Composition comprising biocompatible polymer having tropoelastin cross-linking activity

Abstract

The present invention relates to a composition comprising a biocompatible polymer having tropoelastin cross-linking activity, and a method for producing same, and more specifically, to: a composition having the effects of providing improved elasticity and volume, reducing foreign body sensation, and having improved stability; and a method for producing same.

Description

Composition comprising a biocompatible polymer having tropoelastin cross-linking activity

[0001] The present invention relates to a composition comprising tropoelastin and a biocompatible polymer having tropoelastin crosslinking activity, which provides an improved elasticity effect by crosslinking of tropoelastin after injection into the body.

[0002] As life expectancy increases and interest in beauty grows across all age groups, interest in various plastic surgeries, procedures, and cosmetics is also increasing, leading to rapid technological growth in related fields.

[0003] Recently, there has been a significant increase in interest in cosmetic procedures that provide immediate effects immediately after the procedure, compared to cosmetics, and do not require surgery or anesthesia, such as plastic surgery. Accordingly, new technologies for various procedures, such as fillers, botox, laser treatment, and radiofrequency treatment, are being actively developed.

[0004] Fillers have been developed from the first generation fillers that used collagen extracted from animals, the second generation fillers that use hyaluronic acid, the third generation fillers that use substances that do not easily break down in the body, such as calcium fillers or PMMA, and the fourth generation fillers that use biodegradable polymers such as polycaprolactone.

[0005] The skin is a very complex organ made up of several layers. It is the largest organ in the human body and an important organ that covers the outer surface of the body and is in direct contact with the outside world. All external factors affect the body, and the skin protects the body from these factors.

[0006] The structure of the skin is divided into the epidermis, dermis, and subcutaneous tissue. The skin not only functions as a barrier to prevent external substances from entering the body, but also has a recovery function against external stimuli due to the various fibrous tissues contained within, that is, it has the function of returning to its original state despite external stimuli through elasticity.

[0007] Fillers are injected into various locations, such as the epidermis, dermis, and subcutaneous tissue layers, depending on their purpose and taking into account the characteristics of the skin in each area, such as thickness and movement.

[0008] Collagen fillers and hyaluronic acid fillers are injected into the dermis and subdermal layers of the skin. Collagen, elastin, and hyaluronic acid are the main components of the dermis. Collagen accounts for over 90% of the dermis' total mass, playing a key role in maintaining moisture and forming the skin.

[0009] Elastin functions to interconnect collagen fibers and is responsible for the skin's flexibility, elasticity, and resilience. Therefore, research is currently being conducted on fillers that contain elastin, a component that provides elasticity and improved resilience to the skin, along with collagen, which constitutes the main material of the dermal layer of the skin.

[0010] Korean Patent No. 2226810 relates to a composition for promoting elastin and collagen biosynthesis among connective tissues, and discloses a pharmaceutical composition for promoting elastin or collagen biosynthesis by containing glycine, L-proline, L-alanine, L-valine, L-leucine, and L-lysine hydrochloride and copper (Cu) ions as a divalent metal.

[0011] Korean Patent Publication No. 2023-0012532 relates to a composition comprising tropoelastin cross-linked to hyaluronic acid and a method of using the same, and discloses a method of treating a soft tissue condition by increasing the moisture content in the soft tissue by administering a composition comprising tropoelastin cross-linked to hyaluronic acid.

[0012] Compositions using collagen and hyaluronic acid, which are the main substances that make up the skin's dermis layer, are being widely developed, but the technology for a composition with improved elasticity and maintenance effect using elastin is still minimal, and the development of such technology is urgently needed.

[0013] The present invention aims to provide a composition that provides improved elasticity and volume by including elastin.

[0014] The purpose of the present invention is to provide a composition that has less foreign body sensation and less clumping or inflammation of the filler by crosslinking of body tissue and filler components.

[0015] The present invention also aims to provide a composition that has the effect of shortening the stabilization time after filler treatment, and is stably positioned at the treatment site, thereby improving treatment stability.

[0016] To solve the above-described problem, a composition according to the present invention comprises 20 to 30 wt% of biodegradable polymer particles, 5 to 8 wt% of tropoelastin, 0.1 to 10 wt% of a carrier, 0.1 to 10 wt% of a pain reliever, and the remainder of an injectable vehicle, wherein the biodegradable polymer particles comprise collagen and polycaprolactone, and the biodegradable polymer particles are characterized in that they are coated with lysyl-oxidase.

[0017] In the present invention, the biodegradable polymer particles may be a mixed powder containing collagen and polycaprolactone in a weight ratio of 1:0.5 to 1.

[0018] In the present invention, the lysyl-oxidase may be included in an amount of 0.1 to 3 wt% based on the total weight of the biodegradable polymer particles.

[0019] The present invention also provides a method for producing the above-described composition, comprising the steps of: a) mixing collagen and polycaprolactone and compressing them to form pellets, b) freeze-drying the pellets produced in step a) and then pulverizing them, c) dispersing the pulverized product produced in step b) in water and then drying them to form powder, d) compressing the powder obtained in step c) to form pellets and then producing powder, e) spraying ethanol corresponding to 0.1 to 1% of the weight of the powder on the surface of the powder produced in step d) and mixing it with lysyl-oxidase freeze-dried powder, f) mixing the powder produced in step d) into the dispersion of step e) and then drying them to obtain powder, and g) obtaining a powder comprising 20 to 30 wt% of the powder produced in step f), 5 to 10 wt% of tropoelastin powder, 0.1 to 10 wt% of a carrier, 0.1 to 10 wt% of a pain reliever, and the balance. A step of preparing a composition by mixing an injectable vehicle is included.

[0020] In the method for manufacturing a composition according to the present invention, the powder of step d) may be characterized as being a powder of 400 to 500 mesh.

[0021] The purpose of the composition according to the present invention is to provide a composition that provides improved elasticity and volume compared to existing collagen fillers or hyaluronic acid fillers.

[0022] The purpose of the composition according to the present invention is to provide a composition that has a low foreign body sensation and a long-lasting effect through crosslinking of body tissue and filler components.

[0023] The present invention also aims to provide a composition that has the effect of shortening the stabilization time after filler treatment, and is stably positioned at the treatment site, thereby improving treatment stability.

[0024] Hereinafter, each component of the present invention will be described in more detail so that a person having ordinary skill in the art to which the present invention pertains can easily practice it; however, this is only an example, and the scope of the rights of the present invention is not limited by the following contents.

[0025] As used herein, the terms "preferred" or "preferably" refer to embodiments of the invention that have particular advantages under specific conditions. However, other embodiments may also be preferred under the same or different conditions. Furthermore, the fact that one or more preferred embodiments are preferred does not imply that other embodiments are not useful, nor does it exclude other embodiments within the scope of the invention.

[0026] The term "comprises" as used herein is used to list materials, compositions, devices, and methods useful in the present invention, but is not limited to the listed examples.

[0027] Collagen is the main protein component of the extracellular matrix and is found in large quantities in soft tissues such as skin, tendons, and blood vessels, as well as hard tissues such as bones and teeth. It accounts for about one-third of the total protein in mammals and plays a role in forming the basic structure of tissues and organs through the assembly of cells.

[0028] Elastin is a highly elastic protein found in connective tissue, enabling many body tissues to maintain their shape after stretching or contracting. Elastin helps skin return to its original position when compressed or stretched. Elastin is also an important supportive tissue in vertebrates, requiring mechanical energy for storage.

[0029] Hyaluronic acid is a substance that fills the empty spaces within tissues where collagen and elastin fibrous tissues are densely distributed, and plays a role in maintaining moisture in the skin.

[0030] As we age, the amount of collagen fibers that make up the majority of the dermis decreases. The activity of collagen-synthesizing fibroblasts decreases, diminishing their ability to support the fibers and retain moisture. This reduces skin elasticity and prevents wrinkles from recovering. Collagen in the dermis is rapidly destroyed by collagenase, an enzyme that breaks down collagen, and elastinase, an enzyme that breaks down elastin.

[0031] The present invention aims to provide a composition that fills the constituents of the skin dermis layer while combining with tissue components of existing skin tissue to provide a natural skin texture and excellent volume, and that is quickly stabilized in the body, so that it has a quick effect after the procedure and is stably positioned at the target location, thereby providing excellent procedure stability.

[0032] Specifically, the composition according to the present invention comprises 20 to 30 wt% of biodegradable polymer particles, 5 to 8 wt% of tropoelastin, 0.1 to 10 wt% of a carrier, 0.1 to 10 wt% of a pain reliever, and the remainder of an injectable vehicle, wherein the biodegradable polymer particles comprise collagen and polycaprolactone, and the biodegradable polymer particles are coated with lysyl-oxidase.

[0033] The composition according to the present invention is manufactured as a single mixed powder using collagen and polycaprolactone as biodegradable polymer particles, and coats the surface thereof with lysyl oxidase, which is a tropoelastin cross-linking agent, so that a firm cross-linking bond can be formed with the biodegradable polymer particles through a reaction with tropoelastin contained in the composition, and the cross-linking reaction helps the composition to be more quickly and stably positioned at the treatment site.

[0034] Specifically, the biodegradable polymer particles may use a mixed powder containing collagen and polycaprolactone in a weight ratio of 1:0.5 to 1.

[0035] If the ratio of polycaprolactone is lower than the above ratio, there is a problem that the filler is biodegraded quickly and the filler maintenance period is shortened, and if the ratio is higher than the above ratio, there is a problem that the speed of volume formation is slow and the volume is relatively less than when the ratio is in the above range.

[0036] The above biodegradable polymer particles are composed of a mixed powder in which collagen and polycaprolactone are uniformly mixed, and the shape of the particles is not limited to any shape such as spherical, irregular, or plate-shaped. As described below, they may have an irregular shape as they are manufactured through a pulverization process.

[0037] The surface of the biodegradable polymer particles is coated with lysyl-oxidase. The lysyl-oxidase is physically bound to the surface of the biodegradable polymer particles coated with ethanol, and the coating is achieved by repeating drying and natural cooling. At this time, the temperature of the surrounding environment in which natural cooling is performed may be in the range of 20 to 30°C, and natural cooling may be performed at room temperature (25°C).

[0038] The above lysyl-oxidase may be included in an amount of 0.1 to 3 wt% based on the total weight of the biodegradable polymer particles.

[0039] If the amount exceeds the above, there is a problem of minimal difference in effect compared to the amount added. If the amount is less than the above, there is a problem of reduced elasticity and reduced stability of the procedure. In other words, there may be problems such as not being able to quickly settle after the procedure, moving easily, or losing shape due to movement.

[0040] Meanwhile, the tropoelastin is an elastin precursor and a basic unit of elastin fibers, and is crosslinked by lysyl oxidase. In the present invention, it can be added in an amount of 5 to 8 wt% based on the total weight of the composition.

[0041] If it is added in a smaller amount than the above content, there is a problem that the stability of the procedure is reduced, and if it is added in an amount exceeding the above content, there is a problem that the content of biodegradable polymer particles is relatively small and the expected volume effect when the same amount of filler is injected is reduced.

[0042] Any carrier that can be added to an injectable composition may be used, and for example, may include one or more selected from the group consisting of sodium carboxymethyl cellulose, sodium alginate, gelatin, albumin, collagen, sodium hyaluronic acid, dextran, hydroxyethyl cellulose, hypromellose, glycerin, sorbitol, and propylene glycol.

[0043] The pain reliever may also be any agent that can be added to an injectable composition, and may include, for example, one or more selected from the group consisting of lidocaine, bupivacaine, lignocaine, ropivacaine, cocaine, tetracaine, amethocaine, amylocaine, benzydamine, cinchocaine, levobupivacaine, mepivacaine, oxybuprocaine, prilocaine, procaine, proparacaine, and salts thereof.

[0044] The above-mentioned injectable vehicle may be any vehicle that can be added to an injectable composition, and may include, for example, one or more selected from the group consisting of water for injection, saline solution, polyalcohols, and fatty acids.

[0045] The present invention also provides a method for producing the composition described above, comprising the following steps:

[0046] a) A step of mixing collagen and polycaprolactone, compressing them, and pelletizing them;

[0047] b) A step of freeze-drying and then crushing the pellets manufactured in step a);

[0048] c) a step of dispersing the pulverized material manufactured in step b) in water and then drying it to powder;

[0049] d) a step of compressing the powder obtained in step c) into pellets and manufacturing the same into powder;

[0050] e) a step of spraying ethanol at an amount of 0.1 to 1% by weight of the powder weight onto the surface of the powder prepared in step d) and mixing it with the lysyl-oxidase freeze-dried powder;

[0051] f) a step of mixing the powder prepared in step d) into the dispersion of step e) and drying to obtain a powder; and

[0052] g) A step of preparing a composition by mixing 20 to 30 wt% of the powder prepared in step f), 5 to 10 wt% of tropoelastin powder, 0.1 to 10 wt% of a carrier, 0.1 to 10 wt% of a pain reliever, and the remaining amount of an injectable vehicle.

[0053] Below, the composition of each step is described in more detail.

[0054] The above steps a) to d) are steps for manufacturing biodegradable polymer particles, and the process of dispersing in a dispersion medium, compressing to manufacture pellets, and then grinding is repeated to manufacture the particles so that collagen and polycaprolactone are uniformly present in the powder.

[0055] It is preferable that the mixed powder finally obtained in the above step d) has a particle size of 400 to 500 mesh. If it is manufactured with a finer powder than the above powder, there is a problem that it may clump or be difficult to mix uniformly during the subsequent lysyl-oxidase coating process. If it is manufactured with a powder larger than the above powder, there is a problem that the dispersibility within the composition is poor.

[0056] The above steps e) and f) are steps for coating the surface with lysyl-oxidase, and the step of spraying ethanol in an amount corresponding to 0.1 to 1% of the weight of the mixed powder prepared above and mixing it with the lysyl-oxidase freeze-dried powder is performed. Next, the powder is kept in an oven maintained at a temperature of 30 to 35°C for 5 to 10 minutes, taken out, and allowed to cool naturally. The process of putting it in the oven, taking it out, and allowing it to cool naturally at room temperature is repeated 10 times to obtain a surface-coated powder.

[0057] Finally, in step g), a step of preparing a composition by mixing 20 to 30 wt% of the powder prepared in step f), 5 to 10 wt% of tropoelastin powder, 0.1 to 10 wt% of a carrier, 0.1 to 10 wt% of a pain reliever, and the remaining amount of an injectable vehicle may further include a process of mixing the final mixture, filling the mixture into a container, and sterilizing the mixture to complete the composition into a final product.

[0058]

[0059] Hereinafter, the present invention will be described in more detail based on examples, but this is only an exemplary description for understanding the present invention, and the scope of the present invention is not limited or restricted to the following examples.

[0060]

[0061] [Example 1]

[0062] <Extraction of tropoelastin from porcine adipose tissue>

[0063] Adipose tissue was extracted from the dorsal fat region of 12-month-old female pigs. 1 M NaCl aqueous solution was added to the adipose tissue, stirred at 4°C for 2 days, and then centrifuged at 4°C for 5 minutes to obtain the undissolved residue. The obtained residue was washed with deionized water (DW), and the washed extracellular matrix residue was autoclaved (autoclaved, Company C) at a temperature of 100°C or higher for 45 minutes five times at a pressure of 1.25 kg / cm3 to remove components other than elastin.

[0064] Here, the step of treating with a 0.25 M oxalic acid solution at 90°C for 1 hour was repeated 10 times to hydrolyze the cross-linked elastin component and recover the water-soluble elastin component.

[0065] The supernatant obtained by hydrolysis was dialyzed at 4°C for 2 days to replace the acid solvent with an aqueous solvent. After dialysis, the product was freeze-dried to extract the water-soluble elastin component in the form of a yellow powder with a yield of 100 mg per 100 g of tissue.

[0066] The extracted water-soluble elastin was mixed with 0.01 M sodium acetate solution in a 1:1 (v / v) ratio, incubated at 37°C for 10 minutes, and centrifuged for 20 minutes at the same temperature. The resulting yellow viscous precipitate was recovered with distilled water, and impurities were removed from the recovered precipitate by recrystallization. The supernatant from which impurities had been removed was collected and dialyzed at 4°C for 2 days to remove salt components. After dialysis, the product was freeze-dried to obtain tropoelastin in the form of a yellow powder.

[0067]

[0068] <Manufacturing of Collagen-Polycaprolactone Mixed Powder>

[0069] 10g of collagen and 10g of polycaprolactone were dispersed in a 95% ethanol solution and then placed in an ultrasonic grinder for pulverization. Stirred at 50 rpm, the mixture was distilled under reduced pressure, and compressed into pellets. The resulting pellets were freeze-dried and then pulverized.

[0070] After dispersing the above-mentioned pulverized material in water, the dispersion was stirred at a speed of 10 rpm for 40 minutes, and the water was removed by distillation under reduced pressure at a temperature of 30°C to obtain a powder.

[0071] The obtained powder was compressed again to make pellets, which were then crushed to make 400 mesh powder.

[0072]

[0073] Lysyl-oxidase coating

[0074] Ethanol is sprayed onto 99 wt% of the above mixed powder, 1 wt% of lysyl-oxidase powder is added and mixed, and the mixture is kept in an oven maintained at a temperature of 30 to 35°C for 5 minutes, taken out, and allowed to cool naturally at room temperature. The process of keeping in the oven and then cooling naturally at room temperature is repeated 10 times to obtain a surface-coated powder.

[0075]

[0076] <Composition Manufacturing>

[0077] A composition was prepared by mixing 24 wt% of the surface-coated powder, 6 wt% of tropoelastin powder, 3 wt% of sodium carboxymethylcellulose, 1 wt% of lidocaine, and the remainder of water for injection.

[0078]

[0079] [Comparative Example 1]

[0080] Use commercially available collagen filler (Company A)

[0081]

[0082] [Comparative Example 2]

[0083] Use commercially available collagen filler (Company B)

[0084]

[0085] [Comparative Example 3]

[0086] Use commercially available collagen filler (Company C)

[0087]

[0088] [Comparative Example 4]

[0089] In the above example, when manufacturing a collagen-polycaprolactone mixed powder, the same method was used except that instead of using 10 g of collagen and 10 g of polycaprolactone, 15 g of collagen and 5 g of polycaprolactone were used.

[0090]

[0091] [Comparative Example 5]

[0092] In the above example, when manufacturing a collagen-polycaprolactone mixed powder, the same method was used except that instead of using 10 g of collagen and 10 g of polycaprolactone, 5 g of collagen and 15 g of polycaprolactone were used.

[0093]

[0094] [Comparative Example 6]

[0095] In the above example, the collagen-polycaprolactone mixed powder was manufactured using the same method except that the lysyl-oxidase coating process was not performed.

[0096]

[0097] [Comparative Example 7]

[0098] In the above example, the composition was prepared in the same manner as above, except that instead of using 24 wt% of surface-coated powder, 6 wt% of tropoelastin powder, 3 wt% of sodium carboxymethylcellulose, 1 wt% of lidocaine, and the remainder of water for injection, 20 wt% of surface-coated powder, 10 wt% of tropoelastin powder, 3 wt% of sodium carboxymethylcellulose, 1 wt% of lidocaine, and the remainder of water for injection was used.

[0099]

[0100] [Experimental Method]

[0101] 1. Cytotoxicity test (ISO 10993-5)

[0102] The composition of the example was tested according to ISO 10993-5 (in vitro dissolution and agarose overlay method: Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test).

[0103] The composition of the example was extracted in minimum essential medium (MEM) containing 10% fetal bovine serum (referred to as complete MEM) at 37±1℃ for 24±2 hours, and a negative control (HDPE) and a positive control (natural rubber) were also prepared under the same conditions. Each was added to the maintenance medium of L929 cells grown in a 96-well plate, and after culturing at 37℃ in 5% CO2 for 24 hours, a cytotoxicity evaluation was performed.

[0104] As a result, it was confirmed that the composition of the example did not have cytotoxicity.

[0105]

[0106] 2. Filler Effect Evaluation

[0107] 100 μL (microliter) of the compositions of the control group, examples, and comparative examples 1 to 7 were injected into the dorsal area of ​​SD rats, and the volume change at the injection site was measured for 50 weeks using PRIMOS CR (Canfield). The measurement results are shown in Fig. 1 and Table 3.

[0108]

[0109] (Unit: mm 3 ) Week 4 Week 8 Week 25 Week 50 Control group 000-5 Example 2 202 152 00 182 Comparative example 1 250 183 104 49 Comparative example 2 167 116 8 6 24 Comparative example 3 132 122 9 231 Comparative example 4 232 197 112 96 Comparative example 5 156 17 199 59 Comparative example 6 148 166 152 101 Comparative example 7 144 155 132 122

[0110] 3. Evaluation of treatment stability

[0111] In the experiment of the above 2., the stabilization of the filler was evaluated by two filler treatment specialists (one with 8 years of experience as a dermatologist and the other with 5 years of experience as a dermatologist) for the first 14 days after the procedure.

[0112] Each evaluation was conducted daily from the 7th to the 14th day after the procedure to determine the date on which the filler was deemed to have stably settled, as shown in Table 4 below. If it was not stable by the 14th day, it was marked as 'X'.

[0113] The criteria for evaluating whether the filler is stably positioned are based on expert judgment, but the evaluation focuses on whether the filler is dislodged from the treatment area or does not create sufficient volume.

[0114]

[0115] (Unit: days) Expert A, Expert B Example 10 days, 12 days Comparative Example 1X, X Comparative Example 2X, X Comparative Example 3X, X Comparative Example 4 12 days, 13 days Comparative Example 5X, X Comparative Example 6X, X Comparative Example 7 10 days, 10 days

[0116] Referring to the above experimental results, it can be confirmed that the composition of the example is not cytotoxic. Regarding the filler effect, it can be confirmed that it has a long-lasting effect of maintaining volume compared to commercially available collagen fillers. In particular, it can be confirmed that the composition of the example maintains a similar level of volume up to the 50th week.

[0117] The treatment stability was confirmed to be superior to other samples in Example, Comparative Example 4, and Comparative Example 7, confirming that the filler settles quickly after injection.

[0118] From the above results, it can be confirmed that the composition according to the present invention, such as the example, has excellent filler volume and high treatment stability.

Claims

1. A composition comprising 20 to 30 wt% of biodegradable polymer particles, 5 to 8 wt% of tropoelastin, 0.1 to 10 wt% of a carrier, 0.1 to 10 wt% of a pain reliever, and the remainder of an injectable vehicle, The above biodegradable polymer particles contain collagen and polycaprolactone, A composition wherein the biodegradable polymer particles are coated with lysyl-oxidase.

2. In paragraph 1, The above biodegradable polymer particles are a composition that is a mixed powder containing collagen and polycaprolactone in a weight ratio of 1:0.5 to 1.

3. In paragraph 1, A composition wherein the above lysyl-oxidase is contained in an amount of 0.1 to 3 wt% based on the total weight of the biodegradable polymer particles.

4. A method for manufacturing a composition according to paragraph 1, a) A step of mixing collagen and polycaprolactone, compressing them, and pelletizing them; b) A step of freeze-drying and then crushing the pellets manufactured in step a); c) a step of dispersing the pulverized material manufactured in step b) in water and then drying it to powder; d) a step of compressing the powder obtained in step c) into pellets and manufacturing the same into powder; e) a step of spraying ethanol corresponding to 0.1 to 1% of the powder weight onto the surface of the powder manufactured in step d) and mixing it with the lysyl-oxidase freeze-dried powder; f) a step of mixing the powder prepared in step d) into the dispersion of step e) and drying to obtain a powder; and g) a step of preparing a composition by mixing 20 to 30 wt% of the powder prepared in step f), 5 to 10 wt% of tropoelastin powder, 0.1 to 10 wt% of a carrier, 0.1 to 10 wt% of a pain reliever, and the remaining amount of an injectable vehicle; A method for producing a composition comprising:

5. In paragraph 4, A method for producing a composition, characterized in that the powder of the above step d) is a powder of 400 to 500 mesh.