, which are precursors or precursors used to produce molecular aggregates of the bile acid or bile acid salt according to the present invention, and may also be referred to as “precursors.” That is, the molecules of the bile acid or bile acid salt according to the present invention refer to bile acid or bile acid salt to which no shear stress has been applied.

[0037] In the present invention, terms “patient,” “subject,” “individual,” etc. are used interchangeably herein and refer to any animal or cell thereof, whether in vitro or in situ, capable of conforming to the method described herein. In certain non-limiting embodiments, said patient, subject, or individual is a human.

[0038] In the present invention, the terms “composition” or “pharmaceutical composition” refer to a mixture of at least one compound of the present invention and other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates the administration of said compound into an organism.

[0039] In the present invention, the terms "effective amount," "pharmaceuticalally effective amount," and "therapeutically effective amount" refer to amounts that are non-toxic but sufficient to provide a desired biological result. Such result may be a reduction and / or alleviation of signs, symptoms, or causes of disease, or any other desired alteration of the biological system. In any individual case, an appropriate therapeutic amount may be determined by a person skilled in the art using ordinary experimentation.

[0040] In the present invention, the term "efficacy" refers to the maximum effect (Emax) achieved within the analysis method.

[0041] In the present invention, "treatment" or "treating" is defined as applying or administering a therapeutic agent, i.e., a compound of the present invention (alone or in combination with other pharmaceutical agents), to a patient to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or influence the condition, symptoms of the condition, or potential to progress to the condition considered herein, or to apply or administer a therapeutic agent to a tissue or cell line isolated from the patient (e.g., for diagnosis or ex vivo application), and having the condition, symptoms, or potential to progress to the condition considered herein. The treatment may be specifically tailored or modified based on knowledge obtained from the field of pharmacology.

[0042] In the present invention, the "therapeutically effective amount" is an amount of the compound of the present invention that improves the symptoms of a disease when administered to a patient. The amount of the compound of the present invention constituting the "therapeutically effective amount" may vary depending on the compound, the disease state and its severity, the age of the patient being treated, etc. The therapeutically effective amount may be ordinarily determined by a person skilled in the art in consideration of their knowledge and the present disclosure.

[0043] In the present invention, the term “application as a skin external agent” refers to delivering a drug into the skin by applying a pharmaceutical composition to the outside of the skin, for example, by a non-surgical and non-invasive method, and may also be determined ordinarily by a person skilled in the art in consideration of their knowledge and the present disclosure.

[0044] In the present invention, the term “local fat removal” refers to an act of treating a disease selected from the group consisting of, for example, obesity, fat redistribution syndrome, submental fat (a double chin formed by localized fat accumulation), lower eyelid fat herniation, lipomas, Dercum’s disease, lipodystrophy, Buffalo hump dystrophy, and combinations thereof, and may also be ordinarily determined by a person skilled in the art in consideration of their own knowledge and the present disclosure.

[0045] In the present invention, the term “local fat” refers to localization in an area selected from the group consisting of, for example, abdominal-neck fat, thigh fat, upper arm fat, visceral fat accumulation, fat generated after breast augmentation surgery, chest fat, fat diffused around the arm circumference, under-eye fat, under-chin fat, buttock fat, calf fat, back fat, thigh fat, ankle fat, cellulite, and combinations thereof; additionally, it may be ordinarily determined by a person skilled in the art in consideration of their own knowledge and the present disclosure.

[0046] In the present invention, the term “external skin preparation” refers to a formulation that can be applied to the skin, for example, as any one of the group consisting of gels, creams, ointments, unguents, sprays, thickened formulations, and poultices, and may also be conventionally determined by a person skilled in the art in consideration of their own knowledge and the present disclosure.

[0048] Molecular assembly of bile acids or bile acid salts

[0049] The present invention provides a molecular aggregate in which bile acid molecules or bile acid salt molecules are physically bonded, wherein the molecular aggregate is formed by a composition containing water, and the molecular aggregate in the composition has an aggregated structure.

[0050] In the present invention, the average particle size of the molecular aggregate may be 1.0 to 10 nm or less, preferably 1.5 nm or more, 2.0 nm or more, 7.0 nm or less, 5.0 nm or less, or 3.0 nm or less. The average particle size of the molecular aggregate can be measured through diffraction experiments, preferably using a Zetasizer or Small Angle Neutron Scattering (SANS). Additionally, an image may be measured using Transmission Electron Microscopy. If the average particle size of the molecular aggregate exceeds 10 nm, there is a problem of reduced dispersibility and decreased transparency and transmittance. Furthermore, although there is no specific limit on the lower value of the average particle size of the molecular aggregate, one of approximately 1.0 nm or more may be used.

[0051] As the molecular aggregate according to the present invention is manufactured by applying shear stress to bile acid molecules or bile acid salt molecules, it can have an amorphous form even though it is manufactured in nano-size, and accordingly, not only is size control easy, but skin permeability can also be improved.

[0052] In addition, the molecular assembly according to the present invention may have a pH greater than 8.5 and less than 10. When the pH value satisfies the above range, not only is the skin permeability of the molecular assembly increased, but it can also effectively break down fat after reaching fat cells. Furthermore, unlike substances conventionally used as injectables, the molecular assembly of the present invention is used as a topical agent, so the pH may be relatively high, and storage stability may be further improved as the pH is higher. Specifically, the pH of the molecular assembly may be greater than 8.6, greater than 8.7, greater than 8.8, greater than 8.9, greater than 9.0, greater than 9.1, and less than 9.9, less than 9.8, less than 9.7, less than 9.6, less than 9.5, less than 9.4, and less than 9.3.

[0053] In addition, the molecular aggregate according to the present invention has excellent storage stability. Unlike general nano-sized dispersed particles, which have low storage stability because they are easily exposed to aggregation or Ostwald ripening phenomena, the molecular aggregate and composition of the present invention exhibit excellent stability with minimal changes in appearance, pH, and particle size under accelerated conditions of 40 ± 2℃ / 75 ± 5%.

[0054] Specifically, the molecular aggregate according to the present invention may have a rate of change in concentration of greater than 1 and less than 10% over 12 months under accelerated conditions of 40±2℃ / 75±5%, preferably greater than 1 and less than 5%, and more preferably greater than 1 and less than 4%. In addition, the molecular aggregate according to the present invention may have a rate of change in concentration of greater than 1 and less than 10% over 3 months under accelerated conditions of 40±2℃ / 75±5%, preferably greater than 1 and less than 5%, and more preferably greater than 1 and less than 3%.

[0055] Specifically, the molecular aggregate according to the present invention may have a particle size change rate of greater than 1 and less than 10% over 12 months under accelerated conditions of 40±2℃ / 75±5%, preferably greater than 1 and less than 7%, and more preferably greater than 1 and less than 5%. In addition, the molecular aggregate according to the present invention may have a particle size change rate of greater than 1 and less than 10% over 3 months under accelerated conditions of 40±2℃ / 75±5%, preferably greater than 1 and less than 5%, and more preferably greater than 1 and less than 3%.

[0056] Specifically, the molecular assembly according to the present invention may have a rate of change in pH of greater than 0 and less than 5% over 12 months under accelerated conditions of 40±2℃ / 75±5%, preferably greater than 0 and less than 3%, and more preferably greater than 0 and less than 1.5%. In addition, the molecular assembly according to the present invention may have a rate of change in pH of greater than 0 and less than 5% over 3 months under accelerated conditions of 40±2℃ / 75±5%, preferably greater than 0 and less than 3%, and more preferably greater than 0 and less than 1.5%.

[0057] The above rate of change refers to the average rate of change calculated by averaging the monthly rates of change up to the above period.

[0058] As described above, it can be seen that the stability of the molecular aggregate according to the present invention is excellent.

[0059] Method for manufacturing molecular assemblages

[0060] A molecular aggregate of a bile acid or bile acid salt according to one embodiment of the present invention can be prepared by applying shear stress to a solution containing a bile acid or bile acid salt that is a precursor of said molecular aggregate.

[0061] The shear stress applied to a solution containing a bile acid or bile acid salt, which is a precursor of the above molecular assembly, may be either mechanical shear stress or ultrasonic application.

[0062] The above mechanical shear stress may be applied by passing the solution through a silica-filled column or filter paper. The mechanical shear stress will be explained in detail below.

[0063] According to one embodiment of the present invention, the mechanical shear stress may be applied by passing a solution containing a bile acid or a bile acid salt, which is a precursor of the molecular aggregate, through a column packed with silica. When the solution containing the bile acid or a bile acid salt passes through a column packed with silica or the like, the bile acid or a bile acid salt, which is a precursor of the molecular aggregate, is subjected to very high shear stress by passing through a physically narrow region.

[0064] The above silica may be spherical or angular, but is not limited to its shape.

[0065] The average particle size of the silica may be 1.0 to 50 μm, specifically 1.5 μm or more, 2 μm or more, 40 μm or less, 30 μm or less, 20 μm or less, 10 μm or less, or 5 μm or less. When the size of the silica is less than 1.0 μm or greater than 50 μm, even if a solution containing the bile acid or bile acid salt passes through a column packed with silica, shear stress is not applied, so there may be no change in the molecular aggregate.

[0066] A negative pressure of 0.1 bar to 1.0 bar or 0.2 bar to 0.9 bar may be applied to the bottom of the column filled with silica. If the negative pressure applied to the bottom of the column filled with silica is less than 0.1 bar, the time required for the solution containing the bile acid or bile acid salt to pass through the column increases, which may delay the time required to manufacture the molecular aggregate of the bile acid or bile acid salt according to the present invention. Additionally, if the negative pressure applied to the bottom of the column filled with silica exceeds 1.0 bar, the time required for the solution containing the bile acid or bile acid salt to pass through the column is reduced, which may shorten the time required to manufacture the molecular aggregate of the bile acid or bile acid salt according to the present invention; however, since additional pump equipment is required, the manufacturing cost may increase.

[0067] According to another embodiment of the present invention, the mechanical shear stress may be applied by passing a solution containing the bile acid or bile acid salt through one or more filter papers. When passing through the one or more filter papers, the bile acid or bile acid salt, which is a precursor of the molecular aggregate, is subjected to very high shear stress by passing through a physically narrow region.

[0068] The filter paper may be a single filter paper or two or more filter papers. If the filter paper consists of two or more filter papers, the filter papers may be stacked and arranged. If the filter paper consists of two or more filter papers, a higher shear stress may be provided than with a single filter paper.

[0069] The pore size of the filter paper may be 0.1 to 5.0 microns or 0.3 to 4.5 microns. If the pore size of the filter paper is less than 0.1 microns, the amount of the solution containing the bile acid that passes through or is filtered by the filter paper is too small, so the rate of production of the molecular aggregate of the bile acid or bile acid salt according to the present invention may be reduced, and if the pore size of the filter paper is greater than 5.0 microns, the solution containing the bile acid simply passes through the filter paper, so shear stress may not be effectively applied.

[0070] The above shear stress may be applied using ultrasound. The application of ultrasound will be explained in detail below.

[0071] According to one embodiment of the present invention, the shear stress may be applied by applying ultrasound to a solution containing the bile acid or bile acid salt.

[0072] When the above ultrasound is applied to a solution containing the bile acid or bile acid salt, a pressure wave is generated, and shear stress can be applied to the bile acid or bile acid salt, which is a precursor of the molecular aggregate, by the pressure wave.

[0073] The intensity of the applied ultrasound may be 200 J / sec to 800 J / sec or 400 J / sec to 600 J / sec.

[0074] The energy applied per volume of the above-mentioned ultrasound can be calculated as ultrasound intensity (J / sec) x applied time (sec) / measured volume (ml).

[0075] According to one embodiment of the present invention, the energy applied per volume of ultrasound to a solution containing the bile acid or bile acid salt may be 100 J / ml to 90 kJ / ml.

[0076] If the energy of the ultrasound is less than 100 J / ml, sufficient shear stress is not applied to the solution containing the bile acid or bile acid salt, making it difficult to form molecular aggregates. Additionally, if the energy of the ultrasound exceeds 90 kJ / ml, excessive heat is applied to the solution containing the bile acid or bile acid salt, making it difficult to form molecular aggregates.

[0077] The above ultrasound may be applied at a temperature of 10°C to 80°C for 10 seconds to 60 minutes. If the above ultrasound is applied at a temperature below 10°C, there is no change in the solution containing the bile acid or bile acid salt, and if it is applied at a temperature above 80°C, a phase change occurs in the solution containing the bile acid or bile acid salt, making it difficult to form the molecular aggregate of the bile acid or bile acid salt according to the present invention. Additionally, if the above ultrasound is applied for less than 10 seconds, there is no change in the solution containing the bile acid or bile acid salt, and if it is applied for a time exceeding 60 minutes, the molecular aggregate in the solution containing the bile acid or bile acid salt is deformed, making it impossible to form the molecular aggregate of the bile acid or bile acid salt according to the present invention.

[0078] According to another embodiment of the present invention, a silica-filled column can be combined with an ultrasonic generator by applying the shear stress. The silica-filled column may be placed inside the ultrasonic generator, or the silica-filled column and the ultrasonic generator may be separated and arranged continuously.

[0079] For example, after pouring a solution containing the bile acid or bile acid salt into the silica-filled column, the column can be placed inside an ultrasonic generator to apply ultrasound. Additionally, after applying ultrasound to the solution containing the bile acid or bile acid salt, the solution can be passed through the silica-filled column.

[0081] Pharmaceutical composition for localized fat removal

[0082] The present invention provides a pharmaceutical composition for local fat removal comprising the above molecular aggregate.

[0083] In the present invention, the pharmaceutical composition for local fat removal may be used in any one of the formulations of the group consisting of gel, cream, ointment, unguent, spray, thickened formulation, and poultice.

[0084] In the present invention, the pharmaceutical composition for local fat removal may include, in addition to the molecular aggregate, one or more selected from the group consisting of glycerin, chia seed oil, glucan, hyaluronic acid, honeysuckle extract, collagen, ceramide, lecithin, betaine, trehalose, panthenol, squalane, caprylic / capric triglyceride, butylene glycol, propanediol, pentylene glycol, sodium levulinate, hydrogenated lecithin, and sodium hyaluronate.

[0085] In addition, in the present invention, the pharmaceutical composition for local fat removal may further include, without special limitation, any ingredients used in the industry for use in cream formulations in addition to the above ingredients.

[0086] In the present invention, the pharmaceutical composition for local fat removal may contain the molecular aggregate in an amount of 0.05% to 10.0% by weight, specifically 0.08% or more, 0.1% or more, 0.3% or more, 0.5% or more, 1.0% or more by weight, and 5% or less by weight, 3.0% or less by weight, 2.0% or less by weight, or 1.0% or less by weight.

[0087] In the present invention, the concentration of bile acid or bile acid salt measured in plasma 3 hours after the above-mentioned pharmaceutical composition for local fat removal may be 0.001 to 0.2 μg / ml, specifically 0.01 μg / ml or more, 0.05 μg / ml or more, 0.1 μg / ml or more, and 0.18 μg / ml or less, 0.15 μg / ml or less, 0.12 μg / ml or less.

[0089] The present invention will be explained in more detail below through embodiments. It should be understood that the present invention is not limited to these embodiments.

[0091] [Example]

[0092] Example 1-1. Preparation of a molecular aggregate of deoxycholic acid (DCA)

[0093] 3.6g of deoxycholic acid (DCA) was placed in a 250ml beaker and dissolved in 177g of ethanol. 142g of SYLOID 244 FP was placed in a 3L beaker and stirred at 50rpm using an overhead stirrer. While stirring, 17g of 1M NaHCO3 and 45g of 1M Na2SO4 were slowly added to the silica, followed by the slow addition of the DCA solution. The mixture was stirred for 30 minutes to ensure the added aqueous solution was properly supported on the silica. 1780g of ethanol was placed in a 3L beaker and stirred at 70rpm while slowly adding the silica supported with deoxycholic acid. After the addition was complete, stirring was performed for an additional 45 minutes to allow for sufficient elution time. After stirring was finished, the mixture was filtered sequentially using a 1μm paper filter and a 0.45μm membrane filter. The filtered effluent was 1450g, and it was concentrated to 704g by partially removing ethanol using a rotary evaporator. 1640g of water was placed in a beaker, 0.3g of NaHCO3 was added, and the above first concentrate was added. This diluted solution was concentrated using a rotary evaporator for about 3 hours to obtain 112g of a colorless, transparent liquid. The concentration of this solution is 2.34%, and the pH is 9.22. In this process, the recovery rate of DCA was 69%.

[0095] Example 1-2. Preparation of molecular aggregates of deoxycholic acid (DCA).

[0096] Prepare a DCA solution by placing 2.0g of deoxycholic acid (DCA) and 98.75g of ethanol in a 500mL beaker and stirring with a magnetic stirrer. Add 80g of ethanol to 8.2g of SYLOID 244 FP to thoroughly wet the silica. Prepare 1.3mL of a co-salt aqueous solution by mixing 1M NaHCO3 and 1M Na2SO4 aqueous solutions in a specific ratio. Place a 1.00 μm paper filter in a Büchner funnel, wet the paper filter with ethanol, and then use a pump to adsorb it to the bottom of the funnel before slowly pouring in the prepared wet silica. When about 1cm of ethanol remains on the packed silica, slowly pour in the co-salt aqueous solution and 100g of purified water. Afterward, pour in an additional 100g of ethanol; once some of the ethanol is filtered out, stop the pump and replace the 1000ml filter bottle at the bottom. After operating the pump again, the prepared deoxycholic acid solution is slowly poured in, and an additional 202g of ethanol is added. The resulting effluent is filtered using a 0.45µm membrane filter to obtain 394.2g of filtrate. Using another 2.0L beaker, 920g of a 4 mM NaHCO3 aqueous solution is prepared, and the filtrate is slowly added to dilute it. This diluted liquid is concentrated using a rotary evaporator at 30°C and 180rpm for 2 hours and 25 minutes. The final concentrate yielded a molecular aggregate of deoxycholic acid with a volume of 98.19g and a concentration of 1.92%, with a particle size of 1.36 nm, a pH of 9.15, and a final recovery rate of 94.2%.

[0098] Examples 1-3. Molecular aggregates of sodium deoxycholate (NaDC)

[0099] 18.0 g of sodium deoxycholate (NaDC, Sigma Aldrich #30970) was placed in a 1 L beaker and dissolved in 604 g of water using a magnetic stir bar. 60.5 g of Silica Gel 60 (Merck) was placed in a 400 mL beaker containing 242 g of 0.1 M NaHCO3 and mixed with a spatula. The moistened silica was slowly poured into a vacuum filtration device (pressure 200 mbar) equipped with a vacuum pump and packed. The above NaDC solution was added to the packed silica. Before the silica bed dried, 160 g of water was added in two portions of 80 g each. The total effluent time was 1 hour, and after effluent was completed, the solution was filtered using a 0.45 µm membrane filter. The filtered effluent weighed 808 g. The concentration of this solution was 2.07%, and the pH was 7.67. The recovery rate of sodium deoxycholate in this process was 93%.

[0101] Example 2. Composition comprising a molecular aggregate of deoxycholic acid

[0102] Since the above Example 1 is in an aqueous solution state, it tends to run off when applied to the skin, so there is a possibility that it may not be sufficiently delivered into the skin. Therefore, in order to prepare a formulation of the molecular aggregate prepared in the above Example 1 that can be applied transdermally, a gel-type formulation was prepared by adding 0.7 wt% of Hyaluronic acid (HA) and 2 wt% of 1,2-hexanediol to 0.04 wt% and 0.08 wt% of the molecular aggregate of the above Example 1, respectively, and the test results were confirmed in the verification of the ability to destroy preadipocytes and adipocytes in Experimental Example 3 described later.

[0104] Comparative Example 1. Precursor of deoxycholic acid

[0105] The deoxycholic acid itself, which was not subjected to the process of passing through the silica of Example 1, was used.

[0107] [Experimental Example]

[0108] Experimental Example 1. Measurement of the particle size of deoxycholic acid molecular aggregates

[0109] Since deoxycholic acid has a very low solubility in water of 0.024%, in order to improve solubility and stabilize it in an aqueous solution, in Example 1 above, a colorless, odorless, transparent liquid molecular aggregate of deoxycholic acid was prepared in which deoxycholic acid molecules formed clusters by dissolving deoxycholic acid in ethanol, adding water, and removing ethanol through the process of the present invention. The particle size of the deoxycholic acid molecular aggregate was confirmed to be 1.6 nm through analysis using the following Zetasizer and TEM.

[0111] Zetasizer analysis

[0112] After filtering the deoxycholic acid molecular aggregate of Example 1 to 0.2 μm using a Zetasizer (Malvern, Nano ZSP), the average particle size of 1.6 nm was confirmed from the size distribution by volume by measuring 10 times under the conditions of Table 1 below (Fig. 1).

[0113] Temperature 25 °C Duration Used 80 S Count rate 461.9 Kcps Measurement (set) 10 Cell Description Disposable sizing cuvette Attenuator 8

[0115] TEM analysis

[0116] An EMS FCF300-Cu 50 / pk (Formvar / Carbon 300Mesh, Copper) grid was placed on filter paper, and approximately 20 μl of the sample was dropped onto it using a micropipette. The grid was dried by waiting for at least 15 minutes without negative staining, and the particle size of the deoxycholic acid molecular aggregate of Example 1 was measured using a TEM image. The result confirmed that the particle size was similar to that measured with a Zetasizer (Fig. 2).

[0117] Point resolution Line resolution HR STEM resolution EDS resolution 0.205 nm 0.102 nm 0.16 nm 136 eV

[0119] Experimental Example 2. Stability Test

[0120] ① Materials

[0121] To confirm the storage stability of the molecular aggregate of deoxycholic acid to which the present invention is applied, 292g of DCA200265~67, which has no change in physical properties and was prepared by the same method as in Example 1, was prepared and used as a stability test sample for 12 months.

[0122] Batch No. Sample amount (g) density(%) Particle size (nm) pH division DCA2002JJ65 98 1.92 1.6 9.16 Stability test sample DCA2002JJ66 96 2.35 1.9 9.13 DCA2002JJ67 98 2.28 1.6 9.16

[0124] ① Sample storage method

[0125] 1.7 mL of the sample was taken into a 5 mL serum vial of CyLab, SL / Vi1361 and placed into a single vial. The storage cap was closed, and the vial and storage cap were secured using a capper. Parafilm was wrapped around the space between the vial and the storage cap, and labeled (including substance name, batch No., and test date).

[0126] ② Analysis Method

[0127] Saint

[0128] Observed with the naked eye.

[0129] density

[0130] HPLC analysis was performed as shown in Table 4 below for concentration analysis, and the calibration curve of deoxycholic acid resulting from the analysis is shown in Figure 3.

[0131] Column RP C18 (215x4.6) Mobile phase Water: ACN: 85% H3PO4(50:50:0.1) Flow rate 1.0 mL / min Injection volume 10 μL Run time 17 min Wavelength 195 nm Sample Temp. 30℃ Column Temp. 25℃

[0132] Particle size analysis

[0133] Analysis was performed according to the Zetasizer analysis conditions and method of Example 1.

[0135] pH

[0136] The molecular aggregates of deoxycholic acid were filtered through a 0.45 μm filter and measured using a Mettler Toledo S220.

[0138] ④ Acceleration stability test results

[0139] Saint

[0140] It was a colorless, transparent liquid that maintained the same properties for 12 months under accelerated conditions of 40±2℃ / 75±5%.

[0141] density

[0142] Changes in concentration under accelerated conditions over 12 months are shown in Table 5 below, and no significant changes in concentration were observed. Specifically, changes in concentration were measured at one-month intervals under accelerated conditions with a temperature of 40±2℃ and humidity of 75±5% over 12 months, and the results are shown in Table 5 below. After calculating the difference between the initial (month 0) concentration and the subsequent concentrations, the value divided by the initial concentration was calculated as the rate of change in concentration, and the average values ​​of these rates of change calculated for 3 months and 12 months, respectively, are listed in Table 5 below.

[0143] DCA2022JJ65 DCA2022JJ66 DCA2022JJ67 0 months 1.88% 1.92% 1.93% 1 month 1.93% 1.98% 1.95% 2 months 1.92% 1.99% 1.99% 3 months 2.03% 1.95% 2.02% 6 months 1.90% 1.98% 2.02% 9 months 2.02% 2.02% 2.05% 12 months 2.05% 2.05% 2.10% 3-month average change rate 4.26% 2.78% 2.94% 12-month average rate of change 5.05% 3.91% 4.75%

[0144] particle size

[0145] The change in particle size under accelerated conditions over 12 months is shown in Table 6 below, and there was no significant change in particle size. Specifically, the change in particle size was measured at one-month intervals under accelerated conditions with a temperature of 40±2℃ and humidity of 75±5% for 12 months, and the results are shown in Table 6 below. After calculating the difference between the initial (month 0) particle size and the subsequent particle size, the value divided by the initial particle size was calculated as the rate of change in particle size, and the average values ​​of this rate of change calculated for 3 months and 12 months, respectively, are listed in Table 6 below.

[0146] DCA2022JJ65 DCA2022JJ66 DCA2022JJ67 0 months 1.48 1.92 1.46 1 month 1.54 1.99 1.49 2 months 1.56 1.95 1.52 3 months 1.60 1.97 1.54 6 months 1.55 2.02 1.52 9 months 1.62 2.05 1.58 12 months 1.58 2.06 1.57 3-month average change rate 5.86% 2.60% 3.88% 12-month average rate of change 6.42% 4.51% 5.25%

[0147] pH

[0148] The pH change under 12-month accelerated conditions is shown in Table 7 below, which showed a stable pH without changes over time. Specifically, the pH change was measured at monthly intervals under accelerated conditions with a temperature of 40±2℃ and humidity of 75±5% for 12 months, and the results are shown in Table 7 below. After calculating the difference between the initial (0 months) pH value and the subsequent pH value, the value divided by the initial pH value was calculated as the rate of change in particle size, and the average values ​​of this rate of change calculated for 3 months and 12 months, respectively, are listed in Table 7 below.

[0149] DCA2022JJ65 DCA2022JJ66 DCA2022JJ67 0 months 9.15 9.13 9.15 1 month 8.96 9.13 8.97 2 months 9.03 9.23 9.14 3 months 9.11 9.14 9.16 6 months 9.20 9.31 9.27 9 months 9.32 9.31 9.29 12 months 9.27 9.36 9.33 3-month average change rate -1.28% 0.40% -0.66% 12-month average rate of change -0.02% 1.28% 0.47%

[0150] ④ Final results of the accelerated stability test

[0151] The final results of the acceleration stability test were summarized and listed in Table 8 below.

[0152] Types of exams Test conditions Test cycle Test items result Accelerated testing 40 ± 2℃75 ± 5% 0~12 months Appearance, concentration, particle size, pH No temporal change

[0153] As shown in Table 8, accelerated stability tests were conducted on three batches over a period of 12 months, and no changes over time were observed in all test items, and they were stable.

[0155] Experimental Example 3. Confirmation of the ability to destroy preadipocytes and differentiated adipocytes

[0156] ① test substance

[0157] To compare the ability to destroy preadipocytes and adipocytes, deoxycholic acid and the deoxycholic acid molecular aggregates to which the present invention was applied (DCA2001JJ43, DCA2001JJ83-1) and the deoxycholic acid salt molecular aggregates (DCA2001JJ85-1) were used.

[0158] ② Ability to destroy preadipocytes

[0159] 3T3-L1 preadipocytes in a 96-well plate at a rate of 5 x 10⁶ per well 3After inoculating 96 cells and culturing them in a medium (high glucose DMEM, 10% bovine calf serum, 1% penicillin / streptomycin) for 16 hours, the above drugs were prepared to contain 0.04% and 0.08%, respectively, and treated for 4 hours. 10 μl of the Dojingo CK04-11 cell counting kit-8 was added to each well according to the manual and reacted for 2 hours. The absorbance at 450 nm was measured using a spectrophotometer to compare the ability to destroy preadipocytes, as shown in Figure 4.

[0160] As a result, at 0.04%, the ability to destroy preadipocytes of the deoxycholic acid precursor, the molecular aggregate of deoxycholic acid, and the molecular aggregate of deoxycholic acid salt were the same, but when treated at 0.08%, compared to the precursor at 24.6%, the survival rate of preadipocytes with added molecular aggregate of deoxycholic acid salt decreased by 11.9% to 12.7% (*p=0.008), and the survival rate of the deoxycholic acid molecular aggregate treatment group decreased by 13.8% to 10.8% (#p=0.01), confirming the increased ability to destroy preadipocytes of the deoxycholic acid molecular aggregate to which the technology of the present invention was applied.

[0162] ③ Destructive ability of adipocytes

[0163] According to the Biovision 3T3 L1 differentiation kit manual, 3T3 L1 preadipocytes were cultured to 100% confluence in a culture dish, and six days after replacing the culture medium with differentiation maintenance medium, adipocytes with multiple lipid droplets were induced (Fig. 5). Adipocytes were treated with a 0.07% molecular aggregate of deoxycholic acid (DCA2001JJ43), and 3D images were acquired at 2.5 frames per second using a Tomocube HT-2H microscope. Then, using the imaging software TomoStudio, the multi-point acquisition function was applied to capture changes in the differentiated adipocytes at 25-second intervals for 40 minutes. As a result, it was confirmed that the cell membrane and intracellular structures of the adipocytes, which were observed up to 5 minutes after imaging, rapidly faded after 5 minutes. Furthermore, after 8 minutes, the RI value around the lipid droplets became nearly similar to that of the outer medium, confirming the ability of the differentiated adipocytes to destroy (Fig. 6).

[0165] Experimental Example 4. Preclinical Test

[0166] ① SD rat skin penetration test

[0167] ingredient

[0168] Experiments were conducted to confirm the skin penetration ability and subcutaneous fat tissue removal ability of a molecular aggregate of deoxycholic acid (DCA2002JJ84 prepared by the method of Example 1) in SD rats.

[0170] Experimental method

[0171] The interscapular region (back of the neck near the ear) of 10-week-old SD rats (280–350 g) with sufficient subcutaneous adipose tissue was depilated and anesthetized. Donor cells were attached to the depilated area using tape. After placing 2.5% deoxycholic acid, a precursor, and 500 μl of the deoxycholic acid molecular aggregate of the present invention onto the donor cells, the mixture was applied to the skin continuously for 3, 6, and 9 hours under sustained anesthesia. Skin permeability was confirmed by examining the distribution of deoxycholic acid in the subcutaneous tissue, skin, and plasma, and the ability to disrupt the subcutaneous adipocyte layer was confirmed through tissue staining, as shown at the bottom of Figure 7. A subcutaneous injection group of deoxycholic acid was used as a positive control (1% DCA, 500 μl subcutaneous injection).

[0173] Measurement results of DCA distribution in subcutaneous tissue

[0174] When deoxycholic acid precursors and deoxycholic acid molecular aggregates were applied sequentially to the skin of SD rats, it was confirmed by quantification of deoxycholic acid in the subcutaneous tissue that the amount of skin penetration increased over time in both groups. In the subcutaneous injection group, the amount of DCA in the subcutaneous tissue gradually decreased after injection, whereas in the skin application group, it showed an increasing trend over time, and the increase in the permeability of the deoxycholic acid molecular aggregate was slightly higher than that of the deoxycholic acid precursor (Fig. 7).

[0176] Results of measuring DCA distribution in the skin

[0177] When deoxycholic acid precursors and deoxycholic acid molecular aggregates were applied sequentially to the skin of SD rats, an increase in deoxycholic acid distributed in the skin over time was observed in both groups. On the other hand, in the subcutaneous injection group, deoxycholic acid was most highly distributed in the skin tissue after 6 hours, but showed a lower distribution than the skin application group at 9 hours (Fig. 8).

[0179] Measurement results of DCA distribution in plasma

[0180] The results in Figure 9 show the DCA concentration in rat plasma. DCA was detected in the plasma only in the deoxycholic acid subcutaneous injection group, while no DCA was detected in the plasma of the skin application group. This confirmed that the deoxycholic acid molecular aggregate has a subcutaneous fat removal effect through skin application but does not transfer into the plasma, thus having no safety issues.

[0182] Histological analysis results

[0183] After the group injected with 500 μL of 1% deoxycholic acid subcutaneously, the group applied 500 μL of 2.5% deoxycholic acid subcutaneously, and the group applied 500 μL of 2.5% deoxycholic acid molecular aggregate to the site where Franz cells were attached for 3, 6, and 9 hours, tissue samples (skin, subcutaneous tissue) from the intercapular region were collected, fixed, and subjected to H&E staining and Masson's trichome staining, and tissue changes were investigated through microscopic observation.

[0184] As a result of H&E staining, severe tissue damage was observed in the subcutaneous injection group of deoxycholic acid after the injection. In the skin application group, a decrease in dermis white adipose tissue was observed over time, which was observed more clearly in the group applied with the molecular aggregate of deoxycholic acid (Fig. 10).

[0185] Masson's trichome staining was performed to observe the increase or decrease in collagen within the skin and subcutaneous tissue. As a result, an increase in the area stained blue due to the destruction of fat cells or an increase in collagen was observed in the skin application group (Fig. 11). In the subcutaneous injection group of deoxycholic acid, hematoma and edema occurred in the tissue, and the tissue damage was severe, causing the tissue to harden during sectioning. In the 9-hour treatment group, the sections cracked due to hardening of the fat tissue, making it impossible to obtain proper tissue sections.

[0187] conclusion

[0188] The topical application group showed a tendency for the amount of deoxycholic acid distributed in the skin and subcutaneous tissue to increase over time, and the molecular aggregate of deoxycholic acid resulted in a higher amount of deoxycholic acid distributed in the skin or subcutaneous tissue compared to the group treated with the deoxycholic acid precursor. The concentration of deoxycholic acid in plasma was detected only in the subcutaneous injection group, and histological analysis revealed a decrease in adipose tissue within the tissue in the topical application group over time after administration; this phenomenon was more clearly evident in the group treated with the molecular aggregate of deoxycholic acid. Since the results of the pharmacokinetic study showed that the amount of deoxycholic acid distributed in the subcutaneous tissue accumulated over time, and histological analysis revealed a decrease in dermis white adipose tissue within the skin, it can be concluded that the molecular aggregate of deoxycholic acid effectively penetrated the skin and contributed to the reduction of adipose tissue.

[0190] ② Skin-penetrating lipolytic effect using ob / ob obese mouse model

[0191] ingredient

[0192] A molecular aggregate of 2.52% deoxycholic acid was prepared in the same manner as in Example 1 (DCA2103JJ134-02, 115 g, 95% recovery rate), and formulated after dilution to 1.0% and 2.5% (1.0% SCAI-101, 2.5% SCAI-101) to evaluate fat reduction efficacy in ob / ob obese mice.

[0194] Experimental method

[0195] We intended to verify the fat-reducing effect of the deoxycholic acid molecular aggregate (1.0% SCAI-101, 2.5% SCAI-101) prepared in Example 1 after skin penetration using an ob / ob obese mouse model. The back and abdomen (belly) fur of the ob / ob obese mice were depilated, and 1 cm 2100 μL of a solution (SCAI-101) containing molecular aggregates of deoxycholic acid at concentrations of 1.0% and 2.5% was applied twice daily to two sites each (a total of four sites) to ensure sufficient absorption. Skin permeability and lipolytic efficacy were confirmed as shown in Table 9 by comparing changes in body weight, waist circumference, and abdominal fat volume obtained from micro-CT scans of the mouse abdomen over 4 weeks with a negative control group.

[0196] Test group test substance Number of animals Group 1 Control (Vehicle) 5 Group 2 1.0% SCAI-101 application group 5 Group 3 2.5% SCAI-101 application group 5

[0198] Body weight change

[0199] The body weight of the ob / ob obese mouse control group continued to grow while consuming normal feed, increasing from 46.2±1.13g (week 0) to 51.1±0.89g (week 4) over 4 weeks, and showed a statistically significant increase at weeks 3 and 4 compared to the body weight of the control group at week 0 (p<0.01 and p<0.001, respectively). The group treated with 2.5% concentration also increased from 43.4±1.62g (week 0) to 48.3±1.49g (week 4), and a statistically significant increase was observed at week 4 compared to the body weight at week 0 (p<0.05). However, the group treated with 1.0% concentration decreased from 42.1±2.14g (week 0) to 41.7±4.07g (week 4).

[0200] As shown in the results of Table 10 and Figure 12 below, it was confirmed that the body weight of the control group and the 2.5% application group increased continuously, and no dose-dependent trend was observed when comparing the changes in body weight between the 1.0% and 2.5% application groups.

[0201] Weeks Control 1.0% SCAI-101 application group 2.5% SCAI-101 application group 0 46.2±1.13 42.1±2.14 43.4±1.62 1 46.5±1.08 40.7±2.83 43.7±1.52 2 47.6±1.09 41.0±3.12 45.5±1.65 3 50.7±0.99** 42.3±3.64 47.8±1.61 4 51.1±0.89*** 41.7±4.07 48.3±1.49* Data were expressed as mean ± SEM. The weight of each group was measured weekly for 4 weeks. Paired t-test was used for the comparison of the body weight of the week 0. *, ** and ***: Statistically significant compared with Week 0 (before treatment) (p<0.05, p<0.01 and p<0.001, respectively)

[0203] Waist circumference and change in waist circumference

[0204] As a result of measuring waist circumference while applying the solution twice daily to ob / ob obese mice for 4 weeks, the waist circumference of the control group increased statistically significantly at 1, 2, 3, and 4 weeks compared to 0 weeks (all p<0.001). (Fig. 13 and Table 11)

[0205] Weeks Control 1.0% concentration Dopogun 2.5% concentration Dopogun 0 9.7±0.11 9.4±0.21 9.8±0.14 1 10.3±0.05*** 9.6±0.24# 10.1±0.19 2 10.6±0.09*** 9.8±0.25***, # 10.4±0.26 3 10.7±0.12*** 9.4±0.50# 10.5±0.13* 4 10.8±0.13*** 9.4±0.43# 10.3±0.16 Data were expressed as mean ± SEM.The waist girth of each group was measured weekly for 4 weeks. Paired t-test was used for the comparison of the waist girth of the Week 0. * and ***: Statistically significant compared with Week 0 (before treatment) (p<0.05 and p<0.001, respectively). #: Statistically significant compared with the same week of Control (p<0.05 and p<0.01, respectively). Ordinary ANOVA test, followed by Tukey multiple comparison post hoc test, was used for the comparison of the waist girth (Week 1, Week 3 and Week 4). Kruskal-Wallis test, followed by Dunn’s multiple comparison test, was used for the comparison of the waist girth (Week 2).

[0206] When a molecular aggregate of deoxycholic acid was applied, at a concentration of 1.0%, waist circumference increased slightly compared to week 0, showing a statistically significant increase at week 2 (p<0.001); however, it decreased from week 3 and remained similar to week 0 until week 4 without a statistically significant increase. On the other hand, when compared to the waist circumference of the control group at the same week after application, the 1.0% concentration group showed a statistically significantly shorter duration at weeks 1, 2, 3, and 4 (all p<0.05) (Table 15). Consistent with this, the waist girth gain of the 1.0% concentration group decreased statistically significantly at weeks 1, 3, and 4 compared to the control group at the same week (all p<0.05) (Table 11).

[0207] The 2.5% concentration application group showed a statistically significant increase in waist circumference at week 3 compared to week 0 (p<0.01) (Table 11), but since no statistical significance was recognized in the waist girth gain compared to the control group, it is considered a temporary result (Table 12).

[0208] Weeks Control 1.0% 농도 도포군 2.5% 농도 도포군 1 0.3±0.0 -0.4±0.2* 0.1±0.2 2 0.6±0.1 -0.2±0.2 0.4±0.3 3 0.7±0.1 -0.6±0.5* 0.5±0.1 4 0.8±0.1 -0.6±0.4* 0.3±0.2 Data were expressed as mean±SEM.The waist girth gain of each group was measured weekly for 4 weeks. ANOVA test, followed by Tukey multiple comparison post hoc test, was used for the comparison of the waist girth gain. *: Statistically significant compared with girth gain of every week of Control (p<0.05).

[0210] micro-CT scan abdominal fat volume measurement results

[0211] As a result of measuring fat mass in the waist area (including the application site of the test substance) and abdominal waist circumference (between lumbar vertebrae 3 and 5), the subcutaneous fat masses for the 1.0% and 2.5% concentration application groups were 2,233.2±351.4 mm³ and 2,589.7±93.4 mm³, respectively; these figures were not statistically significant when compared to the control group (2,505.4±279.4 mm³), and the ratios of subcutaneous fat mass to the control group were 89.1±14.03% and 103.4±3.73%, respectively. The subcutaneous fat mass of the 1.0% concentration application group was, on average, 10.9% lower than the control group, but no dose-dependent decreasing trend was observed compared to the 2.5% concentration application group. (Table 13)

[0212] Item Region of Interest Control 1.0% concentration Dopogun 2.5% concentration Dopogun Unit Objective Volume of fat tissue Subcutaneous 2505.4±279.4 2233.2±351.4 2589.7±93.4 ㎜ 3 Visceral 3861.5±289.9 3081.2±571.0 3475.2±108.4 % Fat volume Subcutaneous 100.0±11.15 89.1±14.03 103.4±3.73 % Visceral 100.0±7.51 79.8±14.79 90.0±2.81 Data were expressed as mean±SEM (n=3)

[0213] Evaluation of fat cell tissue thickness in applied skin tissue

[0214] As a result of measuring the thickness of adipocyte tissue on skin directly coated with the molecular aggregate of deoxycholic acid (SCAI-101, DCA WP) prepared in Example 1, the control group and the groups with 1.0% and 2.5% concentrations were 594.6±30.46㎛, 394.3±17.96㎛, and 492.4±11.42㎛, respectively, at 4 weeks after application twice a day, and both the 1.0% and 2.5% concentration groups showed a statistically significant decrease compared to the control group (all p<0.05). The ratio of adipose tissue thickness (% Average) of the groups treated with deoxycholic acid molecular aggregates to the control group was 66.0±2.94% (1.0% concentration group) and 82.6±1.87% (2.5% concentration group), respectively; a statistically significant decrease was confirmed only in the lower concentration group of 1.0% (p<0.05). It was confirmed that the lipolysis and reduction in accumulation in the 1.0% concentration group were superior to those in the 2.5% concentration group. (Fig. 14 and Table 14)

[0215] Group Thickness (㎛) % Average Control 594.6±30.46 100±4.99 1.0% concentration application group 394.3±17.96* 66.0±2.94* 2.5% concentration application group 492.4±11.42* 82.6±1.87 Data were expressed as mean ± SEM (n=20)Kruskal-Wallis test, followed by Dunn's multiple comparison test, was used for the comparison of the thickness of Dermal White Adipose Tissue. *: Statistically significant compared with Control (Vehicle treatment).

[0216] conclusion

[0217] After applying a 1.0% concentration group (1.0% SCAI-101, DCA WP) twice daily for 4 weeks, statistically significant reductions in waist circumference and fat cell thickness were confirmed, thereby confirming the pharmacological effect of reducing or breaking down subcutaneous fat after skin penetration. Unlike the characteristics of deoxycholic acid, which is generally known to be difficult to penetrate the skin, these results can be attributed to the pharmacological action of molecular aggregates of deoxycholic acid that penetrate the skin when applied directly to the skin.

[0218] Compared to the control group, the decrease in body weight, waist circumference, and subcutaneous fat mass is predicted to be the result of the indirect influence caused by the direct fat cell breakdown action of molecular aggregates of deoxycholic acid that have penetrated the skin.

[0219] Therefore, it is determined that the skin penetration lipolytic effect of the 1.0% concentration application group, which is the condition of the present invention, was the most appropriate concentration. However, although the effect and dose-dependent trend of the 2.5% concentration application group were not recognized, it will be possible to maximize the lipolytic effect of a solution containing molecular aggregates of deoxycholic acid through future improvements in appropriate concentration and composition.

Claims

Claim 1 A molecular aggregate in which molecules of deoxycholic acid or sodium salt of deoxycholic acid are physically bonded, wherein the molecular aggregate is prepared by applying shear stress to molecules of deoxycholic acid or sodium salt of deoxycholic acid by passing them through a silica-filled column or filter paper, and wherein the molecular aggregate is formed with a composition containing water, wherein the molecular aggregate in the composition has an aggregated structure and the average particle size of the molecular aggregate is 1.0 to 10 nm or less. Claim 2 delete Claim 3 A pharmaceutical composition for local fat removal comprising the molecular aggregate of claim 1 and used by applying it as a topical agent to the skin, wherein the composition is characterized by being intended to treat a disease selected from the group consisting of obesity, fat redistribution syndrome, submental fat (a double chin formed by local fat accumulation), lower eyelid fat herniation, lipomas, Dercum's disease, lipodystrophy, Buffalo hump dystrophy, and combinations thereof. Claim 4 In paragraph 3, the above composition is a pharmaceutical composition for local fat removal having a pH greater than 8.5 and less than 10. Claim 5 In paragraph 3, the above composition is a pharmaceutical composition for local fat removal, wherein the rate of change of the molecular aggregate concentration over 12 months under accelerated conditions of 40±2℃ / 75±5% is greater than 1 and less than 10%. Claim 6 In paragraph 3, the above composition is a pharmaceutical composition for local fat removal, wherein the rate of change in the molecular aggregate particle size over 12 months under accelerated conditions of 40±2℃ / 75±5% is greater than 1 and less than 10%. Claim 7 In paragraph 3, the above composition is a pharmaceutical composition for local fat removal, wherein the rate of change of pH over 12 months under accelerated conditions of 40±2℃ / 75±5% is greater than 0 and less than 5%. Claim 8 delete Claim 9 delete Claim 10 In paragraph 3, the above composition is a pharmaceutical composition for local fat removal used by applying it as a topical agent to the skin using a non-surgical, non-invasive method. Claim 11 A pharmaceutical composition for localized fat removal according to claim 3, wherein the composition further comprises one or more selected from the group consisting of glycerin, chia seed oil, glucan, hyaluronic acid, honeysuckle extract, collagen, ceramide, lecithin, betaine, trehalose, panthenol, squalane, caprylic / capric triglyceride, butylene glycol, propanediol, pentylene glycol, sodium levulinate, hydrogenated lecithin, and sodium hyaluronate. Claim 12 In paragraph 3, the above composition is a pharmaceutical composition for local fat removal comprising 0.05 to 10 weight% of the molecular aggregate. Claim 13 delete Claim 14 A pharmaceutical composition for localized fat removal according to claim 3, characterized in that the composition is localized to a site selected from the group consisting of abdominal-neck fat, thigh fat, upper arm fat, visceral fat accumulation, fat generated after breast augmentation surgery, chest fat, fat diffused around the arm circumference, under-eye fat, under-chin fat, buttock fat, calf fat, back fat, thigh fat, ankle fat, cellulite, and combinations thereof. Claim 15 A pharmaceutical composition for local fat removal according to paragraph 3, wherein the composition is used as any one formulation of the group consisting of gel, cream, ointment, unguent, spray, thickened formulation, and poultice. Claim 16 A pharmaceutical composition for local fat removal according to claim 3, wherein the concentration of bile acid measured in plasma 3 hours after application to the skin is 0.001 to 0.2 μg / ml.

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