TW201922261A - Injectable composition for localized fat reduction without pain, edema, and side effects, and method for preparing same - Google Patents

Abstract

The present invention relates to a composition for reducing localized fat without a pain, edema, and side effect, and a method for preparing the same. More specifically, the present invention relates to a composition comprising glycocholic acid (or a salt thereof) or taurocholic acid (or a salt thereof) and phosphatidylcholine (PPC) at a particular mixing ratio for reducing localized fat without side effects such as a pain, edema, necrosis of muscle cells, fibroblasts and vascular endothelial cells other than adipocytes, anesthesia of administration sites, extensive swelling, erythema, induration, paresthesia, nodule, pruritus, burning sensation, nerve injury, and dysphagia, and a method for preparing the same. The DCA single injectable preparation or PPC injectable preparation in which DCA is mixed as a solubilizing agent, which is the conventional injectable preparations for reducing localized fat, has been known to accompany such side effects as a pain, anesthesia, extensive swelling, localized edema, erythema, induration, paresthesia, nodule, pruritus, burning sensation, dysphagia, and the like due to necrosis of cells caused inevitably by lysing not only adipocytes but also fibroblasts, vascular endothelial cells and skeletal muscle cells. Thereby, patients have been complaining of pain, discomfort and anxiety, with low compliance. On the other hand, the injectable composition for reducing localized fat according to the present invention is a safe and stable formulation, selectively lyses adipocytes, and has an excellent effect on inducing apoptosis of adipocytes, so that it is an excellent composition improving the compliance and quality of patient's life without above-mentioned side effects.

Description

Local fat-reducing injectable composition without pain, no edema and no side effects and method for preparing same

The present invention relates to a composition suitable for use in the non-surgical reduction of fat in a subject having localized fat deposition using a pharmaceutically active phospholipid choline painless, edema, and no side effects; and a method for preparing the same method. More specifically, the present invention relates to a form of pain relief and side effects (especially muscle cells, fibroblasts, and vascular endothelial cell necrosis other than fat cells; edema; sensory loss of administration site; extensive swelling; erythema; induration; Abnormal; nodule; itching; burning sensation; nerve damage; or difficulty swallowing) a composition and preparation for reducing local fat, the composition comprising: (i) phospholipid choline; and (ii) selected from glycocholic acid (GCA) And at least one of a group consisting of taurine (TCA) and a salt thereof, wherein the composition (ii) has a molar ratio of (i) in the range of 0.7 to 3.0; a kit comprising the same; A method of making the same; and a method of non-surgically removing localized fat deposits using the composition or formulation to relieve pain and side effects.

The present application claims priority to Korean Patent Application No. 10-2017-0146264, filed on Jan.

Locally deposited fat is of particular concern to many people. People with unwanted bumps or bulging fat deposits on the face or body parts look less attractive and look older. It can be caused by aging, lifestyle or genetic quality. In order to overcome this problem, we tried to improve through exercise and diet, but the fat-reducing effect was limited.

Typical surgical cosmetic orthopedic procedures for reducing localized fat deposition include liposuction, liposuction, and liposculpture suction, which is a cosmetic orthopedic surgery that removes large amounts of fat. The minimally invasive cosmetic (non-surgical) procedure is the procedure using a medical device, mesodermal therapy, or a single injection. However, the recovery of surgical procedures can take weeks or months, and some individual subjects (such as smokers and diabetics) may experience considerable delays in recovery, including potential complications and risks, such as causing every 100,000 people. There are 20 fatal side effects of death, general anesthesia, excessive bleeding, visceral damage, bacterial infections, scars, bruises, swelling and pain. In the case of non-surgical methods for its alternatives, there is a potential risk due to the lack of large-scale clinical trials approved under the supervision of health authorities that cannot ensure safety and efficacy. There is a need to develop new clinically available drugs that are not at risk of surgical and non-surgical procedures.

An injectable drug for topical fat loss is a drug injection which is injected into a subcutaneous fat layer to induce loss of fat cells. A typical example is a PPC injection. PPC injection is an abbreviation for polyene phospholipid choline, and there is Essentiale® N iv developed by A. Nattermann GmbH of Germany in the treatment of liver disease in the 1950s, and developed for the prevention and treatment of fat embolism. Lipostabil® N iv, and Lipobean® approved in South Korea (the Republic of Korea) as an adjunct to hepatic coma due to cirrhosis.

During the PPC injection was used to prevent and treat liver disease or fat embolism, in 1988, an Italian doctor Dr. Sergio Maggiori first reported the following test results at the MesoTherapy Society in Paris, France: Xantelasma The disease in which yellow fat is deposited in the eyelids is treated with PPC injection; in 1999, a Brazilian dermatologist Dr, Patricia G. Rittes announced at the Brazil Dermatology Society that the results of using PPC injection to reduce the lower eyelid fat pad were tested. It was confirmed that the use of PPC injection may cause fat loss. Since then, test results have been published regarding the safety of small-scale safety and PPC injections to fat deposition in the abdomen, ribs, thighs, underarms, back, arms, legs, and lipomas.

The PPC injection is a composition in which the main component polyene phospholipid choline is mixed with a solubilizing agent deoxycholic acid. PPC is an essential phospholipid and a major component of biofilms and consists of a hydrophobic tail structure in which five fatty acids are bonded to the hydrophilic head of phosphorus and choline in the glycerol carbon skeleton. It constitutes 55% of the cell membrane and mitochondrial network of the human body. Because it is hardly synthesized in the human body, it is an essential component for the external supply of the body. It is abundantly contained in soybeans, eggs, and the like. And it can be extracted by nucleic acid by physical or chemical methods and purified in high purity.

Deoxycholic acid (DCA) is one of the secondary bile salts, a metabolic byproduct of intestinal bacteria, and is mixed as a solubilizing agent to make poorly soluble PPC a stable injectable composition. The PPC solubilized with DCA was stably dispersed in the form of a mixed microcell system of less than 10 nm. If the PPC is injected without solubilization, it will not dissolve in a single molecule and the desired blood concentration will not be obtained. In addition, blood vessels may become clogged and thrombus may occur, so un-solubilized PPC is not used as an injection. If the drug is not solubilized during intravenous injection and forms a suspended sediment, larger particles will clog the blood vessels, affect blood flow to surrounding tissue near the blocked blood vessel, or damage or stimulate tissue, leading to itching, pain, and epilepsy. Attacks and similar. In severe cases, embolism may occur.

PPC has been considered as a fat-reducing active ingredient based on the fact that Lipostabil® N iv, a composition comprising PPC as a main component and DCA as a solubilizing agent, is used to treat fats that cause adipose tissue to flow into the vein to cause embolism. embolism. However, it has been confirmed that DCA is mixed as a solubilizing agent in a PPC injection to cause necrosis due to a detergent effect, resulting in a decrease in fat cells. Therefore, it has been found that the lipid-lowering active ingredient of the PPC injection is DCA (Non-Patent Document 1). Based on this, in 2015, the FDA approved a DCA single injection without PPC developed by Kythera biopharmaceuticals (a privately held company in the United States) as a cell lysing agent for improving the appearance by reducing fat under the armpit.

However, since DCA single injection or PPC injection solubilized with DCA not only dissolves fat cells (3T3L1 fat cells) but also non-selectively dissolves normal fibroblasts, endothelial cells, and skeletal muscle cells, it is a lytic injection. Non-fat degradation injection (Non-Patent Document 2).

Clinically, a mixture of PPC and DCA has been reported to cause pain (78.4%), hematoma (83.8%), erythema (100%), burning sensation (100%), edema (100%), and induration (66.7%). ), and a single DCA composition was reported to cause pain (100%), bruises (91.9%), erythema (100%), burning sensation (100%), swelling (100%), and induration (89.2%) ( Non-patent document 3). In addition, the results of large-scale clinical trials of DCA single components showed pain (73.6%), hematoma (72.9%), edema (67.8%), sensory loss (65.5%), erythema (35.3%), and swelling (29.1). %), induration (28.3%), itching (16.3%), and nodules (14.3%) (Non-Patent Document 4). These harmful conditions are caused by the following mechanism of action: DCA injected subcutaneously blocks the supply of oxygen to the cells, causing immediate cell swelling and blistering and damage of the cell membrane, resulting in necrosis of cells due to rapid inflammatory reactions (Non-Patent Document 5) .

When cells die in vivo, there is a significant difference between necrosis and apoptosis. Apoptosis refers to active death, in which the expression and activity of various genes and proteins are regulated by intracellular planned signals, and the apoptotic bodies produced by this process are removed by phagocytosis of surrounding cells or macrophages, And the like, so that it does not cause inflammation. On the other hand, necrosis is a sudden death that occurs suddenly due to changes in the external environment, which leads to irregular agglutination of the chromosomes and swelling of the cytoplasm. Finally, cell debris is formed via cellular degradation and is known to cause inflammation (Earnshaw, WC, Curr. Opin. Cell Biol., 7, pp. 337-343, 1995).

Therefore, when a topical reduced fat injection acts as a cell necrosis factor, the inflammatory action affects the surrounding area except for the target site to which the drug is administered, so that it also affects the normally functioning cells and thus kills them. In summary, it is important to selectively and efficiently induce fat lysis and apoptosis of fat cells without the approved side effects of the injection, such as pain, swelling, and sensation. Deficiency, extensive swelling, erythema, induration, paresthesia, nodules, itching, burning sensation, and muscle cell, fibroblast, and vascular endothelial cell necrosis in addition to fat cells. However, there have been no studies and formulations developed from this perspective (Non-Patent Document 6).

To date, there has been an exception treatment with Lipostabil® N iv, Essentiale® N iv (injectable formulations of a mixture of PPC and DCA) for topical fat reduction, and not only for Kybella (DCA single composition) There are many studies on fat loss, and Kybella is the first in the world to receive FDA approval as a lysing drug for appearance improvement. However, prior art and compositions have certain limitations. People who want to reduce fat with non-surgical treatment complain of discomfort and anxiety due to side effects caused by PPC + DCA composition or DCA single composition, such as pain, swelling, loss of sensation, extensive swelling, erythema, induration, and sensation. Abnormalities, nodules, itching, burning sensation, and necrosis of muscle cells, fibroblasts, and vascular endothelial cells other than fat cells. For these reasons, although clinical efficacy has now been validated, drug compliance is low. Therefore, compared with currently available cell-dissolving injections, it is necessary to develop an injection which does not cause pain and swelling due to inflammation and which selectively induces apoptosis and degradation of fat cells without reducing side effects without adverse effects. . The present invention satisfies these needs.

Based on the above facts, the inventors of the present invention studied the fat-reducing effect of PPC, and reported the following test results: a single PPC composition without DCA reduces fat cells only by apoptosis and does not by necrosis, but does not affect Fibroblasts (Non-Patent Document 7). Since then, the present inventors have completed the present invention and studied a selective fat cell-reducing composition based on PPC as an active ingredient which is painless, edema-free and has no side effects. (Non-Patent Document 1) Rotunda AM, Suzuki H, Moy RL, Kolodney M., Detergent effects of sodium deoxycholate are a major feature of an injectable phosphatidylcholine formulation used for localized fat dissolution. Dermatol Surg 30(7): 1001-8( 2004) (Non-Patent Document 2) A. Gupta, Action and comparative efficacy of phosphatidylcholine formulation and isolated sodium deoxycholate for different cell type, Aest Plast Sur, 33: 346-352, 2009 (Non-Patent Document 3) Giovanni Salti, Phosphatidylcholine and Sodum deoxycholate in the treatment of localized fat: A doube-blind, randomized study, Dermatol Surg 34:60-66, 2007 (Non-Patent Document 4) Humphrey et al., ATX-101 for reduction of submental fat: A Phase III randomized controlled Trial, J AM ACAD DERMATOL Vol. 75, No. 4, 788-797, 2016 (Non-Patent Document 5) Duncan, Injectable therapies for localized fat loss: state of the art, Clin Plastic Surg, 1-13, 2011 (Non- Patent Document 6) Duncan, Refinement of Technique in injection lipolysis based on scientifi c studies and clinical evaluation, Clin Plastic Surg 36 195-209 (2009) (Non-Patent Document 7) Dong-Seok Kim, Phosphatidylcholine induces apoptosis of 3T3-L1 adipocytes, Journal of biomedical science, 18:91,1-7, 2011

technical problem

In order to solve the side effects of conventional DCA single injection or PPC injection solubilized with DCA, such as pain, edema and various side effects caused by non-selective cell lysis, especially muscle cells, fibroblasts and blood vessels other than fat cells The serious clinical side effects caused by endothelial cell necrosis are tested by the inventors of the present invention to prepare a safe and stable topical fat-reducing injectable composition which is painless and edema by inducing apoptosis and fat dissolution of fat cells. Reduces fat and selectively reduces fat cells without damaging fibroblasts, vascular endothelial cells, and skeletal muscle cells. Accordingly, the inventors have completed the present invention by confirming the fact that the composition of the present invention prepared by adding taurine (TCA), particularly glycocholic acid (GCA) to phospholipid choline (PPC), at a specific ratio is It is safe and has excellent formulation stability and does not induce cell necrosis, but selectively induces apoptosis and degradation of only adipocytes.

Accordingly, one aspect of the present invention provides a relief of pain and side effects (edema; loss of sensation at the site of administration; extensive swelling; erythema; induration; paresthesia; nodules; itching; burning sensation; nerve damage; dysphagia; a muscle composition, a fibroblast, and a vascular endothelial cell necrosis in addition to an adipocyte, a composition for reducing local fat, the composition comprising: (i) phospholipid choline; and (ii) selected from glycocholic acid (GCA), At least one of a group consisting of taurine (TCA) and a salt thereof, wherein the composition (ii) has a molar ratio of (i) in the range of 0.7 to 3.0.

Another aspect of the present invention provides a formulation for removing localized fat deposits in an individual with reduced pain and side effects, the formulation comprising: (i) phospholipid choline; and (ii) selected from glycocholic acid (GCA) And at least one of a group consisting of taurine (TCA) and a salt thereof, wherein (ii) the molar ratio of (i) in the formulation is in the range of 0.7 to 3.0.

Another aspect of the present invention is to provide a kit comprising: (I) a first container comprising a composition or formulation for removing localized fat deposition having reduced pain and side effects, the composition or formulation And comprising: (i) phospholipid choline; and (ii) at least one selected from the group consisting of glycocholic acid (GCA), taurocholic acid (TCA), and salts thereof, wherein the composition or preparation (ii) The molar ratio of (i) is in the range of 0.7 to 3.0; and (II) the delivery device capable of delivering the composition or formulation to the fat deposition site.

Another aspect of the present invention is to provide a kit comprising: (I) a first container comprising a composition or formulation for removing localized fat deposition having reduced pain and side effects, the composition or formulation And comprising: (i) phospholipid choline; and (ii) at least one selected from the group consisting of glycocholic acid (GCA), taurocholic acid (TCA), and salts thereof, selected from the group consisting of glycocholic acid (GCA), At least one of the group consisting of tauric acid (TCA) and its salt composition is included in the same or lesser weight as the phospholipid choline; and (II) is capable of delivering the composition or formulation to the fat deposition site Delivery device.

Another aspect of the present invention provides a preparation for alleviating pain and side effects (edema; loss of feeling at the administration site; extensive swelling; erythema; induration; paresthesia; nodules; itching; burning sensation; nerve damage; dysphagia; A method for reducing the injectable composition of local fat, except for muscle cells, fibroblasts, and vascular endothelial cell necrosis, the method comprising the steps of: (a) adding an extract selected from the group consisting of glycocholic acid (GCA), and choline (TCA) and at least one of the group consisting of salt to water for injection, followed by dissolution while stirring to obtain a clear mixture; (b) adding a preservative, followed by stirring; (c) adding phospholipid choline, followed by a chamber Stirring at a temperature; and (d) adjusting the total volume of the composition with water, followed by stirring, wherein the at least one selected from the group consisting of glycocholic acid (GCA), taurocholic acid (TCA), and salts thereof, and the phospholipid The molar ratio of choline is in the range of 0.7 to 3.0.

Another aspect of the present invention provides a method for preparing a pharmaceutical composition for non-surgical removal of topical fat deposition having reduced pain and side effects, the method comprising adding phospholipid choline and selected from the group consisting of At least one of a group consisting of cholic acid, taurocholic acid, and a salt thereof,

Wherein at least one selected from the group consisting of glycocholic acid, taurocholic acid and salts thereof is added in the same or lesser weight than the phospholipid choline.

Another aspect of the present invention provides a method of removing localized fat deposition with reduced pain and side effects, the method comprising administering to a subject having localized fat deposition an effective amount of phospholipid choline; and at least one selected from the group consisting of A phospholipid choline solubilizing agent of a group consisting of cholic acid, taurocholic acid and salts thereof.

Another aspect of the present invention is to provide a method for non-surgically removing localized fat deposits in an individual having localized fat deposition with reduced pain and side effects, the method comprising administering a formulation comprising: (i) a phospholipid Choline; and (ii) at least one selected from the group consisting of glycocholic acid, taurocholic acid, and salts thereof.

Another aspect of the present invention is to provide a method of non-surgically removing localized fat deposits in an individual with reduced pain and side effects, the method comprising administering to the individual having localized fat deposition a formulation comprising: i) phospholipid choline; and (ii) at least one selected from the group consisting of glycocholic acid, taurocholic acid and salts thereof, wherein (ii) the ratio of (i) molar ratio is from 0.7 to 3.0 In the range.

In one embodiment, the formulation comprises from 1.0% to about 15.0% phospholipid choline at a concentration of fat dissolved.

In one embodiment, the formulation is an injectable formulation. In another embodiment, the formulation is an injectable preparation for reducing fat cells.

The invention also provides a method of non-surgical reduction in an individual with localized fat deposition.

In one embodiment, the method comprises administering a phospholipid choline composition in the form of a pharmaceutically acceptable formulation having a pH of 6.0 to pH 9.0 comprising solubilized with glycocholic acid or taurocholic acid. A formulation of at least one of them.

In one embodiment, the administration process comprises subcutaneous injection.

In one embodiment, the topical fat deposit is selected from the group consisting of lower eyelid fat bulge, lipoma, lipid dystrophy, and fat deposits associated with cellulite.

In one embodiment, the fat deposition is limited to the underside of the individual's eye, under the armpit (under the armpits), under the arms, hips, calves, back, thighs, ankles, or stomach.

The present invention also provides a kit comprising a formulation comprising at least one of a phospholipid choline composition comprising solubilization with glycocholic acid or taurocholic acid using a non-surgical removal of localized fat deposits in an individual. Written instructions. Technical solution

According to one aspect of the present invention, there is provided a pain relief and side effects (edema; loss of feeling at the administration site; extensive swelling; erythema; induration; paresthesia; nodules; itching; burning sensation; nerve damage; dysphagia; a fat cell extracellular muscle cell, fibroblast, and vascular endothelial cell necrosis) a composition that reduces local fat, the composition comprising: (i) phospholipid choline; and (ii) selected from glycocholic acid (GCA), bovine At least one of a group consisting of cholic acid (TCA) and a salt thereof, wherein the composition (ii) has a molar ratio of (i) in the range of 0.7 to 3.0.

According to another aspect of the present invention, there is provided a formulation for removing localized fat deposits in an individual with reduced pain and side effects, the formulation comprising: (i) phospholipid choline; and (ii) selected from the group consisting of glycocholic acid At least one of (GCA), a group of tauric acid (TCA) and a salt thereof, wherein the (ii) molar ratio of (i) in the formulation is in the range of 0.7 to 3.0.

According to another aspect of the present invention, a kit is provided comprising: (I) a first container comprising a composition or formulation for removing topical fat deposits having reduced pain and side effects, the composition or The formulation comprises: (i) phospholipid choline; and (ii) at least one selected from the group consisting of glycocholic acid (GCA), taurocholic acid (TCA), and salts thereof, wherein the composition or formulation (ii) a molar ratio of (i) in the range of 0.7 to 3.0; and (II) a delivery device capable of delivering the composition or formulation to the fat deposition site.

According to another aspect of the present invention, a kit is provided comprising: (I) a first container comprising a composition or formulation for removing topical fat deposits having reduced pain and side effects, the composition or The formulation comprises: (i) phospholipid choline; and (ii) at least one selected from the group consisting of glycocholic acid (GCA), taurocholic acid (TCA), and salts thereof, selected from the group consisting of glycocholic acid (GCA) At least one of the group consisting of taurine (TCA) and its salt is contained in the same or lesser weight as the phospholipid choline; and (II) capable of delivering the composition or formulation to the fat deposition site Delivery device.

According to another aspect of the present invention, there is provided a preparation for alleviating pain and side effects (edema; loss of feeling at the administration site; extensive swelling; erythema; induration; paresthesia; nodules; itching; burning sensation; nerve damage; dysphagia; And a method for reducing the injectable composition of local fat, in addition to fat cells, muscle cells, fibroblasts and vascular endothelial cell necrosis, the method comprising the steps of: (a) adding an extract selected from the group consisting of glycocholic acid (GCA), bovine At least one of a group consisting of an acid (TCA) and a salt thereof to water for injection, followed by dissolution while stirring to obtain a clear mixture; (b) adding a preservative, followed by stirring; (c) adding phospholipid choline, followed by Stirring at room temperature; and (d) adjusting the total volume of the composition with water, followed by stirring, wherein the at least one selected from the group consisting of glycocholic acid (GCA), tauric acid (TCA), and salts thereof The molar ratio of phospholipid choline is in the range of 0.7 to 3.0.

According to another aspect of the present invention, there is provided a method for preparing a pharmaceutical composition for non-surgical removal of topical fat deposition having reduced pain and side effects, the method comprising adding phospholipid choline and selecting from At least one of the group consisting of glycocholic acid, taurocholic acid, and a salt thereof, wherein at least one selected from the group consisting of glycocholic acid, taurocholic acid, and salts thereof is the same as or smaller than the phospholipid choline The weight is added.

According to another aspect of the present invention, there is provided a method of removing localized fat deposits in an individual with reduced pain and side effects, the method comprising administering to the individual having localized fat deposition an effective amount of phospholipid choline; At least one phospholipid choline solubilizing agent selected from the group consisting of glycocholic acid, tauric acid, and salts thereof.

According to another aspect of the present invention, there is provided a method of non-surgically removing localized fat deposits in an individual having localized fat deposition with reduced pain and side effects, the method comprising administering a formulation comprising: (i) Phospholipid choline; and (ii) at least one selected from the group consisting of glycocholic acid, tauric acid, and salts thereof.

In accordance with another aspect of the present invention, a method of non-surgically removing localized fat deposits in an individual with reduced pain and side effects is provided, the method comprising administering to the individual having localized fat deposition a formulation comprising: (i) phospholipid choline; and (ii) at least one selected from the group consisting of glycocholic acid, taurocholic acid and salts thereof, wherein (ii) the ratio of (i) molar ratio is from 0.7 to Within the scope of 3.0. the term

In the present invention, the term "phosphatidylcholine" means a compound of the IUPAC name 1,2-dimercapto-sn-glycero-3-phosphocholine which is a phospholipid and is referred to as PPC in the present specification.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section.

The article "a/an" is used herein to mean one or more of the grammatical objects of the article (ie, at least one). For example, "an element" means one or more elements.

When referring to a measurable value such as quantity, length of time, etc., the term "about" means containing ± 20%, ± 10%, ± 5%, ± 1% or ± 0.1% of the specified value. Change, as this change is appropriate for the prescribed method.

A disease or condition "alleviated" if the severity of the symptoms of the disease or condition, the frequency of such symptoms is reduced by the patient, or both.

As used herein, the term "bile acid" includes steroid acids (and/or their formic acid anions) and salts thereof, and is found in the bile of animals such as humans. "Deoxycholic acid" is a type of bile salt and refers to the IUPAC name (4R)-4-[(3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12 - Dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[ a] a compound of phenanthrene-17-yl]pentanoic acid, and is referred to as DCA in this specification.

"Glycocholic acid" is a type of bile salt and refers to the IUPAC name 2-[[(4R)-4-[(3R,5S,7R,8R,9S,10S,12S,13R,14S,17R) )-3,7,12-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetrahydrogen a compound of -1H-cyclopenta[a]phenanthrene-17-yl]pentanyl]amino]acetic acid, and is referred to as GCA in the present specification.

"Taurocholic acid" is a type of bile salt and refers to the IUPAC name 2-[[(4R)-4-[(3R,5S,7R,8R,9S,10S,12S,13R,14S,17R) )-3,7,12-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetrahydrogen a compound of -1H-cyclopenta[a]phenanthrene-17-yl]pentanyl]amino]ethanesulfonic acid, and is referred to herein as TCA.

"cholic acid" is a type of bile salt and refers to the IUPAC name (4R)-4-[(3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7 ,12-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopentyl And [a] phenanthrene-yl] valeric acid compound, and is referred to as CA in this specification.

"chenodeoxycholic acid" is a type of bile salt and refers to the IUPAC name ((4R)-4-[(3R,5S,7R,8R,9S,10S,13R,14S,17R)-3 ,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopentyl And [a] phenanthrene-yl] valeric acid compound, and is referred to as CDCA in this specification.

"ursodeoxycholic acid" is a type of bile salt and refers to the IUPAC name (4R)-4-[(3R,5S,7S,8R,9S,10S,13R,14S,17R)-3, 7-Dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta [a] phenanthrene-yl] valeric acid compound, and is referred to as UDCA in this specification.

"Glycodeoxycholic acid" is a type of bile salt and refers to the IUPAC name 2-[[(4R)-4-[(3R,8R,9S,10S,12S,13R,14S,17R)- 3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-ring A compound of pentato[a]phenanthrene-17-yl]pentanyl]amino]acetic acid, and is referred to herein as GDCA.

"Taurodeoxycholic acid" is a type of bile salt and refers to the IUPAC name 2-[[(4R)-4-[(3R,5R,9S,10S,12S,13R,14S,17R)- 3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-ring A compound of pentato[a]phenanthrene-17-yl]pentanyl]amino]ethanesulfonic acid, and is referred to herein as TDCA.

"hyodeoxycholic acid" is a type of bile salt and refers to the IUPAC name (4R)-4-[(3R,5R,6S,8S,9S,10R,13R,14S,17R)-3, 6-Dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta [a] phenanthrene-yl] valeric acid compound, and is referred to as HDCA in this specification.

"lithocholic acid" is a type of bile salt and refers to the IUPAC name (4R)-4-[(3R,5R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10, 13-Dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17- a compound of valeric acid and referred to as LCA in this specification.

"Tauroursodeoxycholic acid" is a type of bile salt and refers to the IUPAC name 2-[[(4R)-4-[(3R,5S,7S,8R,9S,10S,13R,14S, 17R)-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetrahydrogen- A compound of 1H-cyclopenta[a]phenanthrene-17-yl]pentanyl]amino]ethanesulfonic acid, and is referred to herein as TUDCA.

"Dehydrocholic acid" is a type of bile salt and refers to the IUPAC name (4R)-4-[(5S,8R,9S,10S,13R,14S,17R)-10,13-dimethyl -3,7,12-trisyloxy-1,2,4,5,6,8,9,11,14,15,16,17-dodecahydrocyclopenta[a]phenanthrene-17-yl a compound of valeric acid, and is referred to herein as DHCA.

The terms "patient", "subject", "individual" and the like are used interchangeably herein and refer to any animal or cell thereof capable of performing the methods described herein ( Such as in a test tube or in situ). In certain non-limiting embodiments, the patient, individual, or individual subject is a human.

As used herein, the term "composition" or "pharmaceutical composition" may mean at least one compound or composition used in the present invention with, for example, any additional carrier, stabilizer, suspending agent, dispersion. Mixtures of other chemical components of the agents, suspending agents, thickening agents and/or excipients and the like. The pharmaceutical composition facilitates administration of the compound to the organism.

As used herein, the terms "effective amount", "pharmaceutically effective amount" and "therapeutically effective amount" mean a non-toxic and sufficient amount that provides the desired biological result. . The result can be a reduction and/or alleviation of the signs, symptoms or causes of the disease, or any other desired change in the biological system. The appropriate amount of treatment in any particular case can be determined by routine testing by those of ordinary skill in the art.

As used herein, the term "efficacy" may refer to the maximum effect (Emax) achieved within an analysis.

As used herein, "instructional material" includes a publication that can be used to convey the utility of a compound, composition, carrier or delivery system of the invention in a kit for alleviating the various diseases or conditions mentioned herein, Record, schema or any other presentation medium. Alternatively or in the alternative, the instructional material can describe at least one method of ameliorating a disease or condition in a cell or tissue of a mammal. The instructional materials of the kits of the invention may, for example, be attached to a container comprising the identified compound, composition, carrier or delivery system of the invention, or may be shipped with a container containing the compound, composition, carrier or delivery system.

Alternatively, the instructional material can be shipped separately from the container with the intent to be used by the recipient in conjunction with the compound (composition).

The term "local administration" refers to the administration of a pharmaceutical ingredient to or around the muscle or subdermal location of a patient via a non-systemic route. Thus, topical administration excludes administration via a systemic route, such as intravenous or oral administration.

The term "pharmaceutically acceptable" means that their properties and/or substances are acceptable to the patient from a pharmacological/toxicological perspective and relate to composition, formulation, stability, patient acceptance and bioavailability. Physical/chemical perspectives are acceptable for preparation by pharmaceutical chemists. "Pharmaceutically acceptable carrier" means a medium which does not interfere with the effectiveness of the biological activity of the active ingredient and which is non-toxic to the host to which it is administered.

"Therapeutic" treatment is the treatment administered to an individual exhibiting their symptoms or symptoms for the purpose of alleviating or eliminating pathological signs or symptoms.

As used herein, the term "treatment/treating" is defined as the administration or administration of a therapeutic agent to a patient, ie, a compound of the invention (alone or in combination with another pharmaceutical agent); or a tissue or cell line isolated from a patient. Administration or administration of a therapeutic agent (for example, for diagnostic or in vitro use) having the conditions covered herein, the symptoms of the conditions covered herein, or the possibility of suffering from the conditions covered herein, for administration or administration To cure, heal, alleviate, alleviate, alter, compensate, improve, ameliorate, or affect the conditions covered by this document, the symptoms of the conditions covered herein, or the likelihood of suffering from the conditions covered herein. Such treatments may be specifically adjusted or modified based on knowledge gained from the field of pharmacology.

The term "therapeutically effective amount" is the amount of a compound of the invention that ameliorates the symptoms of a disease when administered to a patient. The amount of the compound of the present invention which constitutes a "therapeutically effective amount" will vary depending on the compound, the state of the disease and its severity, the age of the patient to be treated, and the like. A therapeutically effective amount can be routinely determined by one of ordinary skill in the art in view of its own knowledge and the present invention.

Scope: Throughout the present invention, various aspects of the invention may be presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be construed as an inflexible limitation of the scope of the invention. Accordingly, the description of a range should be considered as a For example, a description of ranges such as 1 through 6 should be considered to have specifically disclosed sub-ranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and Individual values within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Unless otherwise stated, the term "%" used in the present invention to mean the content of the composition means the content of w/v%, and unless otherwise stated, means the w/v% value in terms of the total composition.

In the present invention, the symbol "/" in the description of "at least one selected from the group consisting of glycocholic acid or taurocholic acid and its salt/phospholipid choline" is presented in the form of a commonly used form.

Hereinafter, the present invention will be described in detail.

FDA-approved Kybella (DCA 1.0%) (a cell lysing agent for improving appearance) and PPC injectable compositions (eg Lipostabil, Essential, Lipobean) that are solubilized with DCA when administered In the case of local fat loss, it is accompanied by pain and edema caused by inflammatory reaction. In addition, it is a non-selective cytolytic agent that causes necrosis of fibroblasts, skeletal muscle cells, vascular endothelial cells, and adipocytes, and has been reported to cause side effects such as necrosis of the administration site, ulceration, and mandibular paralysis and nerve damage. For this reason, it has been warned not to inject drugs into the salivary glands, lymph nodes, muscles or areas close to them to prevent potential damage to tissues other than fat. In addition, individuals currently receiving Kybella continue to report side effects such as pain, swelling, facial paralysis and skin necrosis.

In this regard, the inventors of the present invention found that a PPC (5.0%) single composition without a solubilizing agent has an equivalent in vitro adipose cell reduction effect of Kybella (DCA 1.0%), but PPC is apoptotic and degraded by apoptosis. The mechanism selectively induces a decrease in adipocytes, and it has been confirmed that it is possible to develop a fat-reducing composition that is painless, edema-free, and has no side effects. However, PPC has limitations in preparing physically or chemically stable and safe injectable compositions because of its poor solubility. Based on this background, the present inventors have studied the composition of an adipocyte reducing agent containing PPC as a main component since 2010. Therefore, PPC dispersed in a high-pressure homogenizer exhibits a time-dependent and concentration-dependent effect of fat cell reduction, but it is limited in industrial use due to low stability.

For this reason, the inventors conducted a formulation test in a physical and chemical manner to prepare a composition capable of safely and stably subcutaneously injecting poorly soluble PPC (Figs. 1 to 3). This study is based on the hypothesis that toxicity can be manifested by the surface activity specificity of bile acids (salts). Since the surface active function inhibits the apoptosis and degradation effects of the fat cells inherent to PPC and induces cell lysis by the necrotic function, it causes local fat loss accompanied by pain, edema, and side effects. Thus, the particular type of surfactant and its use capacity (ratio) pose significant technical hurdles to those skilled in the art.

The results provided below in the various comparative examples, examples, and experimental examples demonstrate the unexpected specific effects of the compositions of the present invention. Briefly, the inventors selected a combination of bile acids (salts) that had no in situ adverse reactions or mild adverse reactions in the assessment of edema, lesions, and inflammation in vivo among selected compositions, according to the test. The results were confirmed to be industrially applicable and safe. Among the selected bile acids (salts), GCA and TCA were selectively selected for adipocytes in the viability of adipocytes, fibroblasts, endothelial cells, and skeletal muscle cells, and then increased by GCA or TCA. The effect of the dissolved PPC composition on apoptosis and degradation of adipocytes was verified. Further, it has been confirmed that the GPC solubilized PPC composite composition of the present invention is not only systemic and has no local toxicity. The results of the clinical trials of the inventors of the present invention revealed the following unexpected findings: the composition is not inferior to Kybella in reducing fat, and the composition is painless, edema free and has no side effects or pain, edema and side effects reduced by 80% or more. Big. This will be described in more detail below.

As a solubilizing agent selected as a solubilizer for PPC (cholic acid (CA), deoxycholic acid (DCA), glycocholic acid (GCA), bovine cholic acid (TCA), chenodeoxycholic acid (CDCA), bear Deoxycholic acid (UDCA), glycodeoxycholic acid (GDCA), taurate (TDCA), hyodeoxycholic acid (HDCA), ursodeoxycholic acid (TUDCA), lithocholic acid (LCA) And dehydrocholic acid (DHCA) test results, it is confirmed that LCA and DHCA can not solubilize PPC (Figures 3A and 3B), and CA, DCA, GCA, TCA, CDCA, UDCA, GDCA, TDCA, HDCA and TUDCA stably solubilizes PPC at specific or greater molar ratios (Figures 2A-2J).

Inflammation, edema, and lesions were tested in vivo using CA, DCA, GCA, TCA, CDCA, UDCA, GDCA, TDCA, HDCA, and TUDCA, which are bile acids tested from the formulation. Among bile acids, DCA is the most potent surfactant and has been reported to cause inflammation, swelling and clinical side effects caused by non-selective cell lysis. Accordingly, the inventors have recognized that additives exhibiting deleterious effects, such as DCA in bile acids, are not suitable as solubilizing agents because they inhibit the intrinsic activity of PPC on apoptosis and degradation of selective adipocytes. Thus, first, in vivo injections of different concentrations of bile acids were administered to study edema, skin lesions, and inflammation, which are representative deleterious examples.

As a result of the in vivo edema test of bile acids (Figs. 4A to 4G), 2 hours after administration, it was confirmed: "None" - PPC solubilized with GCA (1.25-2.5%) or TCA (1.25-2.5%) (2.50-5.0%) composite composition, "mild" - single composition of PPC (1.25-10.0%), GCA (1.0%), TCA (1.0%) and TUCA (1.0%, 2.5%), and PPC (7.5%, 10.0%) + GCA (3.75%, 5.0%) and PPC (7.5%, 10.0%) + TCA (3.75%, 5.0%) composite composition, "moderate" - PPC (12.5%, 15.0) %), UDCA (1.0%), GDCA (1.0%), CDCA (1.0%), CA (1.0%), GCA (2.5%, 5.0%), TCA (2.5%, 5.0%), and TUDCA (5.0%, 7.5%) of a single composition, and a composite composition of PPC (5.0%) + CA (2.5%), PPC (15.0%) + GCA (7.5%) and PPC (15.0%) + TCA (7.5%), Severe and "extremely serious" - other single components and composite compositions.

The PPC (2.5-15.0%) + GCA (1.25-7.50%) composite composition of the present invention (Fig. 4N) and PPC (Fig. 4N) compared to PPC (5.0%) solubilized with DCA (2.2%) showing extremely severe edema. The 5.0%)+GCA (2.5-7.5%) composite composition (Fig. 4P) is a surprising invention of edema. Surprisingly, it was found that the degree of edema observed in the case of GCA or TCA alone was significantly reduced in the PPC + GCA and PPC + TCA composite compositions. However, DCA alone or a composition with 2.2% solubilized 5.0% PPC showed extremely severe edema after administration, indicating that DCA has the effect of inducing cell necrosis and disrupting apoptosis and degradation of PPC-selective selective adipocytes (Fig. 4M).

As a result of the in vivo skin lesion test (Figs. 5A to 5F), 2 hours after administration, it was confirmed that: "None" - PPC (1.25-15.0%), GCA (1.0%), TCA (1.0%), and TUDCA ( 1.0-7.5%) of a single composition, and a composite composition of PPC (2.5-10.0%) + GCA (1.25-5.0%) and PPC (5.0%) + GCA (2.5-5.0%), "mild" - HDCA (1.0%), CA (1.0%), GCA (2.5%, 5.0%) and TCA (2.5%, 5.0%) single composition, and PPC (15.0%) + GCA (7.5%) composite composition , "Moderate" - DCA (1.0%), UDCA (1.0%), TDCA (1.0%), GDCA (1.0%), CDCA (1.0%), CA (2.5%), GCA (7.5%) and TCA ( 7.5%) of the single composition, "severe" and "extremely severe" - other single compositions and composites.

These results are consistent with the edema test results and it has been demonstrated that subcutaneous injection of PPC + GCA or PPC + TCA complex reduces symptoms of the lesion compared to GCA or TCA single composition.

As a result of the in vivo H&E inflammation test (Figures 6A to 6F), it was confirmed that: "None" - PPC (2.5-7.5%), TUDCA (1.0-5.0%) single composition, and PPC (2.5-7.5%) +GCA (1.25-3.75%), PPC (5.0%) + GCA (2.5-7.5%), PPC (5.0%) + TCA (2.5%) and PPC (5.0%) + TUDCA (4.0%) composite composition , "mild" - a single composition of PPC (10.0%, 12.5%), GCA (1.0%), TCA (1.0%) and TUDCA (7.5%), and PPC (10.0%) + GCA (5.0%), Composite composition of PPC (5.0%) + GCA (10.0%), "moderate" - PPC (15.0%), TDCA (1.0%), GDCA (1.0%), CDCA (1.0%), CA (1.0%) And a single composition of GCA (2.5% and higher) and TCA (2.5% and higher), and a composite composition of PPC (15.0%) + GCA (7.5%) and PPC (5.0%) + CA (2.5%) Things, "severe" and "extremely serious" - other single compositions and composites.

These results are consistent with the edema and lesion test results, demonstrating that the toxicity of GCA or TCA single injection when combined with PPC is not toxic or unresolved, but DCA or its equivalent bile acid causes disruption of apoptosis and degradation. Pain, edema, and side effects caused by necrosis of PPC intrinsic activity.

As a result of an in vitro test for in vitro fat cell viability using the solubilizing agents TUDCA, TCA, and GCA solubilized PPC compositions selected based on the above test of bile acids, and in vivo edema, inflammation, and skin lesion test results, The PPC single composition group, the PPC group solubilized with GCA or TCA showed a decrease in adipocyte viability in a time- and concentration-dependent manner (Figs. 7A to 7D). An unusual finding: TUDCA inhibits apoptosis and degradation of adipocytes. In this regard, the results of studies on apoptosis inhibition of cells of TUDCA have been disclosed (Andrew L. Rivard, Administration of Tauroursodeoxycholic acid reduced apoptosis following myocardial infarction in rat, The American Journal of Chinese Medicine, Vol. 35, No. 2 , 279-295, 2007), indicating that PPC acts on cell necrosis and apoptosis in different ways.

Single PPC 5.0%, PPC 5.0% + GCA 2.5%, and PPC 15.0% + TCA 7.5% exhibited an adipocytopening activity similar to DCA 1.0% at 96 hours. That is, the single PPC 5.0% and PPC 5.0% + GCA 2.5% groups exhibited the same fat cell viability as the FDA approved cell lysing agent Kybella (DCA 1.0%), and the fat cells in these experimental groups There was no statistically significant difference in reducing the effect (Fig. 7E). In summary, it is important to have low autotoxicity (such as selective apoptosis of adipocytes intrinsic to PPC and uninterrupted activity of degradation and necrosis (or this toxicity can be offset by PPC)) and can provide composition safety and formulation stability. The bile acid is selected as a solubilizing agent which is selected to prepare PPC as a topical reduced fat injectable composition. That is, it has surprisingly been found that a solubilizing agent which does not have a negative transformation (PPC + DCA) or inhibition (PPC + TUDCA) activity (such as necrosis) against the inherent fat loss effect of PPC should be selected.

As a result of an in vitro fat cell viability test performed on a GCA single composition mixed with a molar ratio of PPC for solubilization, GCA showed a decrease in adipocyte viability in a time- and concentration-dependent manner. There was no statistically significant difference in fat cell viability between the PPC single composition and the PPC composition solubilized with GCA (Figs. 7F to 7H). That is, the apoptosis effect of the PPC single composition and the fat cell of the PPC composition solubilized by GCA is equivalent. To examine the effect of GCA on adipocyte reduction in the composition of the present invention, the fat cell reduction effect of PPC with increasing GCA input was observed. As a result of the 96-hour adipocyte viability test after PPC (5.0%) composite composition solubilized with GCA (2.5-8.75%) and PPC (5.0%) single composition, solubilized with GCA (2.5-7.5%) The effect of the PPC (5.0%) composite composition was statistically insignificant compared to the PPC (5.0%) single composition. That is, the apoptotic effects of the fat cells of these groups are equivalent. However, the PPC (5.0%) treated group solubilized with GCA (8.75%) showed statistically significant differences in the apoptosis effect of adipocytes (Fig. 7I). These results indicate that the inherent positive efficacy of PPC may be adversely affected when the molar ratio of GCA/PPC is 3.04 mol/mol (PPC 5.0% + GCA 8.75%) or greater.

According to previous reports, DCA compositions or PPC compositions solubilized with DCA have been reported to cause clinically fatal side effects due to not only dissolving fat cells but also fibroblasts, skeletal muscle cells and vascular endothelial cells. As a result of observations of the cell viability of the PPC single composition and the PPC complex composition solubilized with GCA, it was surprisingly found that PPC + GCA selectively reduced fat cells, unlike PPC + DCA (Figures 8A to 8D). ). The composition of the present invention is a selective fat cell depletion composition which is safe and fat-reducing without fatal side effects such as skin necrosis, mandibular nerve palsy, difficulty in swallowing, and side effects by the commercially available cell lysing agent Lipostabil® N, Iv (PPC + DCA) and Kybella iv (DCA) caused.

In order to evaluate the reduction of adipocytes due to necrosis or apoptosis and degradation, the effect on apoptosis of adipocytes was observed via apoptosis protease 3 activity and the effect on fat cell fat dissolution was observed via glycerol release.

The PPC single composition and the PPC + GCA composite composition exhibited a time-dependent effect of inducing apoptosis protease-3 activity at a considerable level. However, the PPC + DCA composite composition inhibits apoptosis protease-3 activity compared to PPC or PPC + GCA. Interestingly, DCA 1.0% exhibited a certain level of caspase-3 activity up to 24 hours, but after 48 hours, caspase-3 activity returned to pre-treatment levels. This phenomenon is considered to be due to the fact that the effect of cell apoptosis against cells is only partially observed 24 hours after treatment with the DCA single composition, and then the effect becomes an acellular necrosis pathway by an inflammatory reaction ( Figures 10A and 10B).

Test materials other than DCA 1.0% and PPC 5.0% + GCA 5.0% similarly induced glycerol secretion 24 hours after treatment. At 48 hours after treatment, single PPC, PPC + DCA, single DCA, and single GCA groups exhibited slightly higher cytolytic activity than at 24 hours. In particular, the PPC + GCA group exhibited a much higher apoptotic effect on cells than the PPC single composition (Figures 10C and 10D).

Therefore, the PPC single composition and the PPC + GCA composite composition cause a specific effect of reducing fat cells due to apoptosis and fat dissolution mechanism different from the necrosis mechanism of PPC + DCA. Under this mechanism, it was found that when the composition of the present invention was administered to the subcutaneous fat layer, fat was reduced without pain, edema, and without side effects.

Based on the effect of intracellular fat cell reduction, PPC single composition (2.5%, 5.0%, 10.0%, and 15.0%), PBS (negative control) and Isuprel (positive control), DCA 1.0%, GCA 2.5%, PPC 5.0 %+DCA 2.2%, PPC 5.0%+HDCA 2.5%, PPC 5.0%+UDCA 3.0%, PPC 5.0%+TDCA 2.5%, PPC 5.0%+GDCA 2.5%, PPC 5.0%+CDCA 2.5%, PPC 5.0%+ In vivo H&E histopathology tests were performed in CA 2.5%, PPC 5.0% + TUDCA 4.0%, PPC 5.0% + TCA 2.5%, and PPC (2.5-10.0%) + GCA (1.25-5.0%). Thus, adipose tissue injected with a single DCA or PPC + DCA composite composition exhibited severe inflammation in the administration area, and the cells dissolved due to necrosis and induced significant damage. The DCA single composition exhibited a severe level of inflammation even if DCA was included at a low concentration of 1%, and the inflammatory induction was greater than the inflammatory induction of the GCA single composition. The composite composition of PPC + GCA showed that the fat cells became smaller, the apoptotic cells were clearly observed, and the fat cells became fat cells formed by the fusion of collapsed fat cells, and the degree of inflammation induction at all treatment concentrations. Both were low and morphological features were found to only damage the adipocyte membrane (Figures 11A to 11D).

Results of the formulation test; in vivo edema, inflammation and skin lesion test results; in vitro test of fat cells, muscle cells, fibroblasts, endothelial cell viability test results; and in vivo fat pad H&E histopathology test results, The GPC solubilized PPC composite composition of the present invention has significantly lower local toxicity than the PPC composite composition and the GCA single composition solubilized by the product DCA. To verify these results according to good laboratory practice (GLP), a single dose toxicity study was performed on the beagle dog by calculating the clinical dose. Therefore, it was confirmed that the composite composition of the present invention is non-toxic (Figs. 12A to 12C).

To assess the extent of pain in the body, the distance and speed of movement of the mice were measured after administration of each experimental composition. Except for the PPC single formulation and the GCA + PPC formulation, the distance and speed of movement were significantly reduced in all experimental groups compared to before administration. And the results showed that the distance and speed of movement were unchanged or slightly increased in the group treated with PPC 5.0% single composition, PPC 5.0% + TUDCA 4.0%, PPC 5.0% + GCA 2.5% or PPC 5.0% + TCA 2.5%. . On the other hand, PPC 5.0%+DCA 2.2%, PPC 5.0%+HDCA 2.5%, PPC 5.0%+UDCA 3.0%, PPC 5.0%+TDCA 2.5%, PPC 5.0%+GDCA 2.5%, PPC 5.0%+ The distance and speed of movement in the CDCA 2.5% or PPC 5.0% + CA 2.5% treated group decreased by 20%, and it was judged that the activity was lowered by pain (Figs. 13A and 13B).

Based on the results of the formulation test; in vivo edema, inflammation and skin lesion test results; test tube fat cells, muscle cells, fibroblasts, endothelial cell viability test results; in vivo fat pad H&E histopathology test results; single dose toxicity Test results; and pain test results, assess safety and efficacy before and after administration to human subjects for clinical validation. The results of the investigator's clinical study showed a subcutaneous fat injection, 0.2 cc, 1 cm interval, 6 to 8 mm depth, a total of 50 points, 10 ml dose, 4 weeks after 4 weeks, 12 weeks, visually It was confirmed that the underarm fat was reduced (Fig. 14A). The level of satisfaction reported by the individual was 4 out of 5, and the improvement after comparison with the pre-dose photo was reported to be grade 1.5 (Fig. 14A). In addition, the underarm fat was reduced by 30.36% on CT, from 5.6 mm before administration to 3.9 mm at 12 weeks after the final administration (Fig. 14B).

After administration of the GCA solubilized PPC injectable composition of the present invention, six male and female patients who have received the PPC injection composition (previously commercialized product) solubilized with DCA are subjected to pain, edema, and side effects. Clinical evaluation. After local anesthesia with 9.6% lidocaine cream for 30 minutes or longer, use a syringe equipped with a 13 mm needle in the abdomen and ribs (1.5 cm spacing, 10 to 12 mm depth, each point) 0.5 cc, 50 ml to 100 ml per administration) or in the underarm fat (1.0 cm interval, 6 to 8 mm depth, 0.2 cc per point, 10 ml per dose). The injection contains a 1:1 ratio. An injection of physiological saline and a composition of Lipobean® (PPC 5.0% infusion of DCNa 2.4% solubilized) (PPC 2.5% + DCNa 1.2%), and the composition of the present invention is administered to the individual by the same administration method (using 2.8% GCA solubilized 5.0% PPC injection or 5.0% PPC injection solubilized with 4.0% GCA). As a result of the test, individuals who have received an injection of the PPC composition solubilized with DCNa complained of pain and edema especially at the time of administration and 10 days after administration, and also reported skin lesions such as erythema, hematoma, bruises and local injuries. Such as induration, nodules, itching and burning. Surprisingly, however, individuals receiving the GPC solubilized PPC injection of the present invention were alleviated to a mild level, in particular to the point where pain (Fig. 15A) and edema (Fig. 15B) were substantially absent. That is, the safety was increased by 80% or more as compared with the group receiving the PPC injection solubilized with DCA, and no serious adverse reactions were reported among the individuals (Figs. 15A to 15C). Specifically, side effects such as swelling, hematoma, bruising, erythema, paresthesia, induration, nodules, and itching were not observed, or these side effects were observed at a significantly low level.

In addition, when PPC solubilized with GCA was administered to the abdomen and ribs at a dose of 0.5 cc, 1.5 cm, 12 mm, and a total of 200 points at a dose of 100 ml, visually no skin lesions were observed (Fig. 16). . As shown in Fig. 16, in addition to the bruise caused by the injection needle itself or the vascular damage caused by the blood vessel damage at the time of injection, the erythema was reduced to no or slight. No paresthesia, extensive swelling, induration, paresthesia, nodules, itching, burning sensation, difficulty in swallowing, and the like were observed (Fig. 15C).

In summary, the composition of the present invention is an innovative invention which selectively reduces fat cells by a mechanism of apoptosis and degradation; reduces anxiety and discomfort of the patient; improves drug compliance of the patient; and has formulation stability. And the composition of the present invention does not cause pain and edema caused by inflammation of a conventional injectable composition of PPC administered with DCA or solubilized with DCA; and does not cause a wide range caused by non-selective cytolytic activity Swelling, erythema, discomfort such as bruising and loss of sensation, induration, paresthesia, nodules, itching, burning sensation, nerve damage and difficulty swallowing.

Accordingly, there is provided a composition for reducing localized fat having reduced pain and side effects, the composition comprising: (i) phospholipid choline; and (ii) selected from the group consisting of glycocholic acid (GCA), choline ( At least one of a group consisting of TCA) and a salt composition thereof, wherein the composition (ii) has a molar ratio of (i) in the range of 0.7 to 3.0.

Further, there is provided a composition for reducing local fat with reduced pain and side effects, the composition consisting essentially of: (i) phospholipid choline; and (ii) selected from glycocholic acid (GCA), taurocholic acid At least one of (TCA) and a group of its salt composition, wherein the composition (ii) has a molar ratio of (i) in the range of 0.7 to 3.0.

In particular, there is provided a composition for reducing localized fat having reduced pain and side effects, the composition comprising: (i) phospholipid choline; (ii) selected from glycocholic acid (GCA), choline At least one of (TCA) and a group of its salt composition; and (iii) water (or water for injection), wherein the composition (ii) has a molar ratio of (i) in the range of 0.7 to 3.0.

In addition, a composition for reducing localized fat with reduced pain and side effects is provided, the composition consisting essentially of: (i) phospholipid choline; (ii) selected from the group consisting of glycocholic acid (GCA), bovine cholic acid ( At least one of a group consisting of TCA) and its salt composition; and (iii) water (or water for injection), wherein the composition (ii) has a molar ratio of (i) in the range of 0.7 to 3.0.

The reduced fat injectable composition of the present invention can be applied to a localized area, and is preferably applied to fat, deposited to the abdomen, underarms, forearms, thighs, waist, hips, under the eyes, bra lines, and the like, However, it is not limited thereto and can be applied to neck wrinkles for improvement.

The compositions of the present invention are preferably used for non-surgical removal of localized fat deposits in an individual. The term "non-surgical" refers to a medical procedure that does not require a cut.

The formulation of the composition of the present invention is not particularly limited as long as it is used for the purpose of reducing fat (in detail, reducing local fat), and includes, for example, an injection such as a patch, a deposit, etc., and it is preferably injectable. Composition. That is, the present invention relates to a composition or preparation that can be directly injected non-surgically into a treatment site of a patient requiring fat removal.

The local fat-reducing injectable composition of the present invention is a pharmaceutical composition for treating adipose tissue hyperplasia or hyperproliferative disorders (diseases), and the disorder is not particularly limited as long as it is known in the art as Pathological hyperproliferation or excessive accumulation of adipose tissue, such as obesity (abdominal obesity), lower eyelid escape, lipoma, Dercum's disease, Madelung's neck, fat edema , but not limited to, piezogenic nodule, Xanthosis, lipodystrophy, fat accumulation associated with cellulite, and the like.

The method of administering the injectable composition of the present invention is not limited, but can be administered by a method suitable for the patient in view of the severity of the disease, the age, sex and other conditions of the patient. The route of administration is not particularly limited in the method, but is preferably administered directly to the subcutaneous fat layer (tissue), for example, multiple subcutaneous or intradermal injections at intervals of 0.5 to 2.0 cm in the lattice interval.

In the present invention, the term side effect refers to a side effect of a PPC preparation containing a specific bile salt which is known or commercially available, and is particularly known as a DCA increase known as an injection for reducing local fat. Soluble PPC injections (eg Lipostabil, Essential, Lipobean) or DCA single formulations (eg Kybella). And it refers to the harmful effects on the human body other than the main effect expected to be the therapeutic effect of the drug (in the present invention, the fat-reducing effect). Specifically, in addition to the hematoma and bruise caused by the injection needle itself, the side effect is at least one selected from the group consisting of edema, loss of sensation (especially loss of sensation at the administration site), extensive swelling, erythema, hematoma, Bruises, induration, sensory abnormalities, nodules, itching, burning sensation, difficulty in swallowing, and necrosis of cells other than fat cells (muscle cells, fibroblasts, vascular endothelial cells, etc.) and the like, but are not limited thereto. The injectable composition of the present invention for reducing local fat is characterized in that the local adverse reaction is alleviated to the extent that the side effect is substantially absent.

As used herein, the term "alleviation of pain and side effects" includes alleviation, elimination of pain and side effects, presence at low levels (partial removal), substantial absence (substantial removal), and completeness. There is no such thing as (complete removal).

In the present invention, pain and edema include pain at the time of administration of an injection and pain and edema after administration. The present invention is characterized by a significant reduction in pain and edema (substantially no pain and edema) as opposed to a conventional commercially available DCA single composition involving pain and edema or a PPC injection that is solubilized with DCA. Such painless and edema-free PPC injections are first disclosed in the present invention, and in particular, are characterized by the absence of secondary pain and edema caused by inflammation for more than 10 days and at the time of injection and immediately after injection. Primary pain after administration. In the present invention, the meaning of pain is different from the symptoms caused by needle invasion (for example, nausea, needle-induced bruise, needle-induced hematoma, needle-induced swelling), and it means that DCA single injection or DCA + has been used. PPC can inject pain or edema (inflammation) caused by the characteristics of the composition itself. In one embodiment of the present invention, the PPC + GCA complex preparation of the present invention uniquely shows almost no pain at the time of injection as compared with the commercially available "DCA solubilized PPC injection" used as a control group (Fig. 15A). It is considered that the particle characteristics (microcells, particle size, etc.) of the composite preparation of the present invention are similar to the existing commercial preparations (representative examples of PPC injections solubilized with DCNa) and all the injections are administered at a pH similar to human body, The effects of the compositions of the present invention are difficult to predict from previously known techniques.

Further, the composition of the present invention is characterized in that it specifically causes apoptosis and fat dissolution of fat cells. In a specific embodiment of the present invention, the PPC + GCA complex preparation of the present invention does not substantially affect other cells other than fat cells (such as fibroblasts, skeletal muscle cells, and vascular endothelial cells), and it is confirmed that the cells The induction of apoptosis and fat solubilization is only specifically effective in adipocytes (see Figures 8A to 8D). This effect is comparable to PPC formulations (typically commercially available PPC + DCA formulations) which are solubilized with other bile acids which cause cell necrosis in addition to fat cells. It has been revealed for the first time in the present invention that when GCA and PPC are mixed at a certain ratio, they have an adipocyte-specific (selective) effect.

The injectable preparation is prepared by dissolving the main drug (in the present invention, PPC solubilized with GCA) and, if necessary, other additives in water for injection, filtering the solution with a bacterial filter, sterilizing the solution, and filling the solution. In a vial, ampoule or free field syringe, it is then sealed. Therefore, in the preparation of an injection, water for injection and water can be used to fill the remaining amount. The water for injection is not particularly limited as long as it is a distilled water for injection or a buffer solution for injection intended to be used for diluting a solid injection or a water-soluble injectable solution. For example, a phosphate buffer solution or sodium dihydrogen phosphate (NaH ₂ PO ₄ ) (in the range of pH 3.5 to 7.5)-citric acid buffer solution or the like can be used. The phosphate used herein may be in the form of a sodium salt or a potassium salt, or may be in the form of an acid anhydride or a hydrate, and may be in the form of a citric acid or an acid anhydride or a hydrate. Examples of water for injection include glucose injection, xylitol injection, D-mannitol injection, fructose injection, physiological saline, polydextrose 40 injection, polydextrose 70 injection, amino acid injection, Ringer Ringer's solution and lactic acid-Linger's solution, but are not limited thereto.

In the present invention, phospholipid choline (PPC) is a phospholipid widely found in animals, plants, yeasts and fungi. It is also known as lecithin, polyene phospholipid choline and 3-sn-phospholipid choline and has the basic structure of formula 1. It is a membranous phospholipid of mammals mainly in the brain, nerves, blood cells, egg yolks and the like. In plants, it is contained in soybeans, sunflower seeds, wheat germs, and is rarely found in bacteria. In general, saturated fatty acids are bonded to the 1st position of the glycerol, unsaturated fatty acids are bonded to the 2 position, and most of the thiol groups are C12 to C22 (12 to 22 carbon atoms). <Formula 1>

The phospholipid choline of the present invention has a structure as shown in Formula 1, R1 is a saturated or unsaturated fatty acid having 12 to 22 carbon atoms, and R2 is a saturated or unsaturated fatty acid having 12 to 22 carbon atoms. The saturated or unsaturated fatty acid may be in the form of a straight chain or a branched chain, and the unsaturated fatty acid may comprise monounsaturated or poly (eg, two, three or four) unsaturation. The phospholipid choline of the present invention may be a single compound or a mixture of various compounds which may be R1 and R2 thiol groups having different carbon numbers. Preferably, the phospholipid choline of the present invention may have a molecular weight of from 700 g/mol to 1000 g/mol, and more preferably from 750 g/mol to 800 g/mol.

The phospholipid choline of the present invention can be extracted from any one selected from the group consisting of various animals or plants such as soybean, sunflower seed, wheat germ, and egg yolk. Alternatively, commercially available phospholipid choline of the present invention may be purchased and used, or a product prepared by chemical synthesis methods known in the art may be used.

The phospholipid choline of the present invention is preferably isolated from soybean or egg yolk. In general, the typical structure of phospholipid choline isolated from soybean is as shown in the following formula 2. Moreover, in general, the typical structure of phospholipid choline derived from egg yolk is as shown in the following formula 3. The phospholipid choline used in the present invention may be a single compound composed only of the compound of the following formula 2 or 3, or further a mixture of several compounds based on the R1 and R2 fluorenyl groups having different carbon numbers of the formula 1. The mixture may contain substantially 50% by weight or more, more preferably 70% by weight or more, and most preferably 90% by weight or more of the compound of the following Formula 2 or 3. <Formula 2> <Formula 3>

Most preferably, the phospholipid choline of the present invention may be extracted from soybeans and may be a mixture containing a compound having a structure as shown in Formula 2 in a ratio of 93.0% by weight or more.

In the injectable composition for reducing local fat of the present invention, phospholipid choline is present in a concentration of from 0.625 to 15.0% (w/v), preferably from 1.25 to 12.5% (w/v), based on the total composition. More preferably, it is included in the total composition at a concentration of 2.5 to 10.0% (w/v). Most preferably, phospholipid choline may be included at a concentration of 2.5 to 7.5% (w/v) of the total composition. When the concentration of phospholipid choline is less than 0.625% (w/v), there is no fat dissolution effect (see Figs. 7A to 7D). When the concentration of phospholipid choline is greater than 15% (w/v), it is inconvenient to administer multiple doses to the subcutaneous fat layer due to its high viscosity, and because of the excessive use of solubilizing agents, a moderate abnormality of inflammatory reaction appears. And serious side effects such as pain, swelling and inflammation may occur.

The composition for reducing local fat of the present invention is characterized in that at least one selected from the group consisting of glycocholic acid (GCA), taurine (TCA) and salts thereof (hereinafter abbreviated as (ii)) and phospholipid choline The molar ratio of (PPC, abbreviated as (i) hereinafter) is in the range of 0.7 to 3.0. In other words, (ii) the molar ratio of (i) may be in the range of 0.7 to 3.0, (ii) the molar ratio of (i) may be better in the range of 0.7 to 2.60, and (ii) ratio ( The moir ratio of i) is preferably in the range of 0.7 to 1.73. When they are contained in a molar ratio (mol/mol) of less than 0.7, it is difficult to form stable micelles, resulting in poor formulation stability. Therefore, the lower molar ratio is preferably 0.70 or more, and more preferably 0.76. When the upper limit of the molar ratio is 3.04 or more, pain, edema, and side effects are remarkably manifested as mild or heavier inflammation, moderate or heavier edema, and severe or heavier skin lesions, respectively, due to cell necrosis. Limiting the inherent function of PPC rather than providing positive effects on apoptosis and fat dissolution of adipocytes. These side effects and pain were significantly alleviated at a molar ratio of 3.0 or less. In particular, when it was included in a molar ratio of 2.60 or less, no edema, lesions, and inflammation were observed or mild symptoms were observed. Clinically, mild edema is visible, but this is a level that is substantially painless and has no side effects and is therefore useful as a better range in the present invention. Optimally, when the molar ratio is 1.73 or less, edema and lesions as well as clinical pain and edema caused by inflammation do not occur.

In the present invention, the range of the (ii) molar ratio of (i) includes a minimum or maximum limit value selected from the group consisting of: 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 1.09, 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.09, 1.08, 1.09, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.20, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.50, 1.54, 1.50, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.00, 2.01, 2.02, 2.03, 2.04, 2.05, 2.06, 2.07, 2.08, 2.09, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, 2.23, 2.2 2.42 2.43, 2.44, 2.45, 2.46, 2.47, 2.48, 2.32, 2.33, 2.34, 2.62, 2.63, 2.64, 2.65, 2.66, 2.67, 2.68, 2.69, 2.70, 2.71, 2.72, 2.73, 2.50, 2.52, 2.53, 2.54, 2.55, 2.56, 2.57, 2.58, 2.59, 2.74, 2.75, 2.76, 2.77, 2.78, 2.79, 2.80, 2.81, 2.82, 2.83, 2.84, 2.85, 2.86, 2.87, 2.88, 2.89, 2.90, 2.91, 2.92, 2.93, 2.94, 2.95, 2.96, 2.99, and 3.00. As a preferred embodiment of the invention, the threshold values of 0.76 and 1.39 may be selected from the range of the molar ratios of the invention described above. Thus, it will be apparent to those skilled in the art that a molar ratio range of 0.76 to 1.39 (i.e., all values in the range of 0.76 or greater to 1.39 or less) can be applied to the present invention.

Specifically, the composition for reducing local fat of the present invention is characterized in that it contains "glycylic acid or a salt thereof" in a specific mixing ratio in the composition. Glycoic acid is a bile salt having a molecular weight of about 465.63 g/mol and may be referred to herein as GCA or GC. Glycocholic acid can be used in the form of a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable" means physiologically acceptable and generally does not cause an allergic or similar reaction when administered to a human, and includes, but is not limited to, sodium, potassium or ammonium. salt. Preferably, the glycocholate of the present invention may be sodium glycocholate (GCNa).

Glycyrrhizic acid or a salt thereof can be extracted parenterally according to methods known in the art and can be used commercially or by chemical synthesis methods known in the art.

More specifically, the minimum molar ratio (GCA/PPC) of GCA to PPC for preparing a clear solution capable of microfiltration and a mixed microcell having a diameter of 10 nm or less which can be safely and stably injected subcutaneously. It was 0.76 (PPC 5.0% + GCA 2.2%). At a molar ratio less than the minimum molar ratio, the stability of the formulation is lower due to precipitation. Therefore, it is preferred that the molar ratio of GCA to PPC (GCA/PPC) is in the range of 0.76 to 3.0 (in terms of PPC 5%, GCA 2.2 to 8.65% (w/v)), including glycocholic acid or Its salt, and the specific range refers to the molar ratio mentioned above. When glycocholic acid is used in its salt form, the molar is preferably calculated based only on the glycocholic acid moiety in the glycocholate.

The composition for reducing local fat of the present invention is characterized in that "the composition of the composition" contains "tauric acid or a salt thereof" in a specific mixing ratio. Bovine cholic acid is a bile salt having a molecular weight of about 515.71 g/mol and may be referred to herein as TCA. The taurocholic acid can be used in the form of a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable" means physiologically acceptable and generally does not cause an allergic or similar reaction when administered to a human, and includes, but is not limited to, sodium, potassium or ammonium. salt. Preferably, the glycocholate of the present invention may be sodium glycocholate (TCNa).

The taurocholic acid or its salt can be extracted parenterally according to methods known in the art and can be used commercially or by chemical synthesis methods known in the art.

More specifically, the minimum molar ratio (TCA/PPC) of TCA to PPC for preparing a clear solution capable of microfiltration and a mixed microcell having a diameter of 10 nm or less which can be safely and stably subcutaneously injected. It is 0.78 (PPC 5.0% + TCA 2.5%). At a molar ratio less than the minimum molar ratio, the stability of the formulation is lower due to precipitation. Therefore, it is preferred that the molar ratio of TCA to PPC (TCA/PPC) is in the range of 0.78 to 3.0 (in terms of PPC 5%, TCA 2.5 to 9.57% (w/v)), including glycocholic acid or Its salt, and the specific range refers to the molar ratio mentioned above. When erocholic acid is used in its salt form, the molar is preferably calculated based only on the portion of the bovine cholic acid in the bovine cholate.

At this time, at least one selected from the group consisting of glycocholic acid (GCA), tauric acid (TCA), and a salt thereof is preferably the same or less weight (or weight/volume percentage) as phospholipid choline. (ie %w/v)) is included. For example, the weight ratio based on PPC can be in the range of 1:0.1 to 1. In particular, the weight ratio based on PPC can be 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9 or 1. When the weight standard is based on a molar ratio (GCA/PPC molar ratio or TCA/PPC molar ratio), the molar ratio may preferably be in the range of 0.7 to 1.73, more preferably 0.76 to 1.73.

Preferably, at least one selected from the group consisting of glycocholic acid (GCA), taurocholic acid (TCA), and salts thereof is less than the weight of the phospholipid choline (or weight/volume percent (ie, %) w/v)) is included. For example, the weight ratio based on PPC can range from 1:0.1 to 0.999. In particular, the weight ratio based on PPC can be 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, or 1:0.9. When the weight standard is based on a molar ratio (GCA/PPC molar ratio or TCA/PPC molar ratio), the molar ratio may preferably be 0.7 or more to less than 1.73, more preferably 0.76 or more to less than 1.73. In the range.

When glycocholic acid or taurocholic acid is used in its salt form, the weight is preferably calculated based only on the ratio of the glycocholic acid moiety in the glycocholate or the ratio of the tauric acid moiety in the choline.

At least one selected from the group consisting of glycocholic acid (GCA), taurocholic acid (TCA), and a salt thereof can be used as follows: (A) individual substances (one of glycocholic acid, one of glycocholate, and bovine bile) One of the acid, bovine cholate may be complexed with PPC, or (B) GCA or a salt thereof, and a mixture of TCA or a salt thereof (hereinafter, a GCA-TCA mixture) may be complexed with PPC.

As described above, the composition of the present invention comprising GCA, TCA or a salt thereof and phospholipid choline (PPC) in a specific mixing ratio is characterized by being a non-liposome microcell preparation. That is, the composition of the present invention is characterized in that a phospholipid choline in the form of a cell is present in the composition, which is different from the conventional PPC formulation using a liposome system.

GCA, TCA or a salt thereof is included in the reduced-fat injectable composition of the present invention in a specific dose (or mixing ratio, molar ratio) as described above, so that it is excellent in not only the formulation stability but also different from Conventional solubilizers (especially deoxycholic acid and its salt types) in PPC injectable compositions, such conventional solubilizers cause side effects such as cell necrosis with body pain and edema, hematoma, sensory loss, erythema, swelling, induration , itching, nodules and the like, GCA, TCA or its salt together with PPC induces high-efficiency fat solubilization and apoptosis of adipocytes to substantially eliminate pain and side effects, and exhibits excellent fat-reducing effects (pain and Edema is reduced by 80% or more, and erythema, hematoma, induration, itching, and nodules are alleviated by more than 80%. Accordingly, it is a feature of the invention that the anti-inflammatory agent and/or analgesic component for individual pain management need not be included or combined in the composition.

Meanwhile, the composition of the present invention may further comprise at least one selected from the group consisting of: a preservative; an isotonic agent; and a pH adjuster.

Specifically, the local fat reducing composition of the present invention preferably further comprises at least one selected from the group consisting of 0.1 to 5% (w/v) of a preservative, 0.1 to 10%, based on the total composition. (w/v) isotonic agent and 0.01 to 2% (w/v) pH adjuster.

The preservative may be selected from the group consisting of benzyl alcohol, lidocaine, procaine, and chlorobutanol, but is not limited thereto. More preferably, it is benzyl alcohol. Benzyl alcohol is one of aromatic alcohols and is a colorless transparent liquid. The concentration of benzyl alcohol contained in the injectable composition of the present invention may preferably be from 0.1% (w/v) to 2% (w/v).

The isotonic agent is used to maintain (control) the osmotic pressure in an appropriate manner when the composition of the present invention containing phospholipid choline is administered to the body, and also has an auxiliary effect of further stabilizing the phospholipid choline in the solution. The isotonic agent can be a pharmaceutically acceptable sugar, a salt, or any combination or mixture thereof. Examples thereof include glucose as a sugar, and sodium chloride, calcium chloride, sodium sulfate, glycerin, propylene glycol, polyethylene glycol having a molecular weight of 1,000 or less, and the like as a water-soluble inorganic salt. And more preferably, it may be sodium chloride. They may be used singly or in combination of two or more. The concentration of the isotonic agent is preferably from 0.1% (w/v) to 5% (w/v), and can be adjusted to an appropriate amount so that the solution formulation containing each of the individual mixtures becomes isotonic The solution depends on the type, amount, and the like of the components contained in the composition of the present invention.

The pH adjusting agent of the present invention functions to control the pH of the injectable preparation and includes both acidic and basic substances. Acidic materials include, but are not limited to, hydrochloric acid, acetic acid, adipic acid, ascorbic acid, sodium ascorbate, sodium ethoxide, malic acid, succinic acid, tartaric acid, fumaric acid, and citric acid. Basic substances include, but are not limited to, inorganic bases (such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogencarbonate, magnesium carbonate, calcium carbonate, magnesium oxide, ammonia, synthetic hydrotalcite), organic bases (such as alkaline Amino acids such as lysine, arginine, etc., meglumine, etc.) and the like. In the present invention, the pH adjuster may include an acidic substance and an alkaline substance, respectively, in a separate composition, or two or more substances may be used in combination. More preferably, the pH adjusting agent of the present invention may be sodium hydroxide and/or hydrochloric acid. The amount of the pH adjusting agent to be added may vary depending on the kind and amount of the components of the composition of the present invention, and is preferably 0.01% (w/v) to 1.32% (w/v), more preferably 0.01% (w/). v) to 1% (w/v). The compositions of the present invention are preferably provided in the range of pH 7.0 to pH 7.8, and the type and amount of pH adjusting agent can vary depending on the particular composition of the solution, as generally known to those skilled in the art.

In a preferred form, the present invention provides a composition for reducing localized fat with reduced pain and side effects, the composition consisting of: (i) phospholipid choline; (ii) selected from glycocholic acid (GCA) or bovine At least one of a group consisting of bile acids (TCA) and its salts; (iii) a preservative; (iv) an isotonic agent; (v) a pH adjusting agent; and (vi) residual water, wherein the composition (ii) The molar ratio of (i) is in the range of 0.7 to 3.0. The individual component characteristics, contents, combinations, and the like of the composition can be understood by referring to the above description.

The invention also provides a formulation for removing localized fat deposits in an individual with reduced pain and side effects, the formulation comprising: (i) phospholipid choline; and (ii) selected from the group consisting of glycocholic acid (GCA), bovine cholic acid At least one of (TCA) and a group of its salt composition, wherein the (ii) molar ratio of (i) in the formulation is in the range of 0.7 to 3.0.

The present invention also provides a formulation for removing localized fat deposits in an individual with reduced pain and side effects, the formulation consisting of: (i) phospholipid choline; and (ii) selected from glycocholic acid (GCA), bovine At least one of a group consisting of cholic acid (TCA) and a salt thereof, wherein the ratio of (ii) to (i) in the formulation is in the range of 0.7 to 3.0.

The present invention also provides a formulation for removing localized fat deposits in an individual with reduced pain and side effects, the formulation consisting essentially of: (i) phospholipid choline; (ii) selected from glycocholic acid (GCA), At least one of a group consisting of taurine (TCA) and its salt; and (iii) water (or water for injection), wherein the ratio of (ii) to (i) molar ratio is in the range of 0.7 to 3.0 .

In a preferred form, the present invention provides a formulation for removing topical fat with reduced pain and side effects, the formulation consisting of: (i) phospholipid choline; (ii) selected from glycocholic acid (GCA) or bovine At least one of a group consisting of an acid (TCA) and a salt thereof; (iii) a preservative; (iv) an isotonic agent; (v) a pH adjusting agent; and (vi) a remaining water, wherein the composition (ii) The molar ratio of (i) is in the range of 0.7 to 3.0. The composition, content and characteristics of the specific substances constituting the preparation are the same as those of the composition for reducing the local fat.

The composition for reducing local fat of the present invention and the properties of the preparation of the present invention may be composed of pH 6.8 to pH 7.8.

The unit dose/unit dosage of the composition or preparation of the present invention may be, for example, the following total amounts: 500 ml, 400 ml, 300 ml, 200 ml, 100 ml, 90 ml, 80 ml, 70 ml, 60 Ml, 50 ml, 40 ml, 30 ml, 20 ml, 10 ml, 9 ml, 8 ml, 7 ml, 6, ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml, 0.9 ml, 0.8 ml , 0.7 ml, 0.6 ml, 0.5 ml, 0.4 ml, 0.3 ml, 0.2 ml, 0.1 ml, 0.09 ml, 0.08 ml, 0.07 ml, 0.06 ml, 0.05 ml, 0.04 ml, 0.03 ml, 0.02 ml, 0.01 ml, 0.009 Ml, 0.008 ml, 0.007 ml, 0.006 ml, 0.005 ml, 0.004 ml, 0.003 ml, 0.002 ml, 0.001 ml, 0.0009 ml, 0.0008 ml, 0.0007 ml, 0.0006 ml, 0.0005 ml, 0.0004 ml, 0.0003 ml, 0.0002 ml or 0.0001 ml, to the affected area of the mammal, but is not limited to this. The unit dose will depend in part on the target area, the amount of fat, and the desired result.

Specifically, the unit dose of the composition or preparation of the present invention can be administered to the affected area in a range of from 0.1 ml to 500 ml, preferably from 1 ml to 200 ml, more preferably from 1 ml to 100 ml.

The composition or preparation of the present invention can be administered by administering to a plurality of target sites (points) at a certain time interval by a single administration in the affected area, and the total amount can be referred to via the plurality of target sites. The total amount of dose administered at the time of single administration. For an affected area, the target portion can be set in the range of 1 to 50, preferably 2 to 30, more preferably 3 to 15, or the like. Further, the composition or preparation of the present invention comprises a single administration to a target site on an affected area, and in this case, it is well understood by those skilled in the art that the total amount is calculated based on the amount of a target site. .

Further, the composition or preparation of the present invention may be administered, but not limited to, in a dose range of 0.01 to 20 ml, preferably 0.1 to 10 ml, more preferably 0.02 to 5 ml, most preferably 0.1 to 1 ml per target site.

The composition or preparation of the present invention can be administered to the target site one or more times. In certain embodiments, the compositions of the invention are administered to the target site for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. One or more administrations can be carried out in one hour, one day, one week, one month or one year. Preferably, multiple administrations to a single target site are 10, 9, 8, 7, 6, 5, 4, 3, or 2 or less per year, 10, 9, 8, 7, 6, each month. 5, 4, 3 or 2 or fewer times, 10, 9, 8, 7, 6, 5, 4, 3 or 2 or less per week, 10, 9, 8, 7, 6, 5, 4 per day 3, 2 or less times, 10, 9, 8, 7, 6, 5, 4, 3 or 2 or less per hour. In certain embodiments, the individual is provided with 1-100, 2-50, 3-30, 4-20, or 5-10 administrations at the target site. Such administration can be carried out over a period of one year, six months, five months, four months, three months, two months, one month, three weeks, two weeks, or one week or less.

The compositions or formulations of the present invention can be administered at various levels (depths) below the skin including, but not limited to, for example, 0.1-4 Å, 0.5-3 Å, 1-2 Å below the skin.

The present invention also provides a kit comprising: (I) a first container comprising a composition or formulation for removing localized fat deposition having reduced pain and side effects, the composition or formulation comprising: (i) Phosphocholine choline; and (ii) at least one selected from the group consisting of glycocholic acid (GCA), taurocholic acid (TCA), and salts thereof, wherein (ii) is ratio (i) of the composition or formulation The molar ratio is in the range of 0.7 to 3.0; and (II) a delivery device capable of delivering the composition or formulation to the fat deposition site.

As a more specific example, the present invention provides a kit comprising: (I) a first container comprising a composition or formulation for removing localized fat deposition, having a reduced pain and side effects, the composition or formulation And comprising: (i) phospholipid choline; and (ii) at least one selected from the group consisting of glycocholic acid (GCA), taurine (TCA), and salts thereof, wherein the one selected from the group consisting of glycocholic acid (GCA) At least one of the group consisting of taurine (TCA) and its salt is contained in the same or lesser weight as the phospholipid choline; and (II) capable of delivering the composition or formulation to the fat deposition site Delivery device.

In the kit of the present invention, (I) the composition or formulation contained in the first container is understood by reference to the description of the reduced-fat composition and formulation of the present invention described above. The first container has a volume sufficient to accommodate a unit dose (dose volume) of the composition or formulation of the invention. For example, the first container can be adapted to hold 500 ml, 100 ml, 20 ml, 10 ml, 5 ml, 4 ml, 3 ml, 2 ml or 1 ml solution. In some embodiments, the first container can have from 0.01 ml to about 100 ml, from about 0.1 ml to about 90 ml, from about 0.5 ml to about 80 ml, from about 1 ml to about 70 ml, from about 2 ml to about 60 ml, A volume of from about 3 ml to about 50 ml, from about 4 ml to about 40 ml, from about 5 ml to about 30 ml, from about 6 ml to about 20 ml, and from about 7 ml to about 10 ml. In a more preferred embodiment, the first container is a vial or ampoule having a volumetric capacity of about 1-10 ml.

The kit of the present invention comprises (II) a delivery device for delivering the composition in the first container to the fat deposition site. The particular type of delivery device is not particularly limited, but may preferably be a syringe, and/or may further include another suitable delivery device (e.g., a patch).

The delivery device may have previously been loaded with a unit dose of a composition or formulation of the invention.

The kit of the present invention may further comprise a plurality of containers as the case may be. For example, the kit can further comprise a suitable amount of diluent for diluting the composition or formulation contained in the first container and/or the second container comprising any other second agent. Any other second agent may be selected as a constituent component by a person skilled in the art according to the purpose of the kit, and the kind thereof is not particularly limited, and examples thereof include an antimicrobial agent, a vasoconstrictor, an antithrombotic agent, and an anticoagulation agent. Agents, dispersants, anti-dispersants, penetration enhancers, steroid tranquilizers, muscle relaxants and antidiarrheal agents.

The kit can include a written description of the composition or formulation used to reduce localized fat with reduced pain and side effects. Therefore, the composition or preparation contained in the first container (I) can be administered according to a written description. The written description may provide an indication of use that may depend, for example, on the target site, the mammal to be treated, the desired result, the location of the target site, the concentration of the solution, and the amount of fat deposition. Preferably, the written description is for treating a mammal, such as a human, a dog, a cat or a horse. The written description may also include information on the treatment of other livestock and/or farm animals.

Written descriptions may include information regarding the use of the compositions of the present invention to treat certain target areas, such as under the eyes of a mammal, under the arm, under the arms, hips, calves, back, thighs, ankles, or abdomen. In certain embodiments, written descriptions of the use of the compositions of the invention to treat specific fat deposition associated with orbital fat escape, lipoma, lipid dystrophy, buffalo hump fat dystrophy, or cellulite Instructions.

The written description may include information regarding the amount of (if necessary) dilution of the components of the first container and/or the diluent of the second container. The written description may provide information regarding the proper administration of the compositions or formulations of the invention, such as the frequency or dosage of the administration.

The term "comprising" is used synonymously with "containing" or "being characterized" and does not exclude additional ingredients or steps not mentioned in the compositions and methods. The term "consisting of" excludes additional elements, steps or ingredients not separately described. The term "essentially consisting of" means that, in the context of a composition or method, the term includes the materials or steps described, and does not substantially affect the essential characteristics of the composition or method. Any material or step.

The present invention provides a method for preparing an injectable composition for reducing topical fat having reduced pain and side effects, the method comprising the steps of: (a) adding from glycocholic acid (GCA), bovine cholic acid (TCA) And at least one of the group consisting of its salt composition is sprayed with water, followed by dissolution while stirring to obtain a clear mixture; (b) adding a preservative, followed by stirring; (c) adding phospholipid choline, followed by stirring at room temperature And (d) adjusting the total volume of the composition with water, followed by stirring, wherein the at least one selected from the group consisting of glycocholic acid (GCA), taurocholic acid (TCA), and salts thereof, and the phospholipid choline The molar ratio is in the range of 0.7 to 3.0.

Hereinafter, a method of preparing an injectable composition for reducing local fat of the present invention will be described step by step.

In the step (a), at least one selected from the group consisting of glycocholic acid (GCA), tauric acid (TCA) and salts thereof is added to water for injection, followed by dissolution while stirring to obtain a substantially clear mixture.

At this time, at least one selected from the group consisting of glycocholic acid, taurocholic acid and salts thereof, and combinations and mixtures thereof are as described above in the composition. In step (a), a pH adjusting agent may be pre-added as appropriate.

Step (b) is a step of administering a preservative. In the step (b), any one or both of an isotonic agent and a pH adjuster may be further added and stirred. The components and concentrations of the preservative, isotonic and pH adjusting agents are the same as described above in the description of the composition.

In the present invention, stirring or mixing can be carried out by a known agitating member (agitator), and those skilled in the art can change such as temperature, pressure, time or rotational speed depending on the kind or characteristics of the material to be introduced to improve efficiency. The conditions.

In step (c), phospholipid choline is added to the stirred mixture in step (b), and the mixture is stirred under light-shielding and air-tight conditions until the mixture is solubilized. Stirring can be carried out by means of a stirring member (agitator) known in the art, preferably from 2 to 24 hours, more preferably from 5 to 15 hours. The rotation speed is not limited to this, but can be performed at 100 to 1000 rpm. Through the above process, phospholipid choline can be prepared as a homogeneous particle having a small particle size (particle size of 2 to 10 nm, preferably 2 to 6 nm) in the composition. If the agitation process is carried out for less than 2 hours, the desired particle size and homogeneity cannot be obtained, and if it exceeds 24 hours, it is not economical for the preparation process. Those skilled in the art will also be able to set various process conditions to increase the solubility of the component materials, such as agitating the component materials under conditions such as nitrogen pressure.

In step (d), the total volume is adjusted with water and mixed homogeneously. Water can be replaced with water for injection, which is the same as described above. In step (d), the addition of a pH adjusting agent can be carried out. In this step, in order to ensure product stability according to the distribution of the injection preparation, the pH can be adjusted by using an acid solution which can be used as an injection or a buffer such as a phosphate (pH adjuster), and physical or chemical stability can be prepared. Inject the preparation. The kind or amount of the pH adjusting agent which can be used in the present invention is as described above.

Further, the method may further comprise (e) filtering the stirred solution in step (d) to obtain a filtrate of phospholipid choline having a particle size of 2 to 10 nm.

Step (e) is a step of separating a phospholipid choline molecule having a particle diameter of 2 to 10 nm by filtration at a high concentration. Filtration can be carried out using conventional filtration means known in the art, and filtration can be carried out, for example, by a syringe filter. The particle size is preferably between 2 and 5 nm.

As a preferred embodiment, the present invention provides a method for preparing a pharmaceutical composition for non-surgical removal of topical fat deposition having reduced pain and side effects, the method comprising the addition of phospholipid choline and selected from the group consisting of At least one of a group consisting of cholic acid, taurocholic acid, and a salt thereof, wherein at least one selected from the group consisting of glycocholic acid, taurocholic acid, and a salt thereof is the same as or smaller than the phospholipid choline Weight added. With regard to these specific examples, specific material compositions, mixing ratios, and the like are understood with reference to the description of the compositions and formulations of the invention described above.

The present invention also provides a method of removing localized fat deposition in an individual with reduced pain and side effects, the method comprising administering to the individual having localized fat deposition an effective amount of phospholipid choline; and at least one selected from the group consisting of A phospholipid choline solubilizing agent of the group consisting of acid, tauric acid and salts thereof.

That is, in the above method, phospholipid choline is contained in a pharmaceutically acceptable injectable solution for administering phospholipid choline at a concentration of 0.625 to 15% (w/v) based on the total composition ( The composition, and preferably at a concentration of 1.25 to 12.5% (w/v), and more preferably at a concentration of 2.5 to 10.0% (w/v), is included in the composition. In this case, the unit dosage for the composition for administration of phospholipid choline may be 500 ml, 400 ml, 300 ml, 200 ml, 100 ml, 90 ml, 80 ml, 70 ml, 60 ml, 50 ml. , 40 ml, 30 ml, 20 ml, 10 ml, 9 ml, 8 ml, 7 ml, 6, ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml, 0.9 ml, 0.8 ml, 0.7 ml, 0.6 ml, 0.5 ml, 0.4 ml, 0.3 ml, 0.2 ml, 0.1 ml, 0.09 ml, 0.08 ml, 0.07 ml, 0.06 ml, 0.05 ml, 0.04 ml, 0.03 ml, 0.02 ml, 0.01 ml, 0.009 ml, 0.008 ml , 0.007 ml, 0.006 ml, 0.005 ml, 0.004 ml, 0.003 ml, 0.002 ml, 0.001 ml, 0.0009 ml, 0.0008 ml, 0.0007 ml, 0.0006 ml, 0.0005 ml, 0.0004 ml, 0.0003 ml, 0.0002 ml or 0.0001 ml or more less. Specifically, the unit dose for administering the composition of phospholipid choline to the affected area may be from 0.1 ml to 500 ml, preferably from 1 ml to 200 ml, and more preferably from 1 ml to 100 ml.

In the above method, the phospholipid choline solubilizer and phospholipid choline may be administered in a molar ratio of 0.7 to 3.0, more preferably 0.7 to 2.60, most preferably 0.7 to 1.73 (solubilizer/phosphocholine choline).

The solubilizers of phospholipid choline and phospholipid choline can be administered simultaneously or sequentially. For example, when the components included in the pharmaceutical composition of the present invention are a single composition, they can be administered simultaneously. If the composition is not a single composition, one component can be administered within minutes of or after administration of the other components. Preferably, the phospholipid choline solubilizer and phospholipid choline are administered simultaneously. Regardless of whether the components are administered simultaneously or sequentially, preferably, each of the components is included in a pharmaceutically acceptable injectable solution (composition), and the composition comprising the components does not have to be According to the composition of the present invention as described above, and the method of administering the drug to the individual, the method of satisfying the molar ratio of each component can be used.

In the method, at least one selected from the group consisting of an isotonic agent and a pH adjuster may be administered simultaneously or sequentially with a solubilizer of phospholipid choline and/or phospholipid choline. The specific concentrations of these components and the like can be understood by reference to the above description in this specification.

Preferably, the administration is direct injection into a site where localized fat deposition (accumulation) has occurred, and the injection preferably includes subcutaneous injection, intradermal injection, and the like.

The individual is preferably a mammal. Such mammals include humans or primates (eg, monkeys, chimpanzees, etc.), livestock (eg, dogs, cats, horses, etc.), farm animals (eg, goats, sheep, pigs, cows, etc.) or laboratory animals (eg, Mouse, rat, etc.). The individual may also be a cell, tissue, organ or analog thereof of animal origin. Preferably, it may be a human in need of removal of localized fat deposits (accumulation) and removal includes both cosmetic and therapeutic purposes. As such an example, it may be a patient in need of treatment for a pathological condition (disease) caused by abnormal local fat deposition.

For example, the compositions of the present invention can be used to treat certain fatty conditions in a patient, including lipoma, prolapse, atherosclerosis, madelung throat, fatty edema, phyozoospermia nodules, yellow myocardium Yellow cardioma, fatty dystrophy and cellulite. In certain embodiments, the compositions of the present invention can be used to treat fatty conditions at various sites, such as localized fats in the eyes, tendons, arms, hips, calves, back, thighs, ankles, or abdomen of a mammal. Deposition.

As used herein, the term "treatment" is a concept that relates to inhibiting, eliminating, ameliorating, ameliorating, and/or preventing a disease or a condition or condition caused by the disease.

The invention also relates to a method of reducing the deposition of fat, particularly subcutaneous fat, in a mammal, wherein the invention is preferably used for non-surgical removal of localized fat deposits in an individual. As a specific example, the non-surgical methods of the present invention do not include liposuction, fat shaping procedures, or inhalation of subcutaneous adipose tissue resection.

The method of the present invention is characterized by the relief of pain and side effects (substantially reduced to no level), and a detailed description of pain and side effects can be understood by reference to the above description.

Preferably, the present invention provides a method of non-surgically removing localized fat deposits in an individual having localized fat deposition with reduced pain and side effects, the method comprising administering a formulation comprising: (i) phospholipid a base; and (ii) at least one selected from the group consisting of glycocholic acid, taurocholic acid, and salts thereof. The formulation or the composition contained in the formulation used in the method of the present invention may be a single composition or a formulation of the composition of the present invention as described above. As a further example, the formulation or the composition contained in the formulation does not have to be in accordance with the composition of the invention as described above, and the result of administering to the individual may satisfy the ratio of (ii) the molar ratio of (i) from 0.7 to 3.0, A preferred method of 0.7 to 2.60, preferably 0.7 to 1.73.

As a preferred embodiment, the present invention provides a method of non-surgically removing localized fat deposits in an individual with reduced pain and side effects, the method comprising administering to the individual having localized fat deposition a formulation comprising: (i a phospholipid choline; and (ii) at least one selected from the group consisting of glycocholic acid, taurocholic acid and salts thereof, wherein the composition (ii) has a molar ratio of (i) from 0.7 to 3.0 In the range.

In the above method, the formulation is understood in terms of composition, content, and characteristics with reference to the above description in the specification of the present invention, and the method of removing localized fat deposition of the present invention comprises or consists of fat to an individual (mammal) The deposition site (affected area) is topically administered in a unit dose (total amount) of one or more of the compositions or formulations described above in the specification of the present invention. With regard to the compositions, formulations and unit dosages thereof, reference will be made to the descriptions mentioned above.

The method of the invention relates to a method of reducing subcutaneous fat deposition. Such methods may comprise or consist of administering one or more of the dosage compositions or formulations of the invention topically to a site of deposition of fat in a mammal.

The preparation is administered to an individual in need thereof in an effective amount, and "effective amount" means an amount which exhibits a local fat-reducing effect (including improvement, treatment, and prevention effect on fat deposition diseases). In general, the total amount administered, the unit dose, and the number of treatments will vary depending on the amount of fat in the target site, the location of the target site, the form of the fat composition, and the desired result. In general, the greater the amount of fat to be treated, the greater the amount administered. Since the amount of the composition of the present invention constituting the "therapeutically effective amount" will vary depending on the state of the disease and its severity, the age of the patient to be treated, and the like, the therapeutically effective amount is not limited to the amount described herein, and may be familiar to the general The technicians routinely determine.

The above administration method is understood with reference to the above description, and as a preferred example, it can be administered transdermally or subcutaneously via a subcutaneous injection using a syringe at a target site. The target site may be, for example, 0.1 cm x 0.1 cm to about 5 cm x 5 cm. The compositions of the present invention can be administered to the same target site adjacent or adjacent to the site at various intervals, doses and amounts as described herein. Favorable effect

The injectable composition for reducing localized fat deposition of the present invention comprising a specific mixing ratio of tauric acid or glycocholic acid (or a salt thereof) and phospholipid choline (PPC) has a stable and safe formulation and has extremely large fat cells Specific fat solubilization and adipocyte-specific apoptosis without fat cell necrosis caused by a conventional PPC preparation containing DCA (such as Lipostabil®) and a preparation containing only DCA (such as Kybella). Therefore, the effect of reducing fat cells without side effects such as pain, edema, paresthesia, extensive swelling, erythema, induration, paresthesia, nodules, itching, burning sensation, and muscle cells other than fat cells are excellent. , fibroblasts and vascular endothelial cell necrosis.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are merely illustrative of the invention and are not intended to limit the scope of the invention. material

The materials added to the formulations of the injectable compositions and comparative compositions of the invention are as follows: Phospholipid choline (PPC, S-100, LIPOID GmbH, 784 g/mol, based on oil-based sulfhydryl-linoleyl-glycerol -phosphocholine), cholic acid (CA, New Zealand pharm), deoxycholic acid (DCA, Sigma-Aldrich), sodium deoxycholate (DCNa, New Zealand pham), glycocholic acid (GCA, New Zealand pham) ), sodium glycocholate (GCNa, New Zealand pham), taurocholic acid (TCA, Sigma-Aldrich), sodium taurocholate (TCNa, New Zealand pham), chenodeoxycholic acid (CDCA, Sigma-Aldrich), Ursodeoxycholic acid (UDCA, Sigma-Aldrich), glycodeoxycholic acid (GDCA, Sigma-Aldrich), taurate (TDCA, Sigma-Aldrich), hyodeoxycholic acid (HDCA, Sigma-Aldrich) ), lithocholic acid (LCA, Sigma-Aldrich), dehydrocholic acid (DHCA, Sigma-Aldrich), ursodeoxycholic acid (TUDCA, Tokyo Chemical Industry) benzyl alcohol (Sigma-Aldrich, 0.9% w/v) ), sodium chloride (Sigma-Aldrich, 0.44% w/v), sodium hydroxide (Sigma-Aldrich, 0.04-0.76% w/v), hydrochloric acid (Sigma-Aldr) Ich, 0.001-0.6% w/v) and water for injection. In the added material, an isotonic agent (sodium chloride) was used in such a manner that it was added together with benzyl alcohol in the examples and comparative examples. Analytical device

The apparatus used in the analysis of the injectable compositions and comparative compositions of the present invention is as follows.

The particle size was measured using a nanoparticle analyzer (Microtrac Wave, MICROTRACT, USA). The layer separation due to precipitation was observed with a camera (Nikkon, D5200, AF-P DX NIKKOR 18-55mm f/3.5-5.6G VR lens). Transparency was measured using a spectrophotometer (CM-3600d, KONICA MINOLTA, JAPAN). The pH system was measured with a pH meter (ST3100, OHAUS, GERMANY), and the isotonicity was measured with an osmometer (Vapro 5600, Elitech Group, Tokyo, Japan), and the viscosity was measured using a viscometer (digital viscometer CL- 2, CAS, Korea) analysis.

The composition (Example) of the present invention and the comparative composition (Comparative Example) were prepared as follows, as a PPC-based preparation for reducing local fat according to the type of solubilizer. Hereinafter, the % of the composition means % (w/v). Comparative Example 1 : PPC single injectable preparation

A phospholipid choline single composition (PPC concentration 0.313-15.0%) without a solubilizing agent was prepared as follows. Using a high pressure homogenizer, the preparations contained PPC 3.125 mg (0.3125%), 6.25 mg (0.625%), 12.5 mg (1.25%), 25.0 mg (2.5%), 50.0 mg (5.0%), and 75.0 mg (7.5%), respectively. An injectable composition of 100.0 mg (10.0%), 125.0 mg (12.5%) or 150.0 mg (15.0%) and benzyl alcohol 9 mg (0.9%) in 1 ml. And representative results are shown in Table 1 below. Hereinafter, the % w/v of the composition is expressed as %.

The specific preparation method is as follows. The washing and sterilizing preparation tank is fed with water for injection (at room temperature), and phospholipid choline (PPC) and benzyl alcohol are added thereto, and the mixture is under nitrogen pressure, light shielding and room temperature. Stir at 200 RPM for 2 hours. After the completion of the stirring, the mixture was transferred to an ultrahigh pressure homogenizer (Nano Disperser NLM100, Ilshin Autoclave, Korea) under a nitrogen pressure. Ultrahigh pressure homogenization (dispersion) was performed at 12,000 psi for 7 cycles, and the particles were finely pulverized. Subsequently, the pH is adjusted. After filtration through a 0.2 μm filter, the vial was filled and sealed. Table 1 PPC single injectable preparation dispersed by high pressure homogenizer

As shown in Figure 1A, the PPC 5.0% composition without solubilizer had a hazy appearance immediately after stirring for 24 to 72 hours. One day after the final agitation, the composition had poor formulation stability due to the fact that PPC was not dispersed in water for injection, and industrial use was limited. Further, as shown in FIG. 1B and Table 1, the composition obtained by dispersing 0.625 to 10.0% of PPC by a high-pressure homogenizer without adding a solubilizer exhibited a slight turbidity or turbidity depending on the concentration, and the particle size was 17.16 ± Liposomal system with 5.88 to 16.69 ± 4.51 nm and unstable dispersion. As a result of observing the properties at 30 days after preparation, it was confirmed that PPC precipitated at a concentration of 2.5% or more and was not dispersed in water for injection and was not suitable as an industrial injectable preparation due to low formulation stability (Fig. 1B).

In order to obtain an injectable composition of a clear solution of a cell structure in which PPC is stably dispersed at a particle size of 10 nm or less, various bile acids (BA) (such as DCA, CA, GCA, TCA, CDCA) are prepared by using various concentrations. PPC composite composition prepared by solubilization of UDCA, GDCA, TDCA, HDCA, LCA, DHCA and TUDCA). Further, the composition thereof is shown in detail in the following comparative examples and examples. Comparative Example 2 : PPC injectable preparation solubilized with DCA

As shown in Table 2 below, by adding 50.0 mg PPC (5.0%) and 10.0 mg (1.0%), 15.0 mg (1.5%), 20.0 mg (2.0%), 21.0 mg (2.1%), 22.0 mg, respectively. 2.2%), 23.0 mg (2.3%), 24.0 mg (2.4%), 25.0 mg (2.5%) or 30.0 mg (3.0%) DCA, and 9 mg benzyl alcohol (0.9%) in 1 ml, for preparation Deoxycholic acid (DCA) solubilized phospholipid choline (PPC 5.0%) composition is identical to the composition of previously known Lipostabil® formulations and their analogs. Specifically, the water for injection is placed in a cleaned and sterilized (room temperature) preparation tank, and sodium hydroxide is added to the water for injection. Subsequently, deoxycholic acid and benzyl alcohol were added, stirred and dissolved. Subsequently, phospholipid choline was added thereto, and the mixture was stirred at 200 RPM for about 24 hours under light blocking, sealing, room temperature (25 ° C) and nitrogen pressure. After the completion of the agitation, the pH was adjusted (if necessary with additional sodium hydroxide or hydrochloric acid) and it was filtered through a 0.2 μm filter and filled into a vial and sealed. Table 2 shows the properties of PPC injectable formulations solubilized with various concentrations of DCA. Table 2 PPC injectable formulations solubilized with DCA

The formulation stability of the PPC injectable preparation solubilized with deoxycholic acid (DCA) was evaluated immediately after preparation and 30 days after preparation (after standing at room temperature), and the evaluation results are shown in Fig. 2A. When DCA was added at a PPC of 5% at 2.1% or greater, stable pharmaceutical formulations were observed and the compositions exhibited clear (clear) solution properties (Comparative Examples 2-4 to 2-9). In Comparative Examples 2-1 to 2-3, an unstable formulation was observed due to the precipitation phenomenon. Based on the above comparison, it was concluded that the stable injectable preparation can be prepared based on a PPC of 5% at a DCA of 2.1% or more. In detail, in Comparative Examples 2-1 to 2-3, the phospholipid choline has a particle size of 50.80 ± 330.0 nm to 39.60 ± 26.14 nm, and it is formed into an unstable emulsion or liposome structure which is not a microcell structure. . In Comparative Examples 2-4 to 2-9, a microcell structure of 10 nm or less was formed. As described above, a composition having a molar ratio of DCA to PPC (DCA/PPC) of less than 0.87 is considered unsuitable for use in an injectable formulation because a substantially stable formulation is absent. Example 1 : Preparation of PPC injectable preparation solubilized with GCA

As shown in Tables 3 and 4 below, by adding 50.0 mg PPC (5.0%) and 10.0 mg (1.0%), 15.0 mg (1.5%), 20.0 mg (2.0%), 21.0 mg (2.1%), respectively. 22.0 mg (2.2%), 23.0 mg (2.3%), 24.0 mg (2.4%), 25.0 mg (2.5%), 26.0 mg (2.6%), 27.0 mg (2.7%), 28.0 mg (2.8%), 29.0 Mg (2.9%), 30.0 mg (3.0%), 35.0 mg (3.5%), 40.0 mg (4.0%) or 45.0 mg (4.5%) DCA, and 9 mg benzyl alcohol (0.9%) in 1 ml, A phospholipid choline (PPC)-based composition solubilized with glycocholic acid (GCA) was prepared. Specifically, the water for injection was placed in a cleaned and sterilized (room temperature) preparation tank, and sodium hydroxide (0.04-0.72%) was added to the water for injection. Subsequently, glycocholic acid and benzyl alcohol were added, stirred and dissolved. Subsequently, phospholipid choline was added thereto, and the mixture was stirred at 200 RPM for about 24 hours under light blocking, sealing, room temperature (25 ° C) and nitrogen pressure. After the completion of the agitation, the pH was adjusted (if necessary with additional sodium hydroxide or 0.001-0.6% hydrochloric acid) and it was filtered through a 0.2 μm filter and filled into a vial and sealed. Tables 3 and 4 show the properties of PPC injectable formulations solubilized with various concentrations of GCA. table 3 Table 4

The formulation stability of the PPC injectable composition solubilized with glycocholic acid (GCA) in Tables 3 and 4 immediately after preparation and 30 days after preparation (refrigerated) was evaluated. The results of the evaluation are shown in Figure 2B. When GCA was added at 5.0% or more for PPC 5.0%, it was confirmed that the formulation was stable, and the composition exhibited transparent (clarified) solution properties (Examples 1-1 to 1-12). In Comparative Examples 1-1 to 1-4, it was confirmed that the formulation was unstable due to the precipitation phenomenon. Examples 1-1 to 1-3 were slightly turbid immediately after preparation, but after filtration with a 0.2 μm filter, they exhibited a transparent property, and thus it was confirmed that it can be used as an injection preparation. Therefore, it was concluded that the stable injectable preparation can be prepared based on 5.0% PPC at a GCA of 2.2% or more. The particle size of the compositions of Comparative Examples 1-1 to 1-4 was in the range of 198.2 ± 721.0 nm to 28.2 ± 11.35 nm, that is, the composition was dispersed as an unstable emulsion or liposome, so that it was judged that the preparation was substantially stable. The substance does not exist and is not suitable for use in an injectable preparation. In addition to this, a PPC composite composition (Examples 1-1 to 1-12) to which GCA 2.2% or more (GCA/PPC molar ratio is 0.76 or more) is added, and the size is 10 nm or less. The microcell structure is composed, and a PPC composite composition (Examples 1-7 to 1-12) having a GCA of 2.8% or more (GCA/PPC molar ratio of 0.97 or more) is added by a size of 5 nm or more. The small microcell structure is constructed and found to be suitable for injectable formulations.

Since the viscosity of the PPC composite composition solubilized by GCA was increased in proportion to the PPC concentration, the viscosity of each composition was tested. Specifically, a PPC (2.5-20.0%) composite composition solubilized with GCA (1.4-11.2%) and a GCA (GCA) were studied based on a PPC 5.0% GCA concentration of 2.8% dispersed at a particle size of 5 nm or less. 4.2-12.6%) Characteristics of solubilized PPC 5.0% composite composition. Each of 500 ml or more of the composition was measured at room temperature (25 ° C) for 3 minutes at 60 RPM after the spin needle No. 1. As a result of the test, it was confirmed that the particle size was 4.23 ± 1.69 to 1.37 ± 0.530 nm, and the transparency (660 nm) was measured to be 99.11 ± 0.77%. Table 5 shows the viscosity characteristics of PPC injectable formulations solubilized with various concentrations of GCA. Based on the following Table 5, it was confirmed that when the PPC exceeds 15% (w/v), it is not suitable for administration due to high viscosity. table 5 Example 2 : Preparation of PPC injectable preparation solubilized with TCA

As shown in Tables 6 and 7 below, by adding 50.0 mg PPC (5.0%) and 10.0 mg (1.0%), 15.0 mg (1.5%), 20.0 mg (2.0%), 21.0 mg (2.1%), respectively. 22.0 mg (2.2%), 23.0 mg (2.3%), 24.0 mg (2.4%), 25.0 mg (2.5%), 26.0 mg (2.6%), 27.0 mg (2.7%), 28.0 mg (2.8%), 29.0 Mg (2.9%), 30.0 mg (3.0%), 35.0 mg (3.5%), 40.0 mg (4.0%) or 45.0 mg (4.5%) TCA, and 9 mg benzyl alcohol (0.9%) in 1 ml, A phospholipid choline (PPC)-based composition solubilized with tauric acid (TCA) was prepared. The specific preparation method is the same as the production method of Example 1 mentioned above. Tables 6 and 7 show the characteristics of PPC injectable formulations solubilized with various concentrations of TCA. Table 6 Table 7

The formulation stability of the PPC injectable composition solubilized with tauric acid (TCA) in Tables 6 and 7 immediately after preparation and 30 days after preparation (refrigeration) was evaluated, and the evaluation results are shown in Fig. 2C. When TCA was added at a PPC 5.0% of 2.5% or greater, it was confirmed that the formulation was stable and the composition exhibited clear (clear) solution properties (Examples 2-1 to 2-9). In Comparative Examples 2-1 to 2-7, it was confirmed that the formulation was unstable due to the precipitation phenomenon. Example 2-1 was slightly turbid immediately after preparation, but after filtration with a 0.2 μm filter, it exhibited a transparent property, and thus it was confirmed that it can be used as an injection preparation. Thus, it was concluded that stable injectable formulations can be made based on 5.0% PPC at 2.5% or greater TCA. The composition of Comparative Examples 2-1 to 2-7 has a particle size ranging from 139.6 ± 126.14 nm to 146.1 ± 2908 nm, that is, the composition is dispersed as an unstable emulsion or liposome, so that it is judged that the preparation is substantially stable. The substance does not exist and is not suitable for use in an injectable preparation. In addition to this, a PPC composite composition (Examples 2-1 to 2-9) having a TCA of 2.5% or more (TCA/PPC molar ratio of 0.78 or more) was added, and the size was 10 nm or less. The microcell structure is composed, and a PPC composite composition (Examples 2-4 to 2-9) having a TCA of 2.8% or more (TCA/PPC molar ratio of 0.88 or more) is added by a size of 5 nm or more. The small microcell structure is constructed and found to be suitable for injectable formulations. Comparative Example 3 : PPC injectable preparation solubilized with CA

As shown in Table 8 below, by adding 50.0 mg PPC (5.0%) and 10.0 mg (1.0%), 15.0 mg (1.5%), 20.0 mg (2.0%), 21.0 mg (2.1%), 22.0 mg, respectively. 2.2%), 23.0 mg (2.3%), 24.0 mg (2.4%), 25.0 mg (2.5%) or 30.0 mg (3.0%) CA, and 9 mg benzyl alcohol (0.9%) in 1 ml, for preparation Choline acid (CA) solubilized phospholipid choline (PPC) based composition. The specific preparation method is the same as the production method of Example 1 mentioned above. Table 8

The formulation stability of the PPC injectable composition solubilized with cholic acid (CA) in Table 8 immediately after preparation and 30 days after preparation (refrigeration) was evaluated, and the evaluation results are shown in Fig. 2D. When CA was added at 5.0% or more with respect to PPC 5.0%, it was confirmed that the formulation was stable, and the composition exhibited transparent (clarified) solution properties (Comparative Examples 3-5 to 3-9). In Comparative Examples 3-1 to 3-4, it was confirmed that the formulation was unstable due to the precipitation phenomenon. Comparative Examples 3-5 were almost transparent immediately after preparation and exhibited clear properties after filtration with a 0.2 μm filter. Therefore, it was concluded that the stable injectable preparation can be prepared based on 5.0% PPC at 2.2% or greater CA. The composition of Comparative Examples 3-1 to 3-4 had a particle size ranging from 50.80 ± 330 nm to 583.00 ± 293 nm, that is, the composition was dispersed as an unstable emulsion or liposome, but Comparative Examples 3-5 were 3-9 consists of a microcell structure with a size of 10 nm or less. As described above, it is judged that a substantially stable formulation is not present in a composition having a CA/PPC molar ratio of less than 0.88, and it is not suitable for use in an injectable preparation. Comparative Example 4 : PPC injectable preparation solubilized with CDCA

As shown in Table 9 below, by adding 50.0 mg PPC (5.0%) and 10.0 mg (1.0%), 15.0 mg (1.5%), 20.0 mg (2.0%), 21.0 mg (2.1%), 22.0 mg, respectively. 2.2%), 23.0 mg (2.3%), 24.0 mg (2.4%), 25.0 mg (2.5%) or 30.0 mg (3.0%) CDCA, and 9 mg benzyl alcohol (0.9%) in 1 ml, for preparation A composition based on phospholipid choline (PPC) that is solubilized by chenodeoxycholic acid (CDCA). The specific preparation method is the same as the production method of Example 1 mentioned above. Table 9

The formulation stability of the PPC injectable composition soaked with chenodeoxycholic acid (CDCA) in Table 9 immediately after preparation and 30 days after preparation (refrigerated) was evaluated, and the evaluation results are shown in Fig. 2E. When CDCA was added at a PPC 5.0% of 2.2% or greater, it was confirmed that the formulation was stable and the composition exhibited clear (clear) solution properties (Comparative Examples 4-5 to 4-9). In Comparative Examples 4-1 to 4-4, it was confirmed that the formulation was unstable due to the precipitation phenomenon. Therefore, it was concluded that the stable injectable preparation can be prepared based on 5.0% PPC at 2.2% or greater CA. The composition of Comparative Examples 4-1 to 4-4 had a particle size ranging from 80.05 ± 280.0 nm to 35.73 ± 0.830 nm, that is, the composition was dispersed as an unstable emulsion or liposome, but Comparative Example 4-5 was 4-9 consists of a microcell structure with a size of 10 nm or less. As described above, it is judged that a substantially stable formulation is not present in a composition having a CDCA/PPC molar ratio of less than 0.89, and it is not suitable for use in an injectable preparation. Comparative Example 5 : PPC injectable preparation solubilized with UDCA

As shown in Table 10 below, by adding 50.0 mg PPC (5.0%) and 10.0 mg (1.0%), 15.0 mg (1.5%), 25.0 mg (2.5%), 26.0 mg (2.6%), 27.0 mg, respectively. 2.7%), 28.0 mg (2.8%), 29.0 mg (2.9%) or 30.0 mg (3.0%) UDCA, and 9 mg benzyl alcohol (0.9%) in 1 ml for the preparation of ursodeoxycholic acid (UDCA) a solubilized phospholipid choline (PPC)-based composition. The specific preparation method is the same as the production method of Example 1 mentioned above. Table 10

The formulation stability of the PPC injectable composition solubilized with ursodeoxycholic acid (UDCA) in the evaluation table 10 immediately after preparation and 30 days after preparation (refrigeration) was evaluated, and the evaluation results are shown in Fig. 2F. When UDCA was added with a PPC 5.0% at 2.7% or greater, it was confirmed that the formulation was stable and the composition exhibited clear (clarified) solution properties (Comparative Examples 5-6 to 5-9). In Comparative Examples 5-1 to 5-5, it was confirmed that the formulation was unstable due to the precipitation phenomenon. Comparative Examples 5-6 were almost transparent immediately after preparation and exhibited clear properties after filtration with a 0.2 μm filter. Therefore, it was concluded that the stable injectable preparation can be prepared based on 5.0% PPC at UDCA of 2.7% or greater. The composition of Comparative Examples 5-1 to 5-5 had a particle size ranging from 289.0 ± 265.0 nm to 42.42 ± 250 nm, that is, the composition was dispersed as an unstable emulsion or liposome, but Comparative Examples 5-6 were 5-9 consists of a microcell structure with a size of 10 nm or less. As described above, it is judged that the substantially stable formulation is not present in the composition having a UDCA/PPC molar ratio of less than 1.12, and it is not suitable for use in an injectable preparation. Comparative Example 6 : PPC injectable preparation solubilized with GDCA

As shown in Table 11 below, by adding 50.0 mg PPC (5.0%) and 10.0 mg (1.0%), 15.0 mg (1.5%), 20.0 mg (2.0%), 21.0 mg (2.1%), 22.0 mg, respectively. 2.2%), 23.0 mg (2.3%), 24.0 mg (2.4%), 25.0 mg (2.5%) or 30.0 mg (3.0%) GDCA, and 9 mg benzyl alcohol (0.9%) in 1 ml, for preparation A phospholipid choline (PPC)-based composition that is solubilized by glycodeoxycholic acid (GDCA). The specific preparation method is the same as the production method of Example 1 mentioned above. Table 11

The formulation stability of the PPC injectable composition solubilized with glycodeoxycholic acid (GDCA) in the evaluation table 11 immediately after preparation and 30 days after preparation (refrigeration) was evaluated, and the evaluation results are shown in Fig. 2G. When GDCA was added at a PPC 5.0% of 2.4% or greater, it was confirmed that the formulation was stable and the composition exhibited clear (clarified) solution properties (Comparative Examples 6-7 to 6-9). In Comparative Examples 6-1 to 6-6, it was confirmed that the formulation was unstable due to the precipitation phenomenon. Comparative Examples 6-7 were nearly transparent immediately after preparation and exhibited clear properties after filtration with a 0.2 μm filter. Therefore, it was concluded that the stable injectable preparation can be prepared based on 5.0% PPC at 2.4% or greater of GDCA. The composition of Comparative Examples 6-1 to 6-6 had a particle size in the range of 204.4 ± 1880 nm to 134.8 ± 680.1 nm, that is, the composition was dispersed as an unstable emulsion or liposome, but Comparative Examples 6-7 were 6-9 consists of a microcell structure with a size of 10 nm or less. As described above, it is judged that the substantially stable formulation is not present in the composition having a GDCA/PPC molar ratio of less than 0.87, and it is not suitable for use in an injectable preparation. Comparative Example 7 : PPC injectable preparation solubilized with TDCA

As shown in Table 12 below, by adding 50.0 mg PPC (5.0%) and 10.0 mg (1.0%), 15.0 mg (1.5%), 20.0 mg (2.0%), 21.0 mg (2.1%), 22.0 mg, respectively. 2.2%), 23.0 mg (2.3%), 24.0 mg (2.4%), 25.0 mg (2.5%) or 30.0 mg (3.0%) TDCA, and 9 mg benzyl alcohol (0.9%) in 1 ml, for preparation A phospholipid choline (PPC)-based composition that is solubilized by taurine (OTCA). The specific preparation method is the same as the production method of Example 1 mentioned above. Table 12

The formulation stability of the PPC injectable composition solubilized with taurine cholate (TDCA) in the evaluation table 12 immediately after preparation and 30 days after preparation (refrigeration) was evaluated, and the evaluation results are shown in Fig. 2H. When TDCA was added with PPC 5.0% at 2.3% or greater, it was confirmed that the formulation was stable and the composition exhibited clear (clear) solution properties (Comparative Examples 7-6 to 7-9). In Comparative Examples 7-1 to 7-5, it was confirmed that the formulation was unstable due to the precipitation phenomenon. Comparative Examples 7-6 were nearly transparent immediately after preparation and exhibited clear properties after filtration with a 0.2 μm filter. Therefore, it was concluded that the stable injectable preparation can be prepared based on 5.0% PPC at TDCA of 2.3% or more. The composition of Comparative Examples 7-1 to 7-5 had a particle size in the range of 185.1 ± 1834 nm to 123.5 ± 72.0 nm, that is, the composition was dispersed as an unstable emulsion or liposome, but Comparative Example 7-6 was 7-9 consists of a microcell structure with a size of 10 nm or less. As described above, it is judged that the substantially stable formulation is not present in the composition having a TDCA/PPC molar ratio of less than 0.74, and it is not suitable for use in an injectable preparation. Comparative Example 8 : PPC injectable preparation solubilized with HDCA

As shown in Table 13 below, by adding 50.0 mg PPC (5.0%) and 10.0 mg (1.0%), 15.0 mg (1.5%), 20.0 mg (2.0%), 21.0 mg (2.1%), 22.0 mg, respectively. 2.2%), 23.0 mg (2.3%), 24.0 mg (2.4%), 25.0 mg (2.5%) or 30.0 mg (3.0%) HDCA, and 9 mg benzyl alcohol (0.9%) in 1 ml, for preparation Phosphodeoxycholic acid (HDCA) solubilized phospholipid choline (PPC)-based composition. The specific preparation method is the same as the production method of Example 1 mentioned above. Table 13

The formulation stability of the PPC injectable composition solubilized with hyodeoxycholic acid (HDCA) in Table 13 immediately after preparation and 30 days after preparation (refrigeration) was evaluated, and the evaluation results are shown in Fig. 2I. When TDCA was added with PPC 5.0% at 2.3% or greater, it was confirmed that the formulation was stable and the composition exhibited clear (clarified) solution properties (Comparative Examples 8-6 to 8-9). In Comparative Examples 8-1 to 8-5, it was confirmed that the formulation was unstable due to the precipitation phenomenon. Comparative Examples 8-6 were nearly transparent immediately after preparation and exhibited clear properties after filtration with a 0.2 μm filter. Therefore, it was concluded that the stable injectable preparation can be prepared based on 5.0% PPC at 2.3% or greater HDCA. The composition of Comparative Examples 8-1 to 8-5 had a particle size ranging from 537.2 ± 320 nm to 81.65 ± 24.30 nm, that is, the composition was dispersed as an unstable emulsion or liposome, but Comparative Examples 8-6 were 8-9 consists of a microcell structure with a size of 10 nm or less. As described above, it is judged that a substantially stable formulation is not present in a composition having a HDCA/PPC molar ratio of less than 0.95, and it is not suitable for use in an injectable preparation.

It is unusual to find that the PPC composite composition solubilized with HDCA is cloudy when stored at low temperature (4-8 ° C) and becomes a clear solution at room temperature. It has been inferred that the PPC + HDCA composite composition is poorly formulated during refrigeration and should be stored at room temperature for a long period of time. However, HDCA is not considered to be a solubilizer suitable for quality control of PPC composite compositions, because when PPC + HDCA is stored at room temperature for a long time, lysophosphatidylcholine choline increases, which leads to hemolysis. Comparative Example 9 : PPC injectable preparation solubilized with TUDCA

As shown in Table 14 below, by adding 50.0 mg PPC (5.0%) and 10.0 mg (1.0%), 15.0 mg (1.5%), 20.0 mg (2.0%), 25.0 mg (2.5%), 30.0 mg, respectively. 3.0%), 35.0 mg (3.5%), 40.0 mg (4.0%), 45.0 mg (4.5%) or 50.0 mg (5.0%) TUDCA, and 9 mg benzyl alcohol (0.9%) in 1 ml, for preparation A composition based on phospholipid choline (PPC) that is solubilized by ursodeoxycholic acid (TUDCA). The specific preparation method is the same as the production method of Example 1 mentioned above. Table 14

The formulation stability of the PPC injectable composition solubilized with ursodeoxycholic acid (TUDCA) in the evaluation table 14 immediately after preparation and 30 days after preparation (refrigeration) was evaluated, and the evaluation results are shown in Fig. 2J. . When TUDCA was added at 5.0% or more with respect to PPC 5.0%, it was confirmed that the formulation was stable, and the composition exhibited transparent (clarified) solution properties (Comparative Examples 9-7 to 9-9). In Comparative Examples 9-1 to 9-6, it was confirmed that the formulation was unstable due to the precipitation phenomenon. Therefore, it was concluded that the stable injectable preparation can be prepared based on 5.0% PPC at 4.0% or more of TUDCA. The particle size of the compositions of Comparative Examples 9-1 to 9-6 was in the range of 86.30 ± 850 nm to 22.8 ± 0.980 nm, that is, the composition was dispersed as an unstable emulsion or liposome, but Comparative Examples 9-7 were 9-9 consists of a microcell structure with a size of 10 nm or less. As described above, it is judged that the substantially stable formulation is not present in the composition having a TUDCA/PPC molar ratio of less than 1.29, and it is not suitable for use in an injectable preparation.

Table 15 below shows the preferred BA/PPC molar ratios required to prepare the PPC injectable compositions as described in Comparative Examples 1 to 9 and Examples 1 and 2, various bile acids (BA) and PPC. Best minimum molar ratio (BA/PPC). A bile salt (BA) that can be used to prepare a stable injectable composition of a clear solution, wherein PPC is deoxycholic acid (DCA) when dispersed in a microcell structure of 10 nm or less at low temperature and room temperature. , cholic acid (CA), glycocholic acid (GCA), taurocholic acid (TCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), glycodeoxycholic acid (GDCA), cattle go Oxycholic acid (TDCA) and ursodeoxycholic acid (TUDCA), and hyodeoxycholic acid (HDCA) at room temperature. At the minimum molar ratios mentioned above, it solubilizes the PPC to disperse into the microcell structure of the stable clear solution. The optimal BA/PPC minimum molar ratio for solubilizing PPC with bile acid (BA) was 0.92 ± 0.17. That is, the optimum BA/PPC minimum molar ratio is from 0.74 to 1.29, as shown in Table 15 below. To solubilize PPC 5%, BA should be mixed at least 2.49 ± 0.56% (w/v). Table 15 Comparative Example 10 : PPC injectable formulation solubilized with LCA

A phospholipid choline (PPC 5.0%)-based composition solubilized with lithocholic acid (LCA) 3.0% (w/v) was prepared by the same method as the method of Example 1 mentioned above.

However, as shown in FIG. 3A, when PPC is solubilized under sodium hydroxide, lithocholic acid (LCA) exhibits turbidity and precipitation properties due to gelation. Comparative Example 11 : PPC injectable formulation solubilized with DHCA

Phospholipid choline (PPC 5.0%) prepared by dehydrocholic acid (DHCA) 2.5%, 3.0%, 4.0% or 5.0% solubilization was prepared by the same method as the method of Example 1 mentioned above. ) the composition. However, as shown in FIG. 3B, when pH was adjusted with sodium hydroxide after solubilizing PPC, dehydrocholic acid (DHCA) exhibited hazy properties and precipitation properties such that it was not suitable for the injectable composition. Comparative Examples 12 to 30 : Preparation of PPC Single, BA Single and PPC+BA Composite Compositions

Deoxycholic acid (DCA) single (Comparative Example 12) and bile acid (CA) single at various concentrations (1.0 to 7.5% (w/v) and the like) were prepared in a single composition (Comparative Example) 13), glycocholic acid (GCA) single (Comparative Example 14), bovine cholic acid (TCA) single (Comparative Example 15), chenodeoxycholic acid (CDCA) (Comparative Example 16), ursodeoxycholic Acid (UDCA) single (Comparative Example 17), glycodeoxycholic acid (GDCA) single (Comparative Example 18), taurine (OTCA) single (Comparative Example 19), hyodeoxycholic acid ( HDCA) single (Comparative Example 20) and ursodeoxycholic acid (TUDCA) single (Comparative Example 21) injectable compositions. Specifically, by adding 10.0 mg (1.0%), 25.5 mg (2.5%), 50.0 mg (5.0%) or 75.5 mg (7.5%) of bile acid, and adding 9 mg (0.9%) of benzyl alcohol, respectively, Composition. The method comprises the steps of: placing the water for injection into a cleaned and sterilized (room temperature) preparation tank, and adding sodium hydroxide to the water for injection. Subsequently, bile acid (salt) and benzyl alcohol are added, stirred and dissolved. Subsequently, it was stirred at 200 RPM for about 2 hours under light blocking, sealing, room temperature (25 ° C) and nitrogen pressure. After the completion of the agitation, the pH was adjusted and it was filtered through a 0.2 μm filter and filled into a vial and sealed.

Further, various concentrations of PPC single composition [(PPC 1.25%, 2.50%, 5.0%, 7.5%, 10.0%, 12.5%, and 15.0%) (Comparative Example 22)] were prepared. The specific preparation method was the same as that of Comparative Example 1.

Further, in the form of a composite composition, PPC 5.0%+DCA 2.2% (Comparative Example 23), PPC 5.0%+HDCA 2.5% (Comparative Example 24), PPC 5.0%+UDCA 3.0% (Comparative Example 25) were prepared. ), PPC 5.0% + TDCA 2.5% (Comparative Example 26), PPC 5.0% + GDCA 2.5% (Comparative Example 27), PPC 5.0% + CDCA 2.5% (Comparative Example 28), PPC 5.0% + CA 2.5 % (Comparative Example 29) and PPC 5.0% + TUDCA 4.0% (Comparative Example 30). The specific preparation method was the same as that of Comparative Examples 2 to 9. Examples 3 and 4 : Preparation of PPC composite composition solubilized with GCA or TCA

Preparation of PPC+GCA [(PPC 2.5%+GCA 1.25%, PPC 5.0%+GCA 2.5%, PPC 7.5%+GCA 3.75%, PPC 10.0%+GCA 5.0%, PPC 15.0%+GCA 7.5%, PPC 5.0%+ GCA 5.0%, PPC 5.0%+GCA 7.5%, PPC 5.0%+GCA 10.0%, PPC 5.0%+GCA 15.0%, PPC 5.0%+GCA 20.0%, and PPC 5.0%+GCA 25.0%) (Example 3)] And PPC+TCA[(PPC 2.5%+TCA 1.25%, PPC 5.0%+TCA 2.5%, PPC 7.5%+TCA 3.75%, PPC 10.0%+TCA 5.0%, PPC 15.0%+TCA 7.5%, PC 5.0%+ TCA 5.0%, PPC 5.0%+TCA 7.5%, PPC 5.0%+TCA 10.0%, PPC 5.0%+TCA 15.0%, PPC 5.0%+TCA 20.0%, and PPC 5.0%+TCA 25.0%) (Example 4)] Composition. The specific preparation method is the same as the preparation methods of Examples 1 and 2. Test Example 1 : Comparison of side effects ( inflammation, edema, and skin lesions )

Various concentrations of phospholipid choline (PPC) single composition (Comparative Example 22), bile acid (BA) single composition (Comparative Examples 12 to 21), DCA, HDCA, UDCA, TDCA, GDCA, CDCA were tested. Or edema, skin lesions, and inflammation of the PPC composite composition (Comparative Examples 23 to 29) in which CA was solubilized, and the PPC composite composition (Examples 3 and 4) solubilized with GCA or TCA.

Inflammation, edema, and local or extensive skin lesions at the site of administration are local side effects typically observed with DCA single injectable compositions (such as Kybella) or PPC injectable compositions (such as Lipostabil, Lipobean, etc.) that are well known and used. The following results show the results of edema, inflammation and skin lesions in a subcutaneous injection of a single composition of bile acid (salt), a PPC single composition, and a bile acid solubilized PPC composite. It was observed from the following results that PPC injectable compositions solubilized with specific molar ratios of GCA, TCA or TUDCA resulted in inflammation and edema that were substantially free of inflammation and edema or that resulted in 80% relief. These results are an unpredictable finding based on the efficacy of individual substances. The following test methods and results are described below. Hereinafter, the % of the composition means % (w/v). 1-1 : rat paw edema

To assess the extent of edema, each of the test compositions described above was injected into rat paws. Specifically, male Sprague Dawley rats (6 weeks old) were purchased and used after one week of adaptation. Rats (body weight: 170-200 g) were randomly selected, and rat paw thickness was measured with a caliper before administration of the test composition. To observe edema, 0.1 ml of PBS and various test compositions were administered to rat paws. The paw volume measurement was performed by a caliper just before the injection, immediately after the injection, and 1 or 2 hours after the injection. In addition, when the thickness was equivalently measured, the skin lesion at the injection site was observed by photographing the bottom of the paw (using a 4 x 4 cm scale).

The degree of edema was assessed to grade 0 to 4. The degree of edema immediately after the injection and 1 or 2 hours after the injection was evaluated as compared with that before the injection, in which the grade was completely edema [-, (the degree of swelling was 0% compared with before injection)], mild Edema [+, (1-20% swelling compared with before injection)], moderate edema [++, (20-40% swelling compared with before injection)], severe edema [++ +, (40-60% degree of swelling compared to before injection)] and extremely severe edema [++++, (degree of swelling is 60% or greater compared to before injection)].

The results of edema assessment at 2 hours (when edema is most severe) after administration of a single PPC composition (PPC 1.25%, 2.50%, 5.0%, 7.5%, 10.0%, 12.5%, or 15.0%) are shown in Table 16 below. The “light” grade was extended from PPC 1.25% to 10.0%, and the “moderate” grade was shown in PPC 12.5% and 15.0%. The edema increased to "severe" 2 hours after the administration of DCA 1.0% (Fig. 4A). In summary, the PPC single composition exhibited concentration-dependent edema at high concentrations, but the PPC 15% single composition (ie, the highest concentration) induced a significantly lower grade of edema than DCA 1.0%. Table 16

The results of edema assessment at 2 hours (when edema is most severe) after administration of various concentrations of bile acids (1.0%, 2.5%, 5.0%, 7.5%) are shown in Table 17, and the "mild" level is shown in Among the TUDCA 1.0%, 2.5%, GCA 1.0% and TCA 1.0%, the "moderate" grade was displayed in UDCA 1.0%, GDCA 1.0%, CA 1.0%, GCA 2.5-5.0%, TCA 2.5-5.0%, TUDCA 5.0- Among the 7.5%, and the remaining concentrations of bile acids exhibited "severe" and "extremely severe" edema (Figures 4B to 4K). Table 17

Specifically, as shown in the results of the in-tube fat cell viability test described below, PPC 5% exhibited an ability to reduce fat cells equivalent to 1% of DCA. Comparing the results of bile acids based on a normal and usually required 2.5% concentration of solubilized PPC 5%, the degree of edema was "mild" in the case of TUDCA at 2 hours after administration, and "moderate" in the case of GCA and TCA. In the case of HDCA, TDCA, GDCA, and CA, it is "severe" and is "extremely severe" in the case of DCA, UDCA, and CDCA, as shown in Table 17. Figure 4L shows a single composition of each bile acid compared to the 5.0% single composition injected with PPC, the concentration required to solubilize PPC 5.0% (usually about 2.5%, but for UDCA 3%, for TUDCA 4%) or The results of edema 2 hours after PBS, and the results were similar to Table 17.

In this connection, in order to confirm the degree of inflammation of the PPC composite composition solubilized with various bile acids, the degree of edema was compared 2 hours after administration of the PPC 5.0% composite composition solubilized with each bile acid. As shown in Table 18, it was confirmed that GCA, TCA and TUDCA showed "no edema", CA showed "moderate", and HDCA, UDCA, TDCA, GDCA and CDCA showed "severe" (Fig. 4M). Table 18

Surprisingly, the degree of edema induced by a single composition of GCA (2.5%), TCA (2.5%), and TUDCA (5.0%) was moderate (Table 17), but by GCA (2.5%), TCA. The degree of edema induced by (2.5%) and TUDCA (4.0%) solubilized PPC 5.0% composite composition was reduced to no or mild (Fig. 4M).

To observe the edema according to the concentration change, the time-dependent edema degree of the PPC complex composition solubilized with GCA or TCA was compared. Therefore, it was confirmed that 2 hours after administration, the composite composition corresponding to 2.5-5.0% of PPC exhibited "no edema", and the composite composition corresponding to PPC 7.5-10.0% exhibited "mild" and corresponded to PPC 15.0. The composite composition of % shows "moderate" as shown in Tables 19 and 20 (Figs. 4N to 4O). Table 19 Table 20

In the PPC composite composition solubilized with bile acid, the preparation time for obtaining a stable and clear solution is shortened as the concentration of the bile acid to be added is increased. For this reason, edema of a composition capable of solubilizing the concentration of GCA or TCA of 5.0% of PPC was evaluated. The test material was a composite composition in which PPC 5.0% was mixed with 2.5%, 5.0%, 7.5%, 10.0%, 5.0%, 20.0% or 25.0% GCA or TCA, respectively. As shown in Table 21 and Table 22, the preferred GCA/PPC molar ratio of edema or mild edema was 2.60 mol/mol or less, and the edema was 3.47 mol/mol or less. Mild and moderate, and showed severe edema at a molar ratio greater than 3.47 mol/mol. In addition, the preferred TCA/PPC molar ratio of edema or mild edema is 2.35 mol/mol or less, and the edema is mild and moderate at a molar ratio of 3.13 mol/mol or less. Severe edema was shown at a molar ratio greater than 3.13 mol/mol (Figures 4P and 4Q). Table 21 Table 22

Results of in vivo edema test In summary, PPC 1.25-10.0% of the single composition exhibited "mild" edema 2 hours after administration (when edema was the most severe), as shown in Tables 16-22 and Figure 4 (Figure 4A) Shown in 4Q). Among the bile acids (BA) selected to address the industrial use limitations due to the low formulation stability of the PPC single composition, DCA 2.5%, UDCA 2.5%, CDCA 2.5%, HDCA 2.5%, TDCA 2.5% GDCA 2.5% and CA 2.5% showed "severe" and "extremely severe" edema, with GCA 2.5%, TCA 2.5% and TUDCA 5.0% showing "moderate" edema. However, the PPC 5.0% composite composition solubilized with GCA, TCA or TUDCA showed "no" or "mild" edema. As a result of observing the change in edema according to the concentration and the amount of the mixture, the PPC 2.5-10.0% composite composition solubilized with 1.25-5.0% GCA or TCA exhibited "none" or "mild" edema. a GCA/PPC molar ratio of 3.47 mol/mol or less, preferably 2.60 mol/mol or less; and a TCA/PPC molar ratio of 3.13 mol/mol or less, preferably 2.35 mol/mol or less. Show "no" or "mild" edema. Based on the results of previous studies and the in vitro in vitro test for cell viability (see test 2 described below), it was found that TUDCA was not suitable for solubilizing agents due to its effect of inhibiting apoptosis, and apoptosis was the mechanism of fat cell lysis of PPC. 1-2 : measurement of skin lesions ( erythema )

In order to observe the skin-related harmful condition after the injection of the test composition, when the thickness of the paw was measured in Test Example 1-1, skin lesions were observed by photographing the bottom of the rat paw (using a 4 cm × 4 cm scale) ). The grade of skin lesions was assessed to grade 0 to 4. Skin lesion grades immediately after injection and 1 or 2 hours after injection were compared to those prior to injection, with grades defined as no erythema (-), very mild erythema (visually almost unrecognizable), mild (+ )], significant erythema (moderate, ++), mild severe erythema (severe, +++) and severe erythema (extremely severe, ++++).

First, there was no erythema in all concentration groups of PPC (1.25-15.0%) single composition, as shown in Table 23 (Fig. 5A). Table 23

Subsequently, skin lesion results for individual compositions at various concentrations (BA 1.0%, 2.5%, 5.0%, and 7.5%) are shown in Table 24, 2.5% or greater of DCA, HDCA, UDCA, GDCA, TDCA, and CDCA. And 5.0% or more of CA showed severe and extremely severe erythema. It was confirmed that 5.0% or less of GCA and TCA showed no or mild erythema, and TUDCA showed no erythema (Figs. 5B and 5C). Table 24

Skin lesion results of PPC 5.0% composite composition solubilized with various bile acids As shown in Table 25, PPC 5.0% composite composition solubilized with DCA, HDCA, UDCA or CDCA exhibited severe erythema 2 hours after administration. The PPC 5.0% composite composition solubilized with TDCA, GDCA or CA exhibited moderate erythema, and the PPC 5.0% composite composition solubilized with GCA, TCA and TUDCA showed no erythema (Fig. 5D). Table 25

In order to observe lesions that vary according to concentration, the extent of lesions was compared under various concentrations of PPC complexes solubilized by GCA. As shown in Table 26, no lesion was observed at PPC 10.0% + GCA 5.0% or less, and mild lesions were observed at PPC 15.0% + GCA 7.5% (Fig. 5E). Table 26

The lesions that were able to solubilize the composition of the increased concentration of GCA at 5.0% of PPC were evaluated. As shown in Table 27, the preferred GCA/PPC molar ratio of no lesion or mild lesion was 2.60 mol/mol or less, and the lesion was severe or extremely at a molar ratio of 3.47 mol/mol or more. Serious (Figure 5F). Table 27

Results of in vivo lesion testing In summary, as shown in Tables 23 to 27 and Figure 5, PPC 1.25-10.0% of the single composition exhibited no lesions. Among the bile acids (BA) selected to address the industrial use limitations due to the low formulation stability of the PPC single composition, DCA 2.5%, UDCA 2.5%, CDCA 2.5%, HDCA 2.5%, TDCA 2.5% GDCA 2.5% and CA 5.0% or greater showed "severe" and "extremely severe" lesions, and 5.0% or less of GCA and TCA showed no or mild lesions, and TUDCA showed no lesions. In the case of PPC 5.0% composite composition solubilized with additional bile acids, PPC 5.0% composite composition solubilized with DCA, HDCA, UDCA or CDCA exhibited severe lesions, PPC 5.0 solubilized with TDCA, GDCA or CA The % composite showed moderate lesions, but the PPC 5.0% composite composition solubilized with GCA, TCA or TUDCA showed no lesions. As a result of changes in the concentration and the amount of the lesion, the PPC 2.5-10.0% composite composition solubilized with GCA 1.25-5.0% showed no lesions, and the PPC 15.0% composite composition with GCA 7.5% solubilization exhibited mild lesions. . In addition, it was confirmed that a GCA/PPC molar ratio of 2.60 mol/mol or less exhibited no or mild lesions. 1-3 : H&E staining histology test ( inflammation )

The rats were sacrificed 3 hours after the completion of Test Examples 1-1 and 1-2 to evaluate the degree of inflammation after the test composition was injected. The tissue of the injection area was dissected, fixed with 10% formalin, and then samples were prepared and images were taken using an optical microscope. The degree of inflammation was assessed as follows.

No inflammation (-) indicates that functional tissues (such as sweat glands, blood vessels, and adipose tissue) are well maintained and inflammatory cells are not visible. Mild (+) indicates that the form of functional tissue (sweat glands, blood vessels, adipose tissue, etc.) is well maintained and occasionally inflammatory cells appear. Moderate (++) indicates that the morphology of functional tissues (sweat glands, blood vessels, adipose tissue, etc.) is impaired and inflammatory cells appear in the tissue. Severe (+++) indicates that the morphology of the functional tissue is impaired and the inflammatory cells are increased, and inflammatory cells (such as neutrophils, monocytes, and the like) migrate to the tissues surrounding the blood vessels. Extremely severe (+++) indicates that the morphology of functional tissues is impaired by edema and inflammation, and that inflammatory cells (such as neutrophils, monocytes, and the like) not only increase but also spread to the papillary dermis. Tissue damage was clearly observed.

First, as a result of inflammation assessment of a single composition of all concentrations of PPC (1.25-15.0%), PPC 2.50-7.50% showed "no" inflammation, PPC 10.0-12.5% showed "mild", and PPC 15.0% showed "middle" Degree, as shown in Table 28 below (Fig. 6A). Table 28

Subsequently, as a result of inflammation assessment of various concentrations of bile acids (BA 1.0%, 2.5%, 5.0%, and 7.5%), 1.0% or greater of DCA, HDCA, and UDCA exhibited severe and extremely severe inflammation, 2.5% or greater. TDCA, GDCA, CDCA, and CA demonstrate severe and extremely severe inflammation. And 2.5% or greater of GCA and TCA exhibited moderate inflammation, and high concentrations of TUDCA showed only mild inflammation (Figures 6B and 6C). Table 29

In order to test the degree of inflammation of the PPC composite composition solubilized with bile acid, a PPC 5.0% composite composition solubilized with various bile acids was prepared and inflammation was measured. As shown in Table 29, the PPC composite composition solubilized with DCA, HDCA, UDCA or CDCA exhibited "extremely severe", and the PPC composite composition solubilized with TDCA or GDCA exhibited "severity" and was solubilized with CA. The PPC composite composition shows "moderate". On the other hand, the PPC composite composition solubilized with GCA, TCA or TUDCA uniquely exhibits no inflammation. It was surprisingly found that GCA 2.5% and TCA 2.5% single compositions exhibited moderate inflammation, but PPC 5.0% composite composition with GCA 2.5% or TCA 2.5% solubilization showed no inflammation (Fig. 6D).

In order to examine the degree of inflammation depending on the concentration, the degree of inflammation of various PPC complexes solubilized by GCA was compared. Thus, PPC 7.5% + GCA 3.75% or less showed "no" inflammation, PPC 10.0% + GCA 5.0% showed mild inflammation, and PPC 15.0% + GCA 7.50% showed moderate inflammation (Figure 6E).

In order to examine the degree of inflammation increased according to the amount of the solubilizing agent, a composition containing GCA capable of solubilizing the increased concentration of PPC 5.0% was prepared and inflammation was evaluated. As shown in Table 28, the preferred GCA/PPC molar ratio showing no or mild inflammation was 3.47 mol/mol or less, and it was confirmed that the molar ratio of 3.47 mol/mol or more exhibited extremely severe inflammation. (Fig. 6F).

An overview of in vivo test results for edema, lesions, and inflammation caused by subcutaneous injections to reduce topical fat is as follows. In order to prepare a stable composition form of PPC 5.0%, which has a fat cell reduction efficacy equivalent to 1.0% DCA, bile acids should generally be mixed at a concentration of 2.5%, but it is confirmed that DCA, HDCA, UDCA, TDCA, GDCA, CDCA, and CA cause severe and extremely severe edema and lesions caused by inflammation at 2.5% concentration. In addition, the PPC 5.0% composite composition formulated with the bile acids mentioned above induced significant pain and edema. Thus, their bile acids are not suitable for use as solubilizers in the PPC-based topical fat reducing injectable compositions of the present invention. On the other hand, GCA + PPC formulations and TCA + PPC formulations did not cause pain, edema and lesions, and these findings were unpredictable and unpredictable findings based on a single composition of PPC, GCA or TCA.

In summary, it has been found that the GCA + PPC composite preparation and the TCA + PPC composite preparation of the present invention preferably have a molar ratio of bile acid to PPC of 0.7 to 3.0 (ie, GCA/PPC molar ratio or TCA/PPC molar ratio). ). When the molar ratio (mol/mol) is less than 0.7, the formulation stability is lowered because it is difficult to form stable micelles. Therefore, it is preferred to have a molar ratio of at least 0.7 or more, and more preferably, when the molar ratio is 0.76 or more, it is most advantageous in terms of stability and process time. When the molar ratio exceeds 3.04, side effects such as inflammation, edema, and skin lesions are significantly induced, and the possibility of necrosis is increased, rather than providing a positive effect on apoptosis and fat dissolution of fat cells. These side effects and pain are significantly alleviated at a molar ratio of 3.0 or less. In particular, when the molar ratio is 2.60 or less in the above range, side effects and pain are alleviated to substantially no such side effects and pain. Optimally, when the molar ratio is 1.73 or less, it has been confirmed that an excellent fat-reducing composition containing no inflammation, edema, and lesions is produced. The results of the confirmation of pain are further described in the following test examples.

Further, it has been confirmed that it is more advantageous in terms of pain, edema and side effects, and the absolute content of PPC in the total composition may preferably be 12.5% (w/v) or less, and more preferably 10.0% (w/). v) or smaller. Test Example 2: Comparison of the effect of reducing the cell 2-1: adipocytes (3T3-L1) to reduce the effect of Comparison

Tests were performed to compare the adipocyte-reducing activity of the PPC single composition and the PPC composition solubilized with bile acid. The differentiated 3T3L-1 adipocyte line was used to observe adipocyte-reducing activity, and cell viability was monitored by MTT assay. The following results were obtained by comparing the fat cell viability of each test material. In particular, the mixed composition of TUDCA, GCA or TCA (which is a solubilizer selected according to in vivo test results) and PPC affects the activity of fat cells in the following in-tube test results at an unexpected mixing ratio, and the following data are presented. The unexpected discovery. The materials and methods used in the following experiments are described below. Hereinafter, the % of the composition means % (w/v).

The specific test method is as follows. The differentiated 3T3-L1 adipocytes were cultured under the confluence of 85-92% of cells. Cells were treated with each of the following test compositions and cultured for 0 to 96 hours at 37 °C: PPC single composition (PPC 0.3125%, 0.625%, 1.25%, 2.5%, 5.0%, 7.5%, 10.0%) , 15.0%), DCA single composition (DCA 1.0%, 1.1%, 2.2%), GCA single composition (GCA 1.25%, 2.5%, 5.0%), PPC composite composition solubilized with GCA (PPC 0.3125%) +GCA 0.1563%, PPC 0.625%+GCA 0.3125%, PPC 1.25% +GCA 0.625%, PPC 2.5%+GCA 1.25%, PPC 5.0%+GCA 2.5%, PPC 7.5%+GCA3.75%, PPC 10.0%+ GCA 5.0%, PPC 15.0%+GCA 7.5%, PPC 5.0%+GCA, 3.75%, PPC 5.0%+GCA 5.0%, PPC 5.0%+GCA 6.25%, PPC 5.0%+GCA 7.5%, PPC 5.0%+GCA 8.75%) PPC composite composition solubilized with TCA (PPC 0.3125%+TCA 0.1563%, PPC 0.625%+TCA 0.3125%, PPC 1.25% + TCA 0.625%, PPC 2.5%+TCA 1.25%, PPC 5.0%+ TCA 2.5%, PPC 7.5%+TCA3.75%, PPC 10.0%+TCA 5.0%, PPC 15.0%+TCA 7.5%), PPC composite composition solubilized with TUDCA (PPC 0.3125%+TUDCA 0.25%, PPC 0.625) %+TUDCA 0.5%, PPC 1.25% + TUDCA 1.0%, PPC 2.5%+TUDCA 2.0%, PPC 5.0%+TUDCA 4.0%, PPC 7.5%+TUDCA 6.0%, PPC 10.0%+TUDCA 8.0%, PPC 15.0%+TUDCA 12.0%), PPC composite composition solubilized with DCA (PPC 2.5%+DCA 1.1%, PPC 5.0%+DCA 2.2%, PPC 10.0%+DCA 4.4%) Or PPC composite composition solubilized with other bile acids (PPC 5.0%+HDCA 2.5%, PPC 5.0%+UDCA 3.0%, PPC 5.0%+TDCA 2.5%, PPC 5.0%+GDCA 2.5%, PPC 5.0%+CDCA 2.5%, PPC 5.0%+CA 2.5%). The cells were washed twice with PBS, treated with MTT reagent (50 μl), and allowed to stand at 37 ° C for 2 hours. After removing the supernatant, MTT formazan crystals were dissolved in DMSO, and the absorbance was measured at 540 nm using a microplate reader.

Thus, as shown in Figures 7A through 7D, the PPC single composition and the PPC + GCA composite composition exhibited a similar time- and concentration-dependent manner of adipocyte-reducing activity. And at the same concentration, the PPC + TCA composition exhibited a lower fat cell reduction effect than the PPC single and PPC + GCA compositions. However, treatment with the PPC + TUDCA composition did not reduce adipocyte viability (Figures 7A to 7D).

In addition, as shown in Figure 7E, at 96 hours, PPC 5.0% single composition, PPC 5.0% + GCA 2.5%, and PPC 15.0% + TCA 7.5% exhibited adipocyte-reducing activity similar to DCA 1.0% single composition. . That is, the preparation having the same fat cell-reducing activity as Kybella (DCA 1.0%) approved by the FDA as an appearance-improving cytolytic agent was a composite composition of PPC 5.0% single composition and PPC 5.0% + GCA 2.5% and larger. Things. There were no statistically significant differences between the test groups (Fig. 7E).

The PPC single composition exhibited time and concentration-dependent adipocyte reduction effects, and the PPC + GCA composite composition exhibited similar effects at the same concentration (this means a test group having the same PPC concentration). In contrast, PPC + TCA exhibited a lower fat cell reduction effect at the same concentration, indicating that TCA inhibits the fat cell reduction effect of PPC. Because of this, the "PPC + TCA" formulation should be treated at very high doses to achieve an effect of fat cell reduction similar to the effects of existing commercial products. That is, as shown in Fig. 7E, in order to obtain an adipocytopening effect similar to the effect of an existing preparation such as Kybella (DCA 1.0%), the PPC + TCA preparation needs to be administered at a PPC of 15.0% + TCA of 7.5%. However, the PPC 15% composition solubilized with TCA 7.5% has a problem that it is difficult to dose multiple doses with a 30G injection needle because of a high viscosity of 20 cP or more. In summary, the "PPC + TCA" formulation is considered to have some advantages in terms of side effects. However, considering the economic viability of the industry and other additional issues associated with high-dose dosing, the "PPC + GCA" formulation is superior to the "PPC + TCA" formulation.

In addition, PPC solubilized with TUDCA was found to have no fat cell reduction effect even at high concentrations, as was the case at very high doses (PPC 15% + TUDCA 12%). Therefore, TUDCA was observed to inhibit apoptosis and degradation of adipocytes. In this regard, studies have been reported on the inhibition of cell apoptosis by TUDCA (Andrew L. Rivard, Administration of Tauroursodeoxycholic acid reduced apoptosis following myocardial infarction in rat, The American Journal of Chinese Medicine, Vol. 2, 279-295, 2007). These results indicate that PPC has different effects on adipocyte reduction depending on the choice of solubilizing agent, and can act on cell necrosis and apoptosis in different ways.

Regarding the PPC composite composition (PPC + GCA) solubilized by GCA (which is preferably selected according to the results of the above test) and the PPC composite composition (PPC + DCA) solubilized by DCA (ie, existing commercial products), In addition to the fat cell reducing activity of the PPC single composition itself, the effects of GCA and DCA selected as solubilizing agents on the fat cell reducing activity were observed. The test results are shown in Figures 7F to 7H. After 96 hours, single PPC and PPC + GCA showed similar levels of adipocyte reduction at the same concentration (this means a test group with the same PPC concentration), but a single GCA showed lower fat cell reduction than PPC + GCA. .

PPC + DCA exhibited a higher fat cell reduction effect at the same concentration compared to single PPC, and exhibited a higher fat cell reduction effect than single DCA (Figs. 7F to 7H), but as in Test Examples 2-3 below This effect is attributed to cell necrosis (Figures 10A and 10B). In addition, HDCA, UDCA, CDCA, TDCA, GDCA, and CA (which were found to be toxic in the above test examples (ie, in edema, lesion, and inflammation tests after subcutaneous injection into the body) have been found to be similar to DCA. Other bile acids have the effect of reducing fat cells in a manner that induces cell necrosis rather than apoptosis and degradation of adipocytes, and apoptosis and degradation of adipocytes are an intrinsic mechanism of PPC (Fig. 8D). Therefore, these types of bile acids are not suitable for solubilizing agents because although the fat cell reduction effect appears to be slightly higher, it causes pain, edema, and side effects.

The minimum GCA to PPC molar ratio (GCA/PPC) required to prepare a stable injectable clarified mixed PPC microcell was 0.76 mol/mol, and it required 12 hours or more of agitation and 2 days of operation time. For this reason, increasing the amount of GCA input can shorten the preparation time. However, excessive doses of additives may cause negative effects on the intrinsic pharmacological activity and safety of PPC on apoptosis and degradation of fat cells, thus fixing PPC concentration (5.0%) and increasing GCA concentration (2.5%-8.75%). To observe the effect on the reduction of fat cells.

Therefore, as shown in Figure 7I, when the PPC 5.0% single composition and the PPC + GCA composite composition were compared, the molar ratio of GCA to PPC (GCA/PPC) was 2.60 mol/mol (PPC 5.0% + GCA). 7.50%) or less, there was no statistically significant difference in adipocyte viability between the test groups, and when the molar ratio of GCA to PPC (GCA/PPC) was 3.04 mol/mol (PPC 5.0% + GCA 8.75%) or Larger, there was a statistically significant difference in adipocyte viability between the experimental groups. That is, when the GCA/PPC molar ratio is 3.04 mol/mol (PPC 5.0% + GCA 8.75%) or more, the possibility of adverse effects on the inherent positive activity of PPC increases. 2-2 : Comparison of the viability of fat cells, fibroblasts, skeletal muscle cells, and vascular endothelial cells in the case of a PPC composite composition solubilized with bile acids - Adipocyte specificity of the present invention

According to previous reports, DCA compositions or PPC compositions solubilized with DCA have been reported to cause severe clinical side effects due to not only dissolving fat cells but also fibroblasts, skeletal muscle cells, and vascular endothelial cells. In this aspect, the effects of the PPC + GCA composite composition of the present invention were evaluated.

The specific methods and materials tested were as follows: PPC composite composition (PPC 5.0%+GCA 2.5%, PPC 5.0%+TCA 2.5%, PPC 5.0%+TUDCA 4.0%, PPC 5.0%+DCA 2.2%, PPC 5.0) %+HDCA 2.5%, PPC 5.0%+UDCA 3.0%, PPC 5.0%+TDCA 2.5%, PPC 5.0%+GDCA 2.5%, PPC 5.0%+CDCA 2.5% or PPC 5.0%+CA 2.5%) Treatment of 3T3-L1 Cell viability was measured by MTT assay 72 hours after adipocytes, normal fibroblasts, skeletal muscle cells, and endothelial cells, and the results were calculated as the total percentage of viable cells compared to the untreated control group.

3T3-L1 fat cells (ATCC) in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen) was cultured under conditions of 5% CO ₂ and 37 °C. Adipocytes were allowed to differentiate for 2 or 4 days until the cell confluence reached 100%, and in DMEM medium containing 1 μg/ml insulin, 500 μM methyl-isobutyl-xanthine and 250 nM dexamethasone Differentiation was induced for 3 days (pre-differentiation and post-differentiation adipocytes are shown in Figures 9A and 9B). Subsequently, the medium was replaced with DMEM medium containing 1 μg/ml of insulin (Sigma), and the medium was maintained and changed every 2 to 3 days until the degree of differentiation was maximized. If it took more than 7 days, the cells were kept in normal DMEM medium until the test was started. After incubation for 72 hours with the treated test material, the MTT solution was diluted to 1 mg/ml in PBS, and 50 μL of MTT (Sigma) solution was added to the wells from which the medium had been removed. After incubating the cells for 3 hours at 37 ° C and 5% CO ₂ , the MTT solution was removed. After dissolution by treatment with 200 μL of DMSO (Sigma), MTT analysis (measured at 570 nm) was performed.

Fibroblasts (CCD-986sk, human fibroblasts, based on passage 2) were grown to 85% confluency and the medium (IMDM, 10% FBS, 1% antibiotic mixture) was subsequently removed. After mixing and processing the test materials into the new medium, the medium was removed at the predetermined treatment time and MTT analysis (measured at 570 nm) was performed.

Skeletal muscle cells (C2C12; mouse myocytes, based on passage 2) were grown to 80% confluency and the medium (DMEM, 10% FBS, 1% antibiotic mixture) was subsequently removed. The cells were cultured for 4 days in DMEM containing 2% horse serum. The elongated shape of the cells was observed at 80% or more, and the test materials were mixed and processed in a new medium, the medium was removed at a predetermined treatment time, and MTT analysis (measured at 570 nm) was performed.

Vascular endothelial cells (HUVEC: human endothelium, based on the third passage) were allowed to stand at room temperature for 1 day in a petri dish coated with 1% gelatin. Subsequently, after incubation in a coated petri dish up to 80% confluence, the medium (EGM-Plus, 10% FBS, 1% antibiotic mixture) was removed. After the test materials were mixed and processed in the new medium, the medium was removed at the predetermined treatment time and MTT analysis (measured at 570 nm) was performed. (Note: discard the cells if they are passaged to passage 6 or more).

As a result of observing the viability of various cells, as shown in Figs. 8A to 8D, it was found that the PPC complex composition solubilized with GCA or TCA selectively reduced only fat cells, unlike DCA, UDCA, HDCA, CDCA, TDCA. , PDC composite composition solubilized by GDCA or CA. This indicates that when the composition of the present invention, PPC + GCA, is administered to an actual human, it can specifically reduce only fat cells without adversely affecting human tissues surrounding the fat cells. 2-3 : Analysis of apoptosis protein 3 activity

Apoptotic protease 3 activity assay was performed on the test material to determine whether cell death in MTT assay results was due to necrosis or apoptosis. Apoptotic protease 3 specifically increases in apoptosis and is a marker of apoptosis. The caspase 3 assay kit (colorimetric) from Abcam was used according to the manufacturer's manual, and the test method was as follows: 1× ^{10 5} cells of 3T3-L1 adipocytes were distributed to each well, and the treatment contained PPC 5.0%, PPC A formulation of 5.0% + DCA 2.2%, PPC 5.0% + GCA 2.5%, PPC 5.0% + GCA 5.0%, DCA 1.0%, GCA 1.0% or GCA 5.0% and PBS were subsequently incubated at 37 ° C for 0-48 hours. The cells were then treated with 50 ul of cell lysis buffer (10 mM Tris-HCl, 10 mM NaH ₂ PO ₄ /NaHPO ₄ , pH 7.5, 130 mM NaCl, 1% Triton X-100, and 10 mM sodium pyrophosphate), and It was allowed to stand at 4 ° C for 10 minutes. The supernatant was collected by centrifugation at 1000 x rpm for 1 minute, and protein quantification was performed by the BCA method. 50 μl of reaction buffer (4 mM HEPES, pH 7.5, 10% glycerol, 2 mM dithiothreitol) and 0.5 μl of 4 mM DEVD-p-NA were added to each sample and allowed to react at 37 ° C for 1 hour. . Subsequently, the wavelength was measured by spectral fluorescence measurement at 405 nm.

Therefore, as shown in FIGS. 10A and 10B, PBS did not induce apoptosis protease-3 activity in adipocytes. The PPC single composition and the PPC + GCA composite composition exhibited a time-dependent effect of inducing apoptotic protease-3 activity to a considerable extent. However, the PPC + DCA composite composition inhibits apoptosis protease-3 activity compared to PPC or PPC + GCA. Interestingly, DCA 1.0% exhibited a certain level of caspase-3 activity up to 24 hours, but after 48 hours, caspase-3 activity returned to pre-treatment levels. This phenomenon is considered to be due to the effect of cell apoptosis on the cells 24 hours after the DCA single composition treatment, and then there is a reaction in which the cells become necrotic by subsequent inflammatory reactions. Apoptotic protease-3 activity was observed in the GCA-treated group, but there was no time- or concentration-dependent change. On the other hand, it was confirmed that the apoptosis protease-3 activity was shown to be higher in the GCA + PPC complex preparation in a time- and concentration-dependent manner. When PPC was mixed with DCA, the activity of apoptosis protease-3 induced by the PPC single composition was significantly reduced. These results indicate that the apoptosis-specific effect of the PPC single composition is inhibited by DCA, and the PPC preparation supplemented with DCA induces more adipocyte necrosis, which is associated with inflammation and the like. 2-4 : Fat dissolution analysis

Glycerol activity analysis was performed on the test material to determine whether cell death in the MTT assay was due to necrosis or fat dissolution. Glycerin is a specific marker that is added when fat is broken down. The Abcam Fat Dissolution Analysis Kit (colorimetric) was used according to the manufacturer's manual, and the test method was as follows: 1× ^{10 5} cells of 3T3-L1 adipocytes were distributed to each well, and the treatment contained PPC 5.0%, PPC 5.0%+DCA Formulations of 2.2%, PPC 5.0% + GCA 2.5%, PPC 5.0% + GCA 5.0%, DCA 1.0%, GCA 1.0% or GCA 5.0% and PBS were subsequently incubated at 37 ° C for 0-48 hours. And then induced to dissolve. Add 30 ul of fat lysis assay buffer (137 mM NaCl, 5 mM KCl, 4.2 mM NaHCO ₃ , 1.3 mM CaCl ₂ , 0.5 mM KH ₂ PO ₄ , 0.5 mM MgCl ₂ , 0.5 mM MgSO ₄ , 5 mM glucose, 20 mM Hepes (pH 7.4), 1% BSA, 1 uM Isoproterenol) was added to the medium, the total volume was adjusted to 50 ul, and it was incubated for 20 minutes. After the glycerol assay complex (50 μl) was added thereto, the solution was incubated at room temperature for 30 minutes. The absorbance was measured at OD 570 (using a standard curve for absolute determination).

Therefore, as shown in FIGS. 10C and 10D, PBS does not induce glycerol secretion in adipocytes. At 24 hours, the test material similarly induced glycerol secretion except DCA 1.0% and PPC 5.0% + GCA 5.0%. At 48 hours, single PPC, PPC + DCA, single DCA, and single GCA groups exhibited slightly higher cytolytic activity than 24 hours of activity. In particular, the PPC + GCA group exhibited a much higher apoptotic effect on cells than the PPC single composition. Test Example 3 : Evaluation of efficacy and side effects of injectable preparations in a mouse obesity model induced by a high fat diet .

Male C57BL/6 mice (4 weeks old) were purchased. A high fat diet (Research diet, 60% kcal lipid) was given for 12 weeks to make it highly obese. After that, the single composition of PPC (2.5%, 5.0%, 10.0%, and 15.0%), PBS (negative control), Isuprel (positive control), DCA 1.0% and GCA 2.5%, and PC 5.0%+DCA 2.2%, PPC 5.0%+HDCA 2.5%, PPC 5.0%+UDCA 3.0%, PPC 5.0%+TDCA 2.5, PPC 5.0%+GDCA 2.5%, PPC 5.0%+CDCA 2.5%, PPC 5.0%+CA 2.5%, PPC 5.0%+TUDCA 4.0%, PPC 5.0%+TCA 2.2%, and PPC (2.5-10.0%)+GCA (1.25-5.0%) composites were directly administered to a mouse model of obesity induced by a high-fat diet. In the adipose tissue (subcutaneous fat tissue) of the groin area, and observed a decrease in fat in the living body. Each test material was administered once. 0.2 ml of each test material was injected subcutaneously into the inguinal fat pad of the mouse. Finally, the mice were sacrificed 8 days after the injection. The inguinal fat pad of the sacrificed mice was dissected, and the subcutaneous fat was quickly removed and fixed in a 4% formaldehyde solution. After fixation, the fat pad was washed and dehydrated, treated with a paraffin solution to prepare a paraffin block, stained with hematoxylin and eosin, and observed with an optical microscope.

First, in the tissue in which the PPC single composition was injected, apoptosis and degradation of the adipocytes were induced in a concentration-dependent manner in a concentration range of 2.5% to 10%, and the size of the fat cells in the adipose tissue was decreased. Some of the reduced fat cells stick together, the area of the dead cells is transparent, and the fat cells appear to increase due to the fusion of the degraded fat cells. At a concentration of 15.0%, not only fat cells were reduced, but also macrophages appeared around the fat cells (Fig. 11A).

Subsequently, in the case of a PPC complex composed of bile acid solubilized, apoptosis of adipocytes was clearly observed, showing smaller fat cells, and DCA, CDCA, HDCA, UDCA, GDCA, TDCA or CA for injection. Macrophage-mediated phagocytosis is evident around the fat cells in the adipose tissue of the solubilized PPC composite. In the adipose tissue (negative control group) injected with Isuprel, the size of the fat cells was decreased, the configuration of apoptosis of the cells was observed, and infiltration of cells other than the fat cells was observed to remove the cells of apoptosis. In the adipose tissue in which the DCA single composition or the PPC composition solubilized with DCA was injected, severe inflammation was induced at the site where the composition was administered, and the cells were dissolved by necrosis, and significant damage was induced. The DCA single composition exhibited severe inflammation even when DCA was contained at a low concentration of 1%, and the inflammation-inducing effect was greater than that of the PPC single composition and the PPC + GCA complex composition. In contrast, adipose tissue injected with PBS (negative control) or PPC 5.0% + TUDCA 4.0% had a clear cell membrane boundary and a well formed cell shape, and consisted only of fat cells in the tissue. In the adipose tissue in which PPC 5.0% + TCA 2.5% was injected, the size of the fat cells was reduced as a whole, and infiltration of cells other than the fat cells was observed in a small area to remove the apoptotic cells. In the injection of the PPC of the present invention PMP 5.0% + GCA 2.5%, the inflammation is not induced, the size of the fat cells is reduced, the area of apoptosis is transparent, and the fat cells appear to be enlarged by the fusion of the degraded fat cells ( Figures 11B and 11C).

As shown in Fig. 11D, as a result of examining the effects of the PPC + GCA composite composition of the present invention not only on fat cells but also on the dermis and epidermis after injection into a rat fat pad, dermal and epidermal tissue after PBS injection Good retention and absence of inflammatory cells (such as neutrophils). And there is a clear cell membrane boundary and a well formed cell shape in the adipose tissue, and the adipose tissue is composed only of the fat cells. After injection of the PPC 5.0% single composition, the dermal and epidermal tissues were well retained and there were no inflammatory cells (such as neutrophils). In adipose tissue, the size of fat cells is reduced, and some cell membranes degrade, and some fat cells appear to increase due to the fusion of degraded fat cells. After injection of the PPC 2.5% + GCA 1.25% composite composition, the dermal and epidermal tissues were well retained and there were no inflammatory cells (such as neutrophils). In adipose tissue, the size of fat cells is reduced, and some cell membranes degrade, and some fat cells appear to increase due to the fusion of degraded fat cells. After injection of the PPC 5.0% + GCA 2.5% composite composition, the dermal and epidermal tissues were well retained and there were no inflammatory cells (such as neutrophils). In adipose tissue, the size of the fat cells is reduced, and the area of apoptosis is transparent, and some of the fat cells appear to be enlarged by the fusion of the degraded fat cells. After injection of PPC 10.0% + GCA 5.0% composite composition, the dermis and epidermal tissue were slightly damaged, and some inflammatory cells were present, and slight edema was observed. In adipose tissue, the size of fat cells is reduced, and the phagocytosis of macrophages and nucleic acid fragments due to apoptosis are present in the region of apoptosis, and some fat cells appear to be due to the fusion of degraded fat cells. And increase. After injection of a 2.5% single composition of GCA, the dermal and epidermal tissues were slightly damaged, and inflammatory cells (such as neutrophils and the like) were dispersed, and thus inflammation was clearly observed. And there is a clear cell membrane boundary and a well formed cell shape in the adipose tissue, and the adipose tissue is composed only of the fat cells. Based on the above results, it was confirmed that the inflammatory reaction on the adipocytes, the dermis, and the epidermis was more significantly induced when the GCA single composition was treated than the GCA and PPC composite preparations.

In conclusion, in adipose tissue injected with single DCA or PPC complex composition solubilized with DCA, HDCA, UDCA, CDCA, TDCA, GDCA or CA, severe inflammation was induced at the site of administration, and the cells were dissolved by necrosis and induced significantly. damage. However, in the adipose tissue in which the PPC + GCA composite composition of the present invention is injected, the size of the fat cells is reduced, and the region of apoptosis is transparent. And some fat cells become larger due to the fusion of degraded fat cells. Despite the slight induction of concentration-dependent inflammation, it presents morphological features of damage to the adipocyte membrane. Test Example 4 : Evaluation of toxicity of PPC injectable preparations solubilized with GCA

The toxic effect of the PPC 5.0% injectable formulation solubilized with GCA 2.8% was evaluated by single subcutaneous administration to Miguel dogs. Specifically, the number of tattoos for all animals is checked and their general condition, weight and body temperature are measured upon arrival. During the 12-day isolation and adaptation period after arrival, the general symptoms were observed once a day, body weight was measured once a week, and the health of the animals was checked at the end of the isolation and adaptation period. After termination of the isolation and adaptation period, one male and one female control group, and two male and two female test group animals were separated based on body weight. The test animals were a total of 14 Miguel dogs (male: 5-6 months, 7.05-8.16 kg/female: 5-6 months, 5.83-7.14 kg), 7 males and 7 female dogs, and were based on dosing The weight of the day was calculated for each individual animal and the hair was removed from the neck before administration. The test materials were subcutaneously administered to the left and right sides of the nape of the neck using a disposable syringe (10 ml, 23 G). The test group was set to the low dose group (dose: PPC 90 mg/kg + GCA 50.4 mg/kg, injection volume: 1.8 ml/kg, maximum injection volume/site: 0.8 ml/site), medium dose group (dose: PPC 180) Mg/kg+GCA 100.8 mg/kg, injection volume: 3.6 ml/kg, maximum injection volume/site: 1.6 ml/site), high dose group (dose: PPC 360 mg/kg+GCA 201.6 mg/kg, injection volume) : 7.2 ml/kg, maximum injection volume/site: 3.2 ml/site) and control group (salt saline, injection volume: 7.2 ml/kg, maximum injection volume/site: 3.2 ml/site). The detailed test results are described below.

1) There was or was no death; no death was observed in all tests and control groups during the test period.

2) General symptoms; no symptoms were observed in all tests and control groups during the test period.

3) Body weight change; no abnormal changes were observed in all tests and control groups during the test period.

4) necropsy; no abnormal changes were observed in all tests and control groups.

5) Histopathological examination; very little to mild degree of granulomatous inflammation was observed in the subcutaneous tissue of the male and female high dose groups (see Figures 12A to 12C). As described above, very mild inflammation was observed only in the high dose group in which the injectable preparation of the present invention was injected. However, the degree of inflammation can be considered as having no side effects from the standpoint of setting an appropriate dose concentration when administering to humans and other animals. Test Example 5 : Comparison of pain in vivo

The following results were obtained by measuring the degree of pain induced by a single composition of DCA, GCA or PPC, and a PPC complex composition solubilized with bile acid in vivo by measuring the moving distance and moving speed of the animal. Hereinafter, the % of the composition means % (w/v).

Specifically, 100 ul of a single composition (DCA 1.0%, PPC 5.0% or GCA 2.5%) or a composite composition (PC 5.0% + DCA 2.2%, PPC 5.0% HDCA 2.5%, PPC 5.0) was injected into the mouse paw. %+UDCA 3.0%, PPC 5.0%+TDCA 2.5%, PPC 5.0%+GDCA 2.5%, PPC 5.0%+CDCA 2.5%, PPC 5.0%+CA 2.5%, PPC 5.0%+GCA 2.5%, PPC 5.0%+ Edema was observed after TCA 2.5% or PPC 5.0% + TUDCA 4.0%). After 5 hours after administration, after the edema was confirmed to be the maximum, the Noldus Video Traking system was used to compare the moving distance (cm) and the moving speed (cm/s) for 5 minutes.

As shown in Figures 13A and 13B, the moving distance and moving speed of the PPC 5.0% single composition, PPC 5.0% + TUDCA 4.0%, PPC 5.0% + GCA 2.5%, and PPC 5.0% + TCA 2.5% group did not change or slightly Increase. On the other hand, PC 5.0%+DCA 2.2%, PPC 5.0%+HDCA 2.5%, PPC 5.0%+UDCA 3.0%, PPC 5.0%+TDCA 2.5%, PPC 5.0%+GDCA 2.5%, PPC 5.0%+CDCA 2.5 % and PPC 5.0% + CA 2.5% group movement distance and movement speed decreased by about 20%, and these results were judged to be attributed to the decrease in activity due to pain. This indicates that the injectable formulations of the present invention cause significantly less pain (substantially painless) compared to existing commercial products. Test Example 6 : Clinical evaluation of PPC compositions solubilized with GCA 6-1 : Clinical evaluation of lipid- lowering efficacy

A PPC 5.0% injectable composition solubilized with GCA 2.8% was administered to a patient having a local underarm fat deposit. Specifically, after local anesthesia with 9.6% lidocaine cream for 30 minutes or longer, a 13 mm 30G injection needle was loaded into a 5 cc syringe, and the composition was injected 6 times at 4 week intervals into the underarm fat. (A total of 50 points, 0.2 cc per point and a total of 10 ml, 1.0 cm spacing, and a depth of 6-8 mm). After 12 weeks, standard clinical images, computed tomography (CT) imaging, improvement reported by the investigator, improvement in individual reporting, and individual satisfaction were assessed.

Clinical photographs were taken before and 12 weeks after the final administration, and the individual front, left side perspective, right side perspective, and left and right side photographs were taken under the following conditions: in the front image, the individual was at the Frankfort level. The posture is gazing at the camera, which is the plane where the ear screen points of the individual ears and the lowest part of the eyelids meet. In the fluoroscopic image sheet, after the individual's body is rotated 45 degrees, the individual gaze at the camera in a position in which the face is placed so as to be in line with the tip of the nose and the edge of the eyeball, and the Frankfort level is horizontal. In the side image, the individual body is rotated 90 degrees from the front end position so as to be in line with the tip of the nose and the nose. At this point, it has been confirmed that the opposing eyebrows are invisible and in a correct posture so that the body does not tilt, bend or stretch to the side. And then the individual gaze at the camera in a position in which the line connecting the back of the individual with the back of the head is adjusted to be vertical and the level of the Frankfort level is horizontal. The camera used for photography is a Nikon DSLR camera D5200 with a 60 mm short focal length lens.

CT images were taken before and 12 weeks after the last administration, and the thickness and area of the underarm fat were measured. The individual swallows saliva according to the notification when lying comfortably after wearing the prescribed top and headband.

CT images were taken before and 12 weeks after the last administration, and the thickness and area of the underarm fat were measured. The individual is prohibited from swallowing saliva when the individual is comfortably lying down after wearing the prescribed top and headband. At this time, the CT imaging pillow is of the neck (NECK) type, and the head of the individual is fixed to the laser guide wire passing through the middle of the forehead, nose, ankle and the clavicle. The imaging parameters of CT imaging were scan range (from ear canal to clavicle bottom), section (5.0 mm), skin FOV, matrix size 512×512, rotation time 0.5 sec, and beam collimation 64×0.6 mm. The device was from GE Medical systems and the images were analyzed using Xelis 1.0 6.0 BN 6 3D from Infinity.

The clinical efficacy of the injectable compositions of the present invention is fully demonstrated in Figures 16A and 16B. Figure 14A is a photograph showing a clinical image of an individual before administration and 12 weeks after the final administration, in which a dose of 0.2 cc per point, a total of 50 points, a total of 10 ml, is administered to the individual at 4 week intervals. The PPC composite composition of the present invention which was solubilized by GCA was used, and the reduction of underarm fat was also observed using the naked eye. The level of satisfaction reported by the individual was 4 out of 5, and the improvement was assessed to be 1.5 after comparing the images before administration. Interestingly, the individual is a patient who has previously received a PPC injectable composition (commercially available as Lipobean iv) solubilized with DCNa, and the individual describes a major surprise: the composition of the invention is administered immediately prior to administration. It is painless after and over time.

In addition, Figure 14B shows the results of a quantitative assessment of the amount of fat reduction by local deposition via CT. The thickness of the anterior subtemporal fat of the platysma, 3 cm below the end of the CT sagittal plane, was reduced by 30.36%, and decreased from 5.6 mm before administration to 3.9 mm after 12 weeks of final administration. 6-2 : Clinical evaluation of pain, edema and side effects

After administration of the GPC solubilized PPC injectable composition of the present invention, six male and female patients who have received the PPC injectable composition (previously commercialized) solubilized with DCA are subjected to pain, edema, and side effects. Clinical evaluation. Specifically, after local anesthesia with 9.6% lidocaine cream for 30 minutes or longer, the syringe was loaded into a 13 mm 30G syringe in the abdomen and ribs (1.5 cm interval, 10-12 mm depth, 0.5 cc per point, and a total of 50 ml to 100 ml per dose) or in the underarm fat (1.0 cm interval, 6-8 mm depth, 0.2 cc per point, and a total of 10 ml per dose) Individuals receiving 10 ml of a composition (ie, PPC 2.5% + DCNa 1.2%) diluted with 1:1 ratio of Lipobean iv (5 ml) in an injectable saline solution (5 ml) were administered 10 ml by the same administration method. The composition of the present invention (PPC 5.0% injectable preparation solubilized with GCA 2.8% (molar ratio of GCA to PPC is 0.97) or PPC 5.0% injectable preparation solubilized with GCA 4.0% (GCA and PPC) The ear ratio is 1.39)). After the administration, a questionnaire, 10 cm ruler and blue oil pen were used for the visual analog scale (VAS) pain assessment, and at the time of administration, 1, 3, 7 and 10 days after administration. Assessment of pain, edema, swelling, hematoma, bruises, erythema, loss of sensation, induration, paresthesia, nodules and itching. Pain was assessed by recording the painlessness on the left end with a 10 cm ruler and a planetary pen on the left end and recording the most severe pain at the right end. In the case of edema and swelling, after using the 10 cm ruler to press the administration site and other parts while looking at the mirror, the individual records are 0. None, 1. Mild (2 mm or less), 2. Moderate ( 2-4 mm), 3. Severe (4-6 mm) and 4. Extremely severe (6-8 mm). In the case of hematoma, bruises and erythema, the individual recorded the degree as 0. None, 1. Mild, 2. Moderate, 3. Severe, and 4. Extremely severe by comparison with the attached example image. In the case of sensory loss, induration, paresthesia, nodules and itching as subjective symptoms, after pressing the entire administration site, the individual records the degree as 0. None, 1. Mild, 2. Moderate, 3. Severe and 4. Extremely serious.

As shown in Fig. 15 (Fig. 15C) and Fig. 16, the individual who had received the administration of the PPC injectable composition solubilized with DCNa complained of pain and edema especially at the time of administration and 10 days after the administration, and the individual reported the skin. Lesions, such as erythema, hematomas and bruises, and local adverse events such as induration, nodules, itching, and burning sensation. Surprisingly, however, individuals receiving the administration of the GPC solubilized PPC injectable formulations of the present invention exhibited, in particular, mild (i.e., almost non-existent) pain (Fig. 15A) and edema (Fig. 15B). In particular, as shown in Fig. 15A, it is extremely unusual that the PPC + GCA composite composition of the present invention is almost painless even when injected. Full consideration is given to the fact that a PPC injectable preparation which is solubilized with DCNa (sodium salt of DCA) which is a conventional preparation and used as a comparative example has particle characteristics (microcells, particle size, etc.) similar to those of the composition of the present invention, And their injectable preparations are administered at a pH similar to that of humans, and the effect of the compositions of the present invention is a unique effect that is difficult to predict from previously known techniques.

Further, as shown in Fig. 16, in addition to the bruise caused by the injection needle itself or the hematoma caused by the vascular injury at the time of injection, the hematoma and erythema caused by the test material are administered after the composition of the present invention. Reduce to no or light level. Specifically, as shown in Fig. 15C, there are no side effects such as nerve damage such as sensation loss, extensive swelling, hematoma caused by drugs, bruises, erythema, induration, paresthesia, nodules, itching, burning Sense (warmth), difficulty swallowing, and the like. Considering the test results of in vivo, in vitro and single dose toxicity, the apoptosis and fat degradation effects of the fat cells of the PPC composition solubilized by GCA can be clinically demonstrated in humans with safety and high efficacy. Industrial utilization

As described above, the present invention relates to a composition suitable for use in the non-surgical reduction of fat in a subject having localized fat deposition using a pharmaceutically active phospholipid choline painless, edema, and no side effects; A method for preparing the same. More specifically, one is to alleviate pain and side effects (especially muscle cells, fibroblasts and vascular endothelial cell necrosis other than fat cells; edema; loss of feeling at the site of administration; extensive swelling; erythema; induration; paresthesia; nodules; Itching; burning sensation; nerve damage; or difficulty swallowing) a composition and preparation for reducing local fat, the composition comprising: (i) phospholipid choline; and (ii) selected from glycocholic acid (GCA), bovine At least one of a group consisting of an acid (TCA) and a salt thereof, wherein the composition (ii) has a molar ratio of (i) in the range of 0.7 to 3.0; a kit comprising the same; and a preparation thereof Method; and a method of non-surgically removing localized fat deposits using the composition or formulation to relieve pain and side effects.

A single composition of deoxycholic acid (DCA) or a complex composition of phospholipid choline (PPC) and deoxycholic acid (DCA) (a conventional injectable preparation for reducing local fat) is reported to be induced by fat Necrosis of cells and side effects caused by cell lysis of fibroblasts, endothelial cells, and skeletal muscle cells, such as loss of sensation, extensive swelling, hematoma caused by drugs, bruises, erythema, induration, paresthesia, nodules, itching , burning sensation (temperature sensation), difficulty swallowing and the like. However, since the injectable preparation for reducing local fat of the present invention reduces local fat by selectively inducing fat dissolution and apoptosis of only fat cells, the composition of the present invention not only exhibits a remarkable effect of reducing fat cells, but also The presentation significantly reduces the pain and side effects associated with the infusion of the injectable compositions with conventional cells. Thus, the individual's drug compliance is significantly improved, and ultimately, the quality of life of individuals who want to reduce localized fat is improved. Therefore, industrial utilization is high.

no

1A and 1B show a series of images of a PPC without a solubilizer dispersed by a stirrer and a high pressure homogenizer. Figure 1A shows an image of the composition, which was obtained immediately after the addition of only 5% (w/v) PPC to water for injection followed by stirring with a stirrer for 72 hours, and one day after the final stirring. Figure 1B shows an image of the composition, where the image is immediately after adding various concentrations (0.625% (w/v), 1.25% (w/v), 2.5% (w/v), 5.0% from left to right ( w/v), 7.5% (w/v), 10.0% (w/v) of PPC to water for injection followed by stirring for 1 hour and after dispersion in a high-pressure homogenizer, and 7 days and 30 after preparation of the composition Days are obtained.

Figures 2A through 2J show a series of images of PPC 5.0% compositions solubilized with various concentrations of DCA, GCA, TCA, CA, CDCA, UDCA, GDCA, TDCA, HDCA, and TUDCA, showing the stability of the formulation, where the image is tight It was obtained after the preparation of the composition and 30 days after the preparation.

Figure 2A shows the formulation properties x deoxycholic acid (DCA) concentration % (w/v) in PPC 5% (w/v).

Figure 2B shows the formulation properties x concentration of glycocholic acid (GCA) in PPC 5% (w/v) % (w/v).

Figure 2C shows the formulation properties x concentration (w/v) of taurine (TCA) in PPC 5% (w/v).

Figure 2D shows the formulation properties x concentration (w/v) of cholic acid (CA) in PPC 5% (w/v).

Figure 2E shows the formulation properties x hyodeoxycholic acid (CDCA) in % concentration (w/v) based on PPC 5% (w/v).

Figure 2F shows the formulation properties x ursodeoxycholic acid (UDCA) concentration % (w/v) in PPC 5% (w/v).

Figure 2G shows the formulation properties x concentration of glycodeoxycholic acid (GDCA) in PPC 5% (w/v) % (w/v).

Figure 2H shows the formulation properties x concentration (w/v) of bovine deoxycholic acid (TDCA) in PPC 5% (w/v).

Figure 2I shows the formulation properties x hyodeoxycholic acid (HDCA) concentration % (w/v) in PPC 5% (w/v).

Figure 2J shows the formulation properties x concentration (w/v) of ursodeoxycholic acid (TUDCA) in PPC 5% (w/v).

3A and 3B show an example of a bile acid that does not solubilize PPC and a description of a composite PPC formulation that is solubilized with various concentrations of GCA. Figure 3A shows an image of a PPC composite composition solubilized with lithic acid (LCA), and it is not possible to prepare mixed micelles. Figure 3B shows an image of a PPC composite composition solubilized with dehydrocholic acid (DHCA), and it is not possible to prepare stable mixed micelles.

Figures 4A to 4Q show graphs showing the results of an edema test in which the edema level (thickness of the rat paw (mm)) is immediately followed by injection of 0.1 ml of a single PPC composition, various bile salts (DCA, HDCA, UDCA, TDCA). , a single composition of GDCA, CDCA, CA, GCA, TCA, and TUDCA), a composite composition of various concentrations of PPC solubilized with bile acids (DCA, GCA, TCA, etc.) and PBS (rat thickness (mm) ) Measured after rat paws and measured 1 hour and 2 hours after injection. The test was repeated 4 times for each treatment and was performed by a caliper. In the following, % means %(w/v).

Figure 4A shows the results of a comparison of edema after injection of various concentrations of PPC single composition (1.25-15.0%) and DCA 1% single composition.

Figure 4B shows the results of a comparison of edema after injection of various concentrations of DCA single composition (1.0-7.5%).

Figure 4C shows the results of a comparison of edema after injection of various concentrations of HDCA single composition (1.0-7.5%) and DCA 1% single composition.

Figure 4D shows the results of a comparison of edema after injection of various concentrations of UDCA single composition (1.0-7.5%) and DCA 1% single composition.

Figure 4E shows the results of a comparison of edema after injection of various concentrations of TDCA single composition (1.0-7.5%) and DCA 1% single composition.

Figure 4F shows the results of a comparison of edema after injection of various concentrations of GDCA single composition (1.0-7.5%) and DCA 1% single composition.

Figure 4G shows the results of a comparison of edema after injection of various concentrations of CDCA single composition (1.0-7.5%) and DCA 1% single composition.

Figure 4H shows the results of a comparison of edema after injection of various concentrations of CA single composition (1.0-7.5%) and DCA 1% single composition.

Figure 4I shows the results of a comparison of edema after injection of various concentrations of GCA single composition (1.0-7.5%) and DCA 1% single composition.

Figure 4J shows the results of a comparison of edema after injection of various concentrations of TCA single composition (1.0-7.5%) and DCA 1% single composition.

Figure 4K shows the results of a comparison of edema after injection of various concentrations of TUDCA single composition (1.0-7.5%) and DCA 1% single composition.

Figure 4L shows 2 hours after the injection of PPC 5.0% single composition and the concentration required to solubilize PPC (typically about 2.5%, but for UDCD 3% and for TUDCA 4%) a single composition of bile salts The result of the comparison of edema.

Figure 4M shows the results of a comparison of edema 2 hours after the single composition of bile acid required for the injection of solubilized PPC and the composite composition of 5.0% of PPC solubilized with the above respective bile acids.

Figure 4N shows the results of a comparison of edema after injection of various concentrations of GCA (1.25-7.5%) solubilized PPC (2.5% - 15.0%) composite composition and DCA 1% single composition.

Figure 4O shows the results of a comparison of edema after injection of various concentrations of TCA (1.25-7.5%) solubilized PPC (2.5% - 15.0%) composite composition and DCA 1% single composition.

Figure 4P shows the results of a comparison of edema after injection of a composite composition of PPC 5.0% solubilized with various concentrations of GCA (2.5-25%).

Figure 4Q shows the results of a comparison of edema after injection of a composite composition of PPC 5.0% solubilized with various concentrations of TCA (2.5-25%).

Figures 5A through 5F show a series of images of the administration site, wherein the images are injected with 1.0 ml of PPC (1.25-15.0%) single composition and PBS (Figure 5A), 1.0-7.5% concentration of various types of bile salts (DCA, Single composition of HDCA, UDCA, TDCA, GDCA, CDCA, CA, GCA, TCA and TUDCA) (Fig. 5B and 5C), PPC 5.0% composite composition solubilized with various kinds of bile salts (Fig. 5D), GCA (1.25-7.5%) solubilized PPC (2.5-15.0%) composite composition (Fig. 5E) or PPC (5.0%) composite composition solubilized with GCA (2.5-20.0%) (Fig. 5F) to large Obtained 2 hours after the rat paw. The claw image was acquired using a 4 x 4 cm scale.

Figures 6A through 6F show a series of images of histological tests. Inject 1.0 ml of PPC (1.25-15.0%) single composition and PBS (Figure 6A), 1.0-7.5% concentration of various types of bile salts (DCA, HDCA, UDCA, TDCA, GDCA, CDCA, CA, GCA, TCA) And TUDCA) single composition (Fig. 6B and 6C), PPC 5.0% composite composition solubilized with various kinds of bile salts (Fig. 6D), PPC solubilized with GCA (1.25-7.5%) (2.5-15.0) The rats were sacrificed 3 hours after the composite composition (Fig. 6E) or the PPC (5.0%) complex composition (Fig. 6F) solubilized with GCA (2.5-20.0%). The injection area was cut, fixed with 10% formalin, and subsequently histologically examined using an optical microscope. H & E staining showed inflammation of the treated paw (200 x magnification).

Figures 7A through 7I show a series of images showing the reduction in viability of 3T3-L1 adipocytes treated with test materials. Adipocyte viability was measured by analysis of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The experiment was repeated 3 times for each treatment and the results are expressed as a percentage of viable cells relative to the untreated control. PPC (0.3125-15.0%) single composition, PPC (0.3125-15.0%) composite composition solubilized with TUDCA (0.25-12.0%), PPC (0.3125-15.0) solubilized with TCA (0.1563-7.5%) %) Composite composition or PPC (0.3125-15.0%) complex composition solubilized with GCA (0.1563-7.5%) 24 hours after treatment of differentiated 3T3-L1 adipocytes (Fig. 7A), 48 hours (Fig. 7B) Fat cell viability was measured at 72 hours (Fig. 7C) and 96 hours (Fig. 7D).

Figure 7E shows the display of adipocytes in DCA (1.0%) and PPC (5.0%) as a single composition, and PPC (5.0%) + GCA (2.5%), PPC (5.0-15.0%) + TCA (2.5-7.5%) And PPC (5.0-15.0%) + TUDCA (4.0-12.0%) as a plot of viability after 96 hours of treatment of the composite composition.

Figures 7F to 7H show the expression of adipocytes in PPC (2.5-10.0%), DCA (1.1-4.4%) and GCA (1.25-5.0%) as a single composition, and PPC (2.5-10.0%) + GCA (1.25- 5.0%) and PPC (2.5-10.0%) + DCA (1.1-4.4%) as a plot of viability after 96 hours of treatment of the composite composition.

Figure 7I shows a graph showing the viability of fat cells at 96 hours after treatment with PPC (5.0%) single composition and PPC (5.0%) composite composition solubilized with various concentrations of GCA (2.5-8.75%).

Figures 8A through 8D show PPC composite compositions showing PPC 5.0% single composition and solubilized with bile acid (PPC 5.0% + GCA 2.5%, PPC 5.0% + TCA 2.5%, PPC 5.0% + TUDCA 4.0%, PPC) 5.0%+DCA 2.2%, PPC 5.0%+HDCA 2.5%, PPC 5.0%+UDCA 3.0%, PPC 5.0%+TDCA 2.5%, PPC 5.0%+GDCA 2.5%, PPC 5.0%+CDCA 2.5% and PPC 5.0% +CA 2.5%) A series of images of reduced activity of skeletal muscle cells (Fig. 8A), normal fibroblasts (Fig. 8B), vascular endothelial cells (Fig. 8C) and 3T3-L1 adipocytes (Fig. 8D). Cell viability was measured by analysis of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The experiment was repeated 3 times for each treatment and the results are expressed as a percentage of viable cells relative to the untreated control.

Figures 9A and 9B show the state before (Figure 9A) and after (Figure 9B) differentiation of 3T3-L1 adipocytes. Differentiation media was used to induce differentiation of 3T3-L1 pre-adipocytes (left image). Differentiated adipocytes (right image) were stained with oil red staining and stained at 200x magnification.

Figures 10A to 10D show a series of images showing the following results. It can be seen from the analysis of apoptotic protease 3 activity that the injectable composition of the present invention has an apoptosis-specific effect, rather than necrosis of fat cells; and it is known by measuring the release of glycerol. It has a fat-dissolving effect.

Figures 10A and 10B show the results of apoptotic protease 3 activity. 3T3-L1 adipocytes were applied to each well at 1×10 ⁵ cells, and would contain PPC 5.0%, PPC 5.0%+DCA 2.2%, PPC 5.0%+GCA 2.5%, PPC 5.0%+GCA 5.0%, The DCA 1.0% or GCA (1.0-5.0%) formulation and the PBS control were incubated at 37 ° C for 0-48 hours (3 replicates). Subsequently, the results were measured with a spectrophotometer at a wavelength of 405 nm after 24 hours (Fig. 10A) and 48 hours (Fig. 10B).

Figures 10C and 10D show the results of glycerol release. The treatment of each test material was carried out in the same manner as in Figs. 10A and 10B. The material-treated fat cells were incubated at 37 ° C for 0-48 hours to induce fat solubilization. After incubation for 30 minutes at room temperature, OD570 was measured spectrophotometrically (3 replicates).

Figures 11A through 11D show images showing histological changes observed in fat pads from mice to which test materials were administered. Figure 11A shows the results of injection of a single composition of PPC (2.5-15.0%).

Figure 11B shows injection of PBS, Isuprel or DCA 1.0% as a single composition, and PPC 5.0% + DCA 2.2%, PPC 5.0% + CDCA 2.5%, PPC 5.0% + HDCA 2.5% or PPC 5.0% + UDCA 3.0% as a composite The result of the composition.

Figure 11C shows injection PPC 5.0% + GDCA 2.5%, PPC 5.0% + TDCA 2.5%, PPC 5.0% + CA 2.5%, PPC 5.0% + GCA 2.5%, PPC 5.0% + TCA 2.5% or PPC 5.0% + TUDCA 4.0 % is the result of the composite composition.

Figure 11D shows the results of injection of PBS, PPC 5.0% or GCA 2.5% as a single composition, and PPC (2.5-10.0%) + GCA (1.25-5.0%) as a composite composition.

After the injection, the adipose tissue at the administration site was cut. The dissected tissue was fixed with formaldehyde, impregnated into a paraffin block, and subsequently fragmented on a glass slide. Tissue necrosis, apoptosis and degradation were observed by H&E staining.

Figures 12A through 12C show images of single dose subcutaneous administration of PPC + GCA complex composition to Miguel dogs for observation of toxic effects. After 14 days of administration, a necropsy was performed for histopathological examination, and images were acquired after H&E staining.

For Meglu females and males, Figure 12A shows the results of the low-dose administration group (PPC (90 mg/kg) + GCA (50.4 mg/kg) composite composition), and Figure 12B shows the medium-dose administration group (PPC (180 mg) /kg) +GCA (100.8 mg/kg) composite composition), and Figure 12C shows the results of the high dose administration group (PPC (360 mg/kg) + GCA (201.6 mg/kg) composite composition).

13A and 13B show the results of assessing the degree of pain induction in vivo by measuring the moving distance (cm) and the moving speed (cm/s) of the experimental animals. Specifically, 100 μl of each test material was injected into the rat paw floor, and edema was observed. Therefore, it was confirmed that the most severe edema was observed 2 hours after the injection, and this time point was set as the maximum pain. The movement before and after injection of the test material was compared via distance and time. The Noldus Video Traking system was used to measure the movement before and after the injection, and the movement distance (Fig. 13A) and the moving speed (Fig. 13B) were compared.

Figures 14A and 14B show the results of efficacy of subgingival fat reduction in an individual receiving the GPC solubilized PPC composite composition of the present invention. After topical sensory loss at the site of administration (underarm) with 9.6% lidocaine ointment, 10 ml of PPC 5.0%+GCA 2.8% complex injectable composition (PPC 500 mg + GCA 280 mg) was injected 6 times at 4 week intervals. ) to the underarm fat (50 points total, 0.2 cc per point, 1.0 cm spacing, 6-8 mm depth, and using a 30 G 13 mm injection needle). After 12 weeks, get a series of images. Figure 14A shows images obtained by standard clinical photography methods, and Figure 14B shows a series of images showing reduction in thickness of underarm fat on CT.

Figures 15A through 15C show the results of comparing pain, edema, and deleterious examples after administration of a GPC solubilized PPC complex composition to 6 individuals who have been subjected to injection of a PPC injectable composition solubilized with DCA. The test material was Lipobean iv (PPC 50.0 mg + DCNa 24.0 mg in 1 ml) in a 10 ml solution (ie PPC 25.0 mg + DCNa 12.0 mg) diluted with a 0.9% saline solution at a ratio of 1:1; 10 ml PPC 5.0% solubilized with GCA 2.8% (PPC 50.0 mg + GCA 28.0 mg in 1 ml); and 10 ml PPC 5.0% solubilized with GCA 4.0% (PPC 50.0 mg + GCA 40.0 mg in 1 ml) . Each test material was injected into the underarm fat (total 50 points, 0.2 cc per point, 1.0 cm interval, 6-8 mm depth, and 30 G 13 mm injection needle), and after administration of each material 1. The questionnaire was conducted at 3, 7 and 10 days.

Figure 15A shows the degree of pain measured with 100 mm pain VAS, and Figure 15B shows the scale according to the edema rating (0: no -0 mm, 1: mild - 2 mm, 2: moderate - 4 mm, 3: severe - 6 mm, 4: extremely severe -8 mm) degree of edema, and Figure 15C shows 5 grades of the site of administration (0: absent, 1: mild, 2: moderate, 3: severe, 4: extremely severe) It includes harmful examples of extensive swelling, hematomas, bruises, erythema, nodules and itching.

Figure 16 shows a comparison of a solution diluted with 1:1 ratio of injectable 0.9% saline solution to Lipobean iv (PPC 50.0 mg + DCNa 24.0 mg in 1 ml) (ie PPC 25.0 mg + DCNa 12.0 mg in 1) Images of skin lesions (erythema, bruises, and hematomas) after a PPC complex (GPC 50.0 mg + GCA 40 mg in 1 ml) solubilized with GCA. After using the 9.6% lidocaine ointment to make the local sensation of the site of administration (under the iliac crest) for 30 minutes, 50 ml of each test material was administered to the subcutaneous fat layer of the rib (total 100 points, 0.5 cc per point, 1.5 cm spacing, 10-12 mm depth, and using a 30 G 13 mm needle). Images were taken 2 days after administration. Skin lesions such as erythema, bruises, and hematoma are observed along the drug dispersion area at the site of administration of the PPC injectable formulation solubilized with DCA. However, the PPC complex injectable preparation for GCA solubilization of the present invention shows only hematoma and bruise caused by needle invasion.

Claims (42)

A composition for reducing localized fat having reduced pain and side effects, the composition comprising: (i) phospholipid choline; and (ii) selected from the group consisting of glycocholic acid (GCA), taurocholic acid (TCA), and At least one of the group of salt compositions, wherein the composition (ii) has a molar ratio of (i) in the range of 0.7 to 3.0. The composition according to claim 1, wherein the side effect is at least one selected from the group consisting of muscle cells other than fat cells, fibroblasts, and vascular endothelial cell necrosis; edema; loss of feeling at the administration site; Swollen; erythema; induration; paresthesia; nodule; itching; burning sensation; nerve damage; The composition according to claim 1, wherein the composition (ii) has a molar ratio of (i) in the range of 0.7 to 2.6. The composition of claim 1, wherein the phospholipid choline is included in the composition at a concentration of 0.625 to 15% (w/v). The composition of claim 4, wherein the phospholipid choline is included in the composition at a concentration of 1.25 to 12.5% (w/v). The composition of claim 1, wherein the salt is a sodium salt, a potassium salt or an ammonium salt. The composition of claim 1, wherein the composition further comprises at least one selected from the group consisting of: a preservative; an isotonic agent; and a pH adjuster. The composition of claim 1, wherein the composition is for non-surgical removal of localized fat deposits in an individual. The composition of claim 1, wherein the composition is an injectable composition. The composition of claim 1, wherein the composition specifically induces apoptosis and fat dissolution of the adipocytes. A formulation for removing localized fat deposits in an individual with reduced pain and side effects, the formulation comprising: (i) phospholipid choline; and (ii) selected from the group consisting of glycocholic acid (GCA), taurocholic acid (TCA), and At least one of the group consisting of salt compositions, wherein the ratio of (ii) to (i) in the formulation is in the range of from 0.7 to 3.0. The preparation of claim 11, wherein the preparation is for non-surgical removal of localized fat deposits in an individual. The preparation of claim 11, wherein the phospholipid choline is included in the total preparation at a concentration of 0.625 to 15% (w/v). The preparation of claim 11, wherein the pH of the preparation is in the range of 6.8 to 7.8. The preparation of claim 11, wherein the preparation further comprises at least one selected from the group consisting of: a preservative; an isotonic agent; and a pH adjuster. The preparation of claim 11, wherein the preparation is an injectable preparation. A kit comprising: (I) a first container comprising a composition or formulation for removing topical fat deposits having reduced pain and side effects, the composition or formulation comprising: (i) phospholipid choline And (ii) at least one selected from the group consisting of glycocholic acid (GCA), taurine (TCA), and salts thereof, wherein (ii) the molar ratio of (i) in the composition or formulation is Within the range of 0.7 to 3.0; and (II) a delivery device capable of delivering the composition or formulation to a fat deposition site. The kit of claim 17, wherein the composition or formulation is for non-surgical removal of localized fat deposits in an individual. The kit of claim 17, wherein the composition or formulation is provided in the form of an injectable formulation. The kit of claim 17, wherein the delivery device is preloaded with an injectable formulation of the composition or formulation. The kit of claim 17, wherein the composition or formulation comprises phospholipid choline at a concentration of from 0.625 to 15% (w/v) based on the composition or formulation. The kit of claim 17, wherein the composition or formulation further comprises at least one selected from the group consisting of: a preservative; an isotonic agent; and a pH adjusting agent. The kit of claim 17, wherein the kit comprises a written description of a composition or formulation for the use of the localized fat deposit in the removed individual having reduced pain and side effects. A kit comprising: (I) a first container comprising a composition or formulation for removing topical fat deposits having reduced pain and side effects, the composition or formulation comprising: (i) phospholipid choline And (ii) at least one selected from the group consisting of glycocholic acid (GCA), taurine (TCA), and salts thereof, wherein the one selected from the group consisting of glycocholic acid (GCA), taurocholic acid (TCA), and At least one of the group of salt constituents is included in the same or lesser weight as the phospholipid choline; and (II) a delivery device capable of delivering the composition or formulation to the fat deposition site. A method for preparing an injectable composition for reducing topical fat having reduced pain and side effects, the method comprising the steps of: (a) adding from the group consisting of glycocholic acid (GCA), taurocholic acid (TCA) and salts thereof At least one of the group consisting of water for injection, followed by dissolution while stirring to obtain a clear mixture; (b) adding a preservative, followed by stirring; (c) adding phospholipid choline, followed by stirring at room temperature; d) adjusting the total volume of the composition with water, followed by stirring, wherein the at least one selected from the group consisting of glycocholic acid (GCA), tauric acid (TCA) and salts thereof and the phospholipid choline The ratio is in the range of 0.7 to 3.0. The method of claim 25, wherein the phospholipid choline is included in the composition at a concentration of 0.625 to 15% (w/v). The method of claim 25, wherein the at least one selected from the group consisting of glycocholic acid (GCA), taurine (TCA), and salts thereof, and the phospholipid choline have a molar ratio of 0.7 to 2.6. In the range. The method of claim 25, wherein the preservative is benzyl alcohol. The method of claim 25, wherein the step (b) further comprises adding an isotonic agent. The method of claim 25, wherein the composition specifically induces apoptosis and fat dissolution of the adipocytes. A method for preparing a pharmaceutical composition for non-surgical removal of topical fat deposits having reduced pain and side effects, the method comprising adding phospholipid choline and selected from the group consisting of glycocholic acid, taurocholic acid and salts thereof At least one of the group consisting of at least one selected from the group consisting of glycocholic acid, taurocholic acid and salts thereof is added in the same or lesser weight than the phospholipid choline. The method of claim 31, wherein the phospholipid choline is included in the composition at a concentration of 0.625 to 15% (w/v). The method of claim 31, wherein the composition further comprises at least one selected from the group consisting of: a preservative; an isotonic agent; and a pH adjusting agent. A method of removing localized fat deposition in an individual with reduced pain and side effects, the method comprising administering to the individual having localized fat deposition an effective amount of phospholipid choline; and at least one selected from the group consisting of glycocholic acid, bovine A phospholipid choline solubilizing agent of a group consisting of an acid and a salt thereof. The method of claim 34, wherein the phospholipid choline solubilizer and the phospholipid choline molar ratio are in the range of 0.7 to 3.0. The method of claim 34, wherein the topical fat deposit is removed in a non-surgical manner. The method of claim 34, wherein the side effect is at least one selected from the group consisting of: edema; muscle cells other than fat cells, fibroblasts and vascular endothelial cell necrosis; loss of sensation at the administration site; extensive swelling ; erythema; induration; paresthesia; nodules; itching; burning sensation; nerve damage; and difficulty swallowing. The method of claim 34, wherein the administering step comprises subcutaneous injection. A method of non-surgically removing topical fat deposits in a subject having localized fat deposition with reduced pain and side effects, the method comprising administering to the individual having localized fat deposition a formulation comprising: (i) phospholipids Choline; and (ii) at least one selected from the group consisting of glycocholic acid, tauric acid, and salts thereof. The method of claim 39, wherein the ratio of (ii) to (i) molar ratio in the formulation is in the range of 0.7 to 3.0. The method of claim 39, wherein the formulation is in the form of an injectable composition. A method of non-surgically removing localized fat deposits in an individual with reduced pain and side effects, the method comprising administering to a subject having localized fat deposition a formulation comprising: (i) phospholipid choline; and (ii) And at least one selected from the group consisting of glycocholic acid, taurine, and a salt thereof, wherein the composition (ii) has a molar ratio of (i) in the range of 0.7 to 3.0.