Method for producing filler containing DNA fraction and filler thus obtained

Abstract

FIELD: fillers.SUBSTANCE: method for producing a filler. A method for producing a filler comprises: a) mixing polynucleotides having a molecular weight of 1800 to 2300 kDa with a solvent to obtain a solution of polynucleotides and then mixing said solution, wherein the polynucleotides are uniformly distributed in the solvent; b) removing bacteria from the resulting polynucleotide solution using a sterile filter; c) mixing non-crosslinked hyaluronic acid having a molecular weight of 100 to 5000 kDa and a pH modifier with a bacteria-free polynucleotide solution, wherein the polynucleotide is contained in a concentration of 1 to 1.5 wt. / vol.% and non-crosslinked hyaluronic acid is contained in a concentration of 0.1 to 1 wt. / vol.% relative to the total volume of the final filler, and is characterized in that steps a)–c) are performed sequentially, wherein the filler has a complex viscosity in the range of 0.3 to 3 Pa⋅ with and is suitable for reducing mild wrinkles according to the Wrinkle Severity Rating Scale (WSRS) or crow's feet; wherein step c) of mixing hyaluronic acid and a pH modifier comprises: c-1) mixing non-cross-linked hyaluronic acid with the bacteria-free polynucleotide solution obtained in step b) to obtain a mixture; c-2) mixing a pH modifier with the obtained mixture.EFFECT: reduction in the possibility of external contamination of the filler during production and a reduction in mild wrinkles according to the Wrinkle Severity Rating Scale (WSRS) or crow's feet.8 cl, 2 dwg, 7 tbl, 8 ex

Description

Field of technology

[0001] The present invention relates to a method for producing a filler containing a DNA fraction and to a filler obtained thereby.Background of the invention

[0002] The structure of soft tissues in the human body is maintained by proteins such as collagen, elastin, and the extracellular matrix, which contains glycosaminoglycans. Various factors cause soft tissue defects. In these cases, the original shape of the defective soft tissue is restored or corrected through the surgical or non-surgical introduction of biological tissue or synthetic chemical compounds.

[0003] Filler is injected for restoration or correction using a medical device that utilizes the principle of autonomously maintaining its volume through physical restoration via subcutaneous injection for temporary improvement, volume restoration, etc. around wrinkles in adults. Typically, the filler injection device consists of a syringe filled with an injectable solution and connected to an injection needle, which are used together.

[0004] Currently available filler products are mainly based on hyaluronic acid. To enhance the chemical and structural stability of hyaluronic acid and maintain its physicochemical properties in vivo, a method of stabilizing hyaluronic acid with the addition of a cross-linking agent has long been used (International Patent Publication No. WO / 2019 / 130360). However, cross-linking agent components degraded in vivo can be recognized as foreign substances, leading to inflammatory reactions. As a result, a step of removing the cross-linking agent by washing is necessary during the process, and testing for residual cross-linking agent is unavoidable, which poses a challenge to the effectiveness of the production process.

[0005] Furthermore, for hyaluronic acid-based fillers, syringes filled with the gel are generally sterilized using high-temperature sterilization methods such as autoclaving. However, this sterilization process can cause a decrease in viscoelasticity. If the hyaluronic acid content is high or cross-linked hyaluronic acid is used to correct this problem, patients may experience severe pain during treatment, which reduces patient satisfaction and makes the treatment uncomfortable for practitioners.

[0006] On the other hand, to compensate for the functional aspects of hyaluronic acid-based fillers, research is being conducted on fillers consisting of polynucleotides and the like. However, during the sterilization process required during production, there is a problem: the functionality of the polynucleotides mixed in the actual production process is insufficient compared to their quantity due to polynucleotide denaturation. Furthermore, the amount of polynucleotides causes pain during treatment, which reduces patient satisfaction. Description of the invention. Technical problem

[0007] The inventors of the present invention seek to solve the problems of the prior art. As a result, when using a specific production method for producing a filler, the advantages of the process enable increased production efficiency and the production of a viscoelastic filler despite the low amounts of its constituent components. Furthermore, the inventors have proven that the filler obtained by this method exhibits superior effects in improving skin thickness, reducing skin wrinkles, collagen formation capacity, and so on, compared with conventional products, while causing low pain during injection, ensuring high satisfaction of patients and practitioners. As a result, the inventors of the present invention have completed the present description.

[0008] The present description provides a method for producing a filler, which comprises: a) mixing a DNA fraction with a solvent to obtain a solution; b) removing bacteria from the resulting solution using a sterile filter; and c) mixing hyaluronic acid and / or a pH modifier with the bacteria-free solution.

[0009] The present description provides a filler obtained by the production method described above.

[0010] This description provides a syringe pre-filled with the filler described above.Technical solution

[0011] The present invention will now be described in detail. All combinations of the various elements described herein are within the scope of the present invention. Furthermore, the scope of the present invention is not limited by the detailed description below.

[0012]

[0013] The present description provides a method for producing a filler, which includes: a) a step of mixing a DNA fraction with a solvent to obtain a solution; b) a step of removing bacteria from the obtained solution using a sterile filter; and c) a step of mixing hyaluronic acid and / or a pH modifier with the bacteria-free solution.

[0014] Each step of the production method will be described in detail below.

[0015] In the present invention, the mixing step (a) is a step of mixing the DNA fraction with a solvent to obtain a solution in which the DNA fraction is dissolved.

[0016] In the present invention, "DNA fraction" means a chain-type polymer formed by covalently linking the third carbon atom of a pentose sugar constituting a nucleotide to a phosphoric acid group of another nucleotide. A nucleotide is a molecule to which one phosphoric acid, one sugar, and one base are independently linked, and is a unit constituting a nucleic acid comprising adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U).

[0017] In the present invention, the DNA fraction can promote the production of extracellular matrix (ECM), which is the intercellular matrix, and can activate the healing ability of the skin in the human body. Therefore, the skin can restore its own deteriorated and weakened regenerative capacity, and the DNA fraction can therefore serve to improve the functionality of the skin itself.

[0018] As used herein, the DNA fraction may include the DNA fraction itself, its salt, or a combination of both. Furthermore, the DNA fraction may be synthesized directly or purchased for use from a commercially available product.

[0019] In the present invention, the DNA fraction may be a polynucleotide (PN), a polydeoxyribonucleotide (PDRN), or a mixture thereof, and comprises fragments thereof. According to embodiments of the present invention, the DNA fraction may be a polynucleotide, but is not limited thereto.

[0020] In the present invention, the polynucleotide salt may be, for example, a sodium polynucleotide, a potassium polynucleotide, a calcium polynucleotide, a magnesium polynucleotide, a zinc polynucleotide, etc. Furthermore, the polydeoxyribonucleotide salt may be, for example, a sodium polydeoxyribonucleotide, a potassium polydeoxyribonucleotide, a calcium polydeoxyribonucleotide, a magnesium polydeoxyribonucleotide, a zinc polydeoxyribonucleotide, etc. However, the polynucleotide salt and the polydeoxyribonucleotide salt are not limited to them. According to embodiments of the present invention, the polynucleotide salt may be, but is not limited to, a sodium polynucleotide.

[0021] In the present description, the DNA fraction may have a molecular weight of 1800 kDa to 6000 kDa, preferably in the range of 1800 kDa to 2300 kDa, and more preferably in the range of 2000 kDa to 2300 kDa. However, the molecular weight of the DNA fraction is not limited to this.

[0022] When the molecular weight of the DNA fraction is less than the range specified above, the viscoelasticity of the finished filler may be extremely low, and the filler may not function as a physical support. When the molecular weight of the DNA fraction exceeds the range specified above, the molecular weight itself may cause problems such as reduction in extraction speed, filter clogging, and so on when using a sterile filter, thereby reducing the extraction efficiency.

[0023] In the present invention, the DNA fraction can be mixed to be included at a concentration of 0.5% (w / v) to 2% (w / v) and preferably 1% (w / v) to 1.5% (w / v) relative to the total volume of the final filler, but is not limited thereto.

[0024] When mixing DNA mixtures below the above range, the effects of the DNA fraction, such as skin regeneration, increased skin density, and stimulation of collagen production, may be insignificant. Furthermore, the viscoelasticity required for physical support may be insufficient. When including DNA mixtures in amounts exceeding the above range, the increased viscoelasticity of the prepared filler may cause pain during injection into the skin, resulting in low patient satisfaction and discomfort for practitioners.

[0025] Any solvent capable of dissolving the DNA fraction without causing a decrease in the efficacy of the preparation when using a sterile filter can be used as the solvent in step (a) of mixing according to the present invention. However, according to embodiments of the present invention, the solvent is preferably water for injection.

[0026] In the experimental example to be described later, it was specifically confirmed that by using water for injection as a solvent, the bacteria removal rate in the removal step (b) in which a sterile filter is used was fast, and clogging of the sterile filter was prevented, thereby greatly improving the production efficiency.

[0027] In the present invention, the mixing step (a) may further include stirring the solution after mixing the DNA fraction with the solvent so that the DNA fraction can be uniformly (or homogeneously) distributed in the solvent.

[0028] Stirring can increase the bacterial removal rate using a sterile filter in step (b). Furthermore, filter clogging caused by DNA fraction aggregation can be prevented, resulting in increased recovery efficiency.

[0029] In the present description, the removing step (b) is a step of removing bacteria from the resulting solution in which the DNA fraction is dissolved, using a sterile filter.

[0030] In this specification, the "sterile filter" used during the bioprocess or the filling and formulation processes means a filter mainly made of PES, PVDF, nylon or PTFE as a raw material.

[0031] In the present invention, the sterile filter may have a pore size suitable for removing harmful bacteria. According to embodiments of the present invention, the sterile filter may have a pore size of 0.2 μm, but is not limited thereto.

[0032] In the present invention, the order of the removal step (b), in which a sterile filter is applied, is important for the integrity of the resulting filler and the production efficiency. Therefore, for the purposes of the present invention, the removal step (b) must be performed before mixing the hyaluronic acid and / or pH modifier. Instead of applying each sterile filter to the solution in which the DNA fraction is dissolved and the solution in which the hyaluronic acid and / or pH modifier are dissolved, and then mixing the solutions, mixing the hyaluronic acid and / or pH modifier after applying the sterile filter to the solution in which the DNA fraction is dissolved is advantageous in terms of filtration speed.

[0033] In the present invention, the mixing step (c) is a step of mixing hyaluronic acid and / or a pH modifier with a bacteria-free solution.

[0034] In this description, "hyaluronic acid" is a biopolymer material, a linear polysaccharide composed of glucuronic acid and acetylglucosamine, which is one of the glycosaminoglycans present in the extracellular matrix (ECM), synovial fluid of joints, and cartilage scaffolds. Hyaluronic acid has viscoelastic properties and can thus be used as a skin additive, etc. In addition, hyaluronic acid is a material with excellent biocompatibility and has no immune problems when used in vivo.

[0035] In the present invention, the term "hyaluronic acid" encompasses all hyaluronic acid, its salts, or combinations thereof. Furthermore, hyaluronic acid can be synthesized directly or purchased for use from a commercially available product.

[0036] The hyaluronic acid salt may be, for example, sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, or cobalt hyaluronate, but is not limited thereto. According to embodiments of the present invention, the hyaluronic acid salt may be sodium hyaluronate, but is not limited thereto.

[0037] In the present description, hyaluronic acid may have a molecular weight in the range of 100 kDa to 5000 kDa, preferably in the range of 500 kDa to 1200 kDa, and more preferably in the range of 700 kDa to 1200 kDa.

[0038] When the molecular weight of hyaluronic acid is less than the above range, the viscosity of the final filler may be extremely low, and the filler may not function properly as a physical support. When the molecular weight of hyaluronic acid exceeds the above range, the viscosity of the final filler may be extremely high, which is unsuitable for the intended use of the present invention, such as reducing mild wrinkles, and may cause pain upon injection into the skin.

[0039] In the present invention, the hyaluronic acid may be non-crosslinked (or not crosslinked).

[0040] For existing fillers, a cross-linking agent is used to prevent bioenzyme-induced degradation of hyaluronic acid and improve elasticity. Cross-linking agents are mainly produced using chemical cross-linking agents such as 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), and the like. Due to the side effects and safety concerns of such chemical cross-linking agents, a washing step is required during the production process. Furthermore, there is the problem of having to test for residual amounts of cross-linking agent, which reduces the effectiveness of the production method.

[0041] The filler of the present invention is produced using non-cross-linked hyaluronic acid, so a washing step or a step for checking the residual amount of a cross-linking agent, which is not necessarily required when using a cross-linking agent, is not required. Therefore, the efficiency of the production method can be maximized. Furthermore, despite using non-cross-linked hyaluronic acid, the filler produced by the production method of the present invention can ensure an in vivo degradation rate that is no less than that of existing products. As a result, the filler of the present invention can have a longer duration of tissue repair and provide better collagen formation than existing products, enabling its effective use as an injectable filler in vivo.

[0042] In the present invention, hyaluronic acid can be mixed to be included at a concentration of 0.1% (w / v) to 1% (w / v) relative to the total volume of the final filler.

[0043] When the hyaluronic acid concentration is below the above range, the viscosity of the finished filler may be extremely low, and the filler may not function as a physical support. If the hyaluronic acid concentration exceeds the above range, the viscosity of the finished filler may be extremely high, causing pain upon injection into the skin. Furthermore, the filler may be unsuitable for the intended use of the present invention, such as reducing fine wrinkles.

[0044] In the present invention, “pH modifier” means a material used to regulate or stabilize pH.

[0045] The pH modifier may be, for example, sodium chloride, sodium monohydrogen phosphate, sodium dihydrogen phosphate, or a mixture thereof, but is not limited to them.

[0046] In the present invention, the pH modifier may comprise a concentrated pH modifier dissolved in a solvent such as water for injection, etc. That is, in the present invention, the pH modifier itself may be mixed with the bacteria-free solution in the removal step (b). Alternatively, the pH modifier may be dissolved in a solvent such as water for injection, etc., concentrated, and then mixed with the bacteria-free solution in the removal step (b).

[0047] In the mixing step (c) of the present invention, at least one agent selected from hyaluronic acid and a pH modifier can be mixed. For example, in the mixing step (c), hyaluronic acid or a pH modifier can be mixed, or both hyaluronic acid and a pH modifier can be mixed.

[0048] When mixing both hyaluronic acid and a pH modifier in the mixing step (c) of the present invention, the hyaluronic acid and the pH modifier are mixed in stages. Alternatively, the hyaluronic acid and the pH modifier may be first mixed and then mixed with the bacteria-free solution in the removing step (b).

[0049] According to embodiments of the present invention, in the case of mixing hyaluronic acid and a pH modifier in a stepwise manner, the mixing step (c) may include: c-1) a step of mixing hyaluronic acid with the bacteria-purified solution obtained in the removing step (b) to obtain a mixture; and c-2) a step of mixing a pH modifier in the obtained mixture.

[0050] In the case of mixing the pH modifier in the mixing step (c) of the present invention, the pH modifier can be mixed in an amount that allows the obtained filler to have a pH in the range of 6.0 to 8.0.

[0051] In the present invention, since steps (a)-(c) are performed sequentially, a drying step is not required, unlike the conventional method in which the bacteria-free solution is dried and then redissolved to obtain a fraction. As a result, the efficiency of the production method can be maximized while maintaining sterile conditions by minimizing the possibility of external contamination due to the reduced number of steps.

[0052] The production method of the present invention may further include mixing an anesthetic agent, wherein the anesthetic agent is preferably mixed after the mixing step (c).

[0053] In the present description, the term "anesthetic agent" means a component used to relieve itching and pain caused by skin inflammation. Examples of the anesthetic agent may include procaine, chloroprocaine, tetracaine, prilocaine, lidocaine, mepivacaine, bupivacaine, ropivacaine, etidocaine, lidocaine hydrochloride, benzocaine, oxetacaine, and the like. According to one embodiment of the present invention, the anesthetic agent is preferably lidocaine or a salt thereof (e.g., lidocaine hydrochloride monohydrate), but is not limited thereto.

[0054] In the present description, lidocaine used for local anesthesia can also have the effect of improving the elasticity of fibrous tissue in combination with a polynucleotide and / or hyaluronic acid.

[0055] In the present invention, the anesthetic agent can be mixed to include it at a concentration of 0.2% (w / v) to 0.4% (w / v) relative to the total volume of the final filler. According to embodiments of the present invention, the anesthetic agent can be mixed at a concentration of 0.3% (w / v). When the anesthetic agent is included below the above range, the anesthetic effect may be low. When the anesthetic agent is added in doses exceeding the above range, the anesthetic effect may last a long time, and therefore patients may have difficulty quickly returning to daily life through a quick and simple procedure. In addition, toxicity may cause side effects.

[0056] According to another aspect of the present invention, there is provided a filler obtained by the production method of the present invention.

[0057] In the present invention, the filler may contain a DNA fraction at a concentration of 0.5% (w / v) to 2% (w / v) and hyaluronic acid at a concentration of 0.1% (w / v) to 1% (w / v) relative to the total volume of the final filler.

[0058] If the filler obtained by the production method of the present invention contains a DNA fraction and hyaluronic acid in concentrations below the above-mentioned ranges, the viscosity of the finished filler may be extremely low, and the filler may not function as a physical support. If the filler obtained by the production method of the present invention contains a DNA fraction and hyaluronic acid in concentrations exceeding the above-mentioned ranges, the viscosity of the finished filler may be extremely high, causing pain upon injection into the skin. Furthermore, the filler may be unsuitable for the intended use of the present invention, for example, for reducing fine wrinkles.

[0059] In the present invention, the filler may have a complex viscosity of 0.3 Pa⋅s to 3 Pa⋅s, preferably 1 Pa⋅s to 3.0 Pa⋅s, at a temperature of 25°C when measured with a rheometer. According to embodiments of the present invention, the complex viscosity may be in the range of 1 Pa⋅s to 1.5 Pa⋅s, but is not limited thereto.

[0060] In the present invention, the filler may have a storage modulus G' of 5 Pa or higher at a temperature of 25°C when measured by a rheometer.

[0061] In the present invention, “complex viscosity (η*)” is the result of a complex interaction between viscosity and elasticity, and means a mathematical expression represented by the sum of real and imaginary numbers.

[0062] In the present invention, the "storage modulus" related to elasticity means a measure of stored energy, indicating that the higher the storage modulus, the greater the force of returning to the original state.

[0063] Despite the low amounts of DNA fraction and hyaluronic acid, the filler obtained by the production method of the present invention can obtain a complex viscosity and storage modulus for the intended use of the present invention.

[0064] In this description, the filler can be used for tissue restoration. In particular, the filler can be used to reduce mild wrinkles or contour the face, more specifically, to reduce mild wrinkles.

[0065] Wrinkles, assessed in clinical studies by the Wrinkle Severity Rating Scale (WSRS), can be divided into 5 grades: none, mild, moderate, severe and severe.

[0066] Traditional botulinum toxin has therapeutic limitations in the treatment of mild wrinkles. Furthermore, due to the viscosity of existing filler products, their direct injection into the dermal layer, which plays a key role in collagen production, is limited. Therefore, achieving the appropriate complex viscosity and storage modulus is important for use as injectable dermal fillers. In particular, fillers used for wrinkle smoothing must have an appropriate complex viscosity depending on the severity of the wrinkles, from mild to severe.

[0067] The filler obtained by the production method of the present invention exhibits a complex viscosity in the range of 0.3 Pa⋅s to 3.0 Pa⋅s and can thus be effectively used for reducing mild wrinkles. Furthermore, compared with fillers injected into the entire face, the filler of the present invention exhibits low injection pressure, causing little pain even when applied to thin skin such as the eye area. According to the experimental examples described below, it was confirmed that, despite the low amount of DNA fraction compared with conventionally available filler products, the filler of the present invention can provide superior skin regeneration, collagen formation, etc., as well as a natural appearance compared with those of existing filler products.

[0068] The present invention uses a filler to reduce crow's feet. The skin around the eyes is extremely thin, thus sensitive to microstimulation and is an area prone to wrinkle formation. Typically, wrinkles around the eyes include crow's feet wrinkles, which form at the outer corners of the eyes, and wrinkles under the eyes. The filler of the present invention has a relatively low viscosity and can thus be effectively applied to mild wrinkles formed on thin skin tissue, such as the eye area.

[0069] The filler of the present invention can be administered to a subject in an effective amount by subcutaneous injection, intradermal injection, etc.

[0070] In this description, "effective amount" means an amount of filler sufficient to exert beneficial cosmetic or therapeutic effects from filler injection. The effective amount may be determined by the operator based on factors such as the subject's age, gender, sensitivity, injection time, wrinkle condition, and other factors well known in the medical field.

[0071] Furthermore, filler injections can be performed, for example, once, several times and / or over one or more extended periods of time.

[0072] In the present invention, "subject" means an individual or patient in need of treatment for a particular condition or disease. The subject may be a mammal and is preferably a human.

[0073] In the present invention, the filler may be a mesofiller administered through mesotherapy.

[0074] In the present invention, "mesotherapy" in which the filler is directly injected into the target area means an injection therapy designed to minimize side effects, to improve the effectiveness and prolong the duration by injecting the filler in an extremely small amount into the middle layer of the skin (dermal layer of the skin) using a microsyringe.

[0075] In the present invention, "mesofiller", a term formed by merging the words mesotherapy and filler, is used for the purpose of smoothing wrinkles by injecting filler between the dermal layer of the skin and fibrous tissue, and can exhibit the effects of skin regeneration, improving elasticity, and reducing fine lines.

[0076] According to a further aspect of the present invention, there is provided a syringe pre-filled with a filler obtained by the production method of the present invention.

[0077] Unless otherwise stated, the detailed description regarding the filler and pre-filled syringe in this document is the same as that mentioned in the method for producing the filler. Therefore, a separate description will be omitted to avoid unnecessary complexity in the description of the invention. Advantages

[0078] In the method for producing a filler according to the present invention, the filler thus obtained can provide viscoelasticity necessary for physical support.

[0079] Furthermore, the method for producing a filler according to the present invention can prevent changes in the DNA fraction content during the bacterial removal step. Thus, despite the low DNA fraction content compared to existing products, the resulting filler has excellent effects in improving skin thickness, reducing wrinkles, and enhancing collagen formation capacity.

[0080] In addition, the method for producing the filler of the present invention uses a low content of DNA fraction, so that the resulting filler causes less pain compared with currently available fillers and thus ensures patient satisfaction while providing comfort to practitioners.

[0081] Furthermore, the method for producing the filler of the present invention uses non-cross-linked hyaluronic acid, so steps such as washing to remove the cross-linking agent, checking the residual amount of the cross-linking agent, etc. are unnecessary. As a result, the efficiency of the production method is excellent, and the stability of the resulting filler is excellent, since no inflammatory response due to the use of the cross-linking agent was detected. Description of graphic materials

[0082] Fig. 1 shows a graph comparing the in vivo degradation rates of the filler sample from Example 3 and the product of other manufacturers from Experimental Example 7.

[0083] Fig. 2 shows the images of the filler samples of Example 2 and Comparative Examples 4 to 6 in Experimental Example 8, where Fig. 2(a) shows the image immediately after production, and Fig. 2(b) shows the image after keeping the filler samples at a temperature of 35°C for 2 weeks. Optimal embodiment of the invention

[0084] Next, the present invention will be described in detail with reference to embodiments. However, the embodiments of the present invention are disclosed for illustrative purposes only and should not be construed as limiting the present invention. Furthermore, unless otherwise specified, all terms used herein have the same meaning as commonly understood in the technical field to which the present invention pertains.

[0085]

[0086] Example 1 and Comparative Examples 1-3

[0087] 6.25g of sodium polynucleotide (manufacturer: HTL, molecular weight: 1800kDa to 2300kDa) were dissolved in water for injection or phosphate-buffered saline to obtain each sodium polynucleotide-containing solution at a concentration of 1.25% (w / v). Each solution had a volume of 500mL and a pH ranging from 6.0 to 8.0. Afterwards, each solution was either untreated or processed by wet sterilization (at 121°C for 20 minutes) or a sterile filter (0.2μm, PES) to obtain fillers.

[0088] The solvents and processing methods used in each of the Examples and Comparative Examples are shown in Table 1 below.

[0089]

[0090] [Table 1] Solvent Processing method Example 1 Water for injection Sterile filter Comparative example 1 Water for injection Unprocessed Comparative example 2 Water for injection Wet sterilization Comparative example 3 Phosphate buffered saline Sterile filter

[0091]

[0092] Experimental Example 1. Analysis of Rheological Properties

[0093] A rheometer (KNX2210, MALVERN, UK) was used to analyze the rheological properties of fillers obtained using solvents and processing methods. The results are presented in Table 2 below.

[0094] Tissue repair filler functions as a physical support after injection into the skin, so the storage modulus (G') and complex viscosity (η*) values ​​are regarded as the most important factors.

[0095]

[0096] [Table 2] G* (Pa) G' (Pa) G” (Pa) η* (Pa⋅s) δ (˚) Example 1 3,39305 1,3815 3,099 0,54005 65,975 Comparative example 2 0,08717 0,086865 0,0071915 0,01387 -4,805 Comparative example 3 7,8095 6,3585 4,5345 1,243 35,49

[0097]

[0098] As shown in Table 2 above, the filler of Comparative Example 2 obtained by the wet sterilization method showed a significantly lower complex viscosity than the filler of Example 1 obtained by the sterile filter in the production method, which confirmed that the filler of Comparative Example 2 was not suitable for use as a tissue repair filler required to function as a physical support after injection into the skin.

[0099] Furthermore, in the case of Comparative Example 3, which used phosphate-buffered saline as the solvent, the storage modulus and complex viscosity were extremely high. In particular, the filtration rate during bacterial removal was extremely slow, and filter clogging occurred quickly. For this reason, the filler of Comparative Example 3 was unsuitable for use in the method for producing a filler of the present invention.

[0100] [1091Experimental Example 2. Measurement of sodium polynucleotide content

[0102] To confirm changes in sodium polynucleotide content in accordance with the processing methods performed on the obtained fillers, HPLC was used to measure its content. The results are presented below in Table 3.

[0103]

[0104] [Table 3] Sodium polynucleotide content (%) Example 1 107,72 Comparative example 1 106,73 Comparative example 2 94,87

[0105]

[0106] As shown in Table 3 above, the filler of Example 1, obtained using a sterile filter in the production method, demonstrated a sodium polynucleotide content that was not inferior to that of the untreated group, confirming that the method of using a sterile filter had virtually no effect on the sodium polynucleotide content. On the other hand, in Comparative Example 2, which employed a wet sterilization method, a significant reduction in the sodium polynucleotide content was confirmed.

[0107]

[0108] Example 2 and Comparative Examples 4-6

[0109] (1) Obtaining Example 2

[0110] 6.25g sodium polynucleotide (manufacturer: HTL, molecular weight: 1800kDa to 2300kDa) was dissolved in water for injection, and then the solution was pre-treated with a sterile filter (0.2μm, PES).

[0111] Non-cross-linked sodium hyaluronate (molecular weight of 700 kDa to 1200 kDa) was then completely dissolved in the solution, and then concentrated phosphate-saline solution was added to make the resulting solution have a volume of 500 ml and a pH in the range of 6.0 to 8.0. As a result, the filler of Example 2 was obtained, containing sodium polynucleotide and non-cross-linked sodium hyaluronate at final concentrations of 10 mg / ml and 3 mg / ml, respectively.

[0112]

[0113] (2) Obtaining comparative examples 4-6

[0114] Sodium polynucleotide (manufacturer: HTL, molecular weight: 1800 kDa to 2300 kDa) and non-cross-linked sodium hyaluronate (molecular weight: 700 kDa to 1200 kDa) were mixed with phosphate-buffered saline and dissolved to obtain the filler of Comparative Example 4.

[0115] In addition, sodium polynucleotide (manufacturer: HTL, molecular weight: 1800 kDa to 2300 kDa) and non-cross-linked sodium hyaluronate (molecular weight: 700 kDa to 1200 kDa) were mixed with phosphate-buffered saline and dissolved. Then, using wet sterilization or a sterile filter (0.2 μm, PES) to remove bacteria, the filler of Comparative Example 5 (wet sterilization) and the filler of Comparative Example 6 (bacteria-free) were independently obtained, respectively.

[0116] Each of the fillers in Comparative Examples 4-6 contained sodium polynucleotide and non-cross-linked sodium hyaluronate at final concentrations of 10 mg / mL and 3 mg / mL, respectively.

[0117]

[0118] Experimental Example 3. Analysis of Rheological Properties

[0119] A rheometer (KNX2210, MALVERN, UK) was used to analyze the rheological properties of the fillers according to their production methods. The results are presented in Table 4.

[0120]

[0121] [Table 4] G* (Pa) G' (Pa) G” (Pa) η* (Pa⋅s) δ (˚) Example 2 8,094 5,712 5,734 1,288 45,11 Comparative example 5 0,1084 0,07653 0,07679 0,01725 45,1 Comparative example 6 6,682 4,365 5,0585 1,0635 49,22

[0122]

[0123] As shown in Table 4 above, the viscoelastic properties of the fillers of Comparative Examples 5 and 6, obtained by wet sterilization and a sterile filter, respectively, deteriorated after the sodium polynucleotide and non-cross-linked sodium hyaluronate were simultaneously dissolved, compared with the filler of Example 2, which was obtained stepwise after dissolving the sodium polynucleotide in water for injection. In particular, the filler of Comparative Example 5, obtained by sterilization, exhibited significantly low viscosity, confirming that the filler of Comparative Example 5 was unsuitable for use as a tissue repair filler required to function as a physical support after injection into the skin.

[0124] In addition, in the case of Comparative Example 6, the filtration speed was significantly slower in the bacteria removal process, and the filter clogging occurred quickly, which confirmed that there was a problem with the efficiency of the production process.

[0125]

[0126] Experimental Example 4. Confirmation of sodium polynucleotide content

[0127] To confirm changes in the content of the main component in accordance with the production methods of the finished fillers, HPLC was used to measure the sodium polynucleotide content. The results are presented below in Table 5.

[0128]

[0129] [Table 5] Sodium polynucleotide content (%) Example 2 106,92 Comparative example 4 107,02 Comparative example 5 101,23 Comparative example 6 104,04

[0130]

[0131] As shown in Table 5 below, the filler of Example 2 obtained by the production method of the present invention exhibited a sodium polynucleotide content that was not inferior to that of Comparative Example 4, which did not separately remove bacteria after simple mixing, and had virtually no effect on the sodium polynucleotide content. On the other hand, in the case of Comparative Examples 5 and 6, which included sterilization and bacterial removal, respectively, a decrease in the sodium polynucleotide content was confirmed after mixing the sodium polynucleotide and non-cross-linked sodium hyaluronate.

[0132] This means that when produced, the content of sodium polynucleotide, the active ingredient contained in the final filler, may vary depending on the technological process.

[0133]

[0134] Experimental Example 5. Measurement of injection pressure

[0135] Whether the filler obtained by the production method of the present invention exhibited high adaptability to patients when used for injection was tested by analyzing the injection pressure (injection force). The results are presented in Table 6 below.

[0136] The injection pressure was measured by a tensile strength tester (JSV H1000, JISC, Japan) using a 33-G injection needle at a testing speed of 5 mm / min and 30 mm / min.

[0137]

[0138] [Table 6] 5 mm / min 30 mm / min Example 2 4.19 N 10.38 N

[0139]

[0140] As shown in Table 6 above, the filler obtained by the production method of the present invention was confirmed to have low injection pressure. This confirms that the filler causes less pain during injection and improves practitioner comfort.

[0141]

[0142] Example 3

[0143] 6.25g of sodium polynucleotide (manufacturer: HTL, molecular weight: 1800kDa to 2300kDa) was completely dissolved in water for injection and then filtered to remove bacteria. Then, non-cross-linked sodium hyaluronate (molecular weight: 700kDa to 1200kDa) and concentrated phosphate-saline solution were added to it so that the resulting solution had a volume of 500mL and a pH in the range of 6.0 to 8.0. As a result, the filler of Example 3 was obtained, containing sodium polynucleotide and non-cross-linked sodium hyaluronate at final concentrations of 10mg / mL and 3mg / mL, respectively.

[0144]

[0145] Experimental Example 6. Evaluation of Wrinkle Reduction and Collagen Production

[0146] In Experimental Example 6, a comparative evaluation of wrinkle reduction and collagen production was conducted through animal efficacy evaluation.

[0147] Specifically, the resulting filler from Example 3 was injected into SKH-1 hairless mice. The injection site volume was then measured for one month during histopathological evaluation. A commercially available polynucleotide filler (containing 2% sodium polynucleotide) served as a control group.

[0148] The results are shown below in Table 7.

[0149]

[0150] [Table 7] Total skin thickness* (µm) Epidermal thickness (µm) Epidermal microfolds (microfold / mm) Collagen fibers (% / mm 2 ) Example 3 4,93 24,89 15,6 10,80 Product from other manufacturers 4,76 21,55 17,55 6,81

[0151] (*The total leather thickness is described on a scale of 1 / 100)

[0152]

[0153] As shown in Table 7 above, it was confirmed that the filler obtained by the production method of the present invention has an excellent effect of improving skin thickness and reducing skin wrinkles, as well as collagen formation, compared with the products of other manufacturers.

[0154] On the other hand, no residual material was detected in the filler of Example 3, obtained by the production method of the present invention, after injection. Furthermore, foreign body reactions and adverse reactions caused by the injection of Example 3 were almost nonexistent during the test period (1 month), confirming that the stability was also good.

[0155]

[0156] Experimental Example 7. Evaluation of Degradation Rate and Volume in Vivo

[0157] In Experimental Example 7, a comparative evaluation of the in vivo degradation rate of the filler obtained by the production method of the present invention and the existing filler was carried out.

[0158] Specifically, the resulting filler from Example 3 was injected into SKH-1 hairless mice, and changes in the volume of the injected solution were observed over 30 hours. A commercially available polynucleotide filler (containing 2% sodium polynucleotide) served as a control group. The results are shown in Fig. 1.

[0159] As confirmed in Fig. 1, the time to achieve complete degradation was predicted using a trend line after 30 hours of observation. As a result, the predicted time to achieve complete degradation was approximately 34 hours in Example 3. Although the polynucleotide concentration in the filler of Example 3 was lower than that of another currently marketed product, which had a predicted time to achieve complete degradation of approximately 37 hours, there was no significant difference in terms of the in vivo degradation rate.

[0160] Furthermore, when filler volume changes were observed macroscopically using a folliscope and PRIMOS, the volume of fillers from other manufacturers was small while maintaining the injected shape. On the other hand, with the filler from Example 3, the volume was small while the injected shape naturally expanded laterally, confirming that the filler from Example 3 possessed aesthetic stability suitable for use as an injectable dermal filler.

[0161]

[0162] Experimental Example 8. Evaluation of the effect depending on the order of the mixing step of the polynucleotide and non-cross-linked hyaluronic acid and the sterilization step.

[0163] In Experimental Example 8, a comparative evaluation of the effect was carried out depending on the order of the step of mixing the polynucleotide and non-cross-linked hyaluronic acid and the sterilization step using the filler of Example 2 and the fillers of Comparative Examples 4 to 6.

[0164] Specifically, whether the filler of Example 2 and the filler samples of Comparative Examples 4-6 degraded or not was evaluated by comparing immediately after their production and keeping each of the filler samples at a temperature of 35°C for 2 weeks. The results are shown in Fig. 2.

[0165] As confirmed in Fig. 2(b), the filler sample from Comparative Example 4 degraded after two weeks. In addition, the filler samples from Example 2 and Comparative Examples 5 and 6 did not degrade.

[0166] However, when comparing the production method, there was a delay in filtering the filler sample of Comparative Example 6 obtained by filtering the polynucleotide solution and non-cross-linked hyaluronic acid to remove bacteria, which took longer to obtain than the filler of Example 2. In the case of the filler sample of Comparative Example 5, wet sterilization had to be carried out at a temperature of 121°C for 20 minutes, which took longer to obtain than the filler of Example 2.

[0167] Thus, it was confirmed that the filler sample of Example 2, obtained by rapidly filtering the polynucleotide solution to remove bacteria and then mixing the solution with non-cross-linked hyaluronic acid, shortened the preparation time while ensuring superior integrity compared with the filler samples of Comparative Examples 5 and 6.

Claims

1. A method for obtaining a filler, including: a) mixing polynucleotides having a molecular weight of 1800 to 2300 kDa with a solvent to obtain a solution of polynucleotides and then mixing said solution, wherein the polynucleotides are uniformly distributed in the solvent; b) removing bacteria from the resulting polynucleotide solution using a sterile filter; and c) mixing non-crosslinked hyaluronic acid having a molecular weight of 100 to 5000 kDa and a pH modifier with a bacteria-free polynucleotide solution, wherein the polynucleotide is present in a concentration of 1 to 1.5% (w / v) and non-crosslinked hyaluronic acid is present in a concentration of 0.1 to 1% (w / v) relative to the total volume of the final filler, characterized in that steps a)-c) are performed sequentially, wherein the filler has a complex viscosity in the range of 0.3 to 3 Pa⋅s and is suitable for reducing mild wrinkles according to the Wrinkle Severity Rating Scale (WSRS) or crow's feet; and wherein step c) of mixing hyaluronic acid and a pH modifier comprises: c-1) mixing non-crosslinked hyaluronic acid with the bacteria-free polynucleotide solution obtained in step b) to obtain a mixture; and c-2) mixing a pH modifier with the resulting mixture.

2. The method according to claim 1, characterized in that the polynucleotide has a molecular weight in the range from 1800 to 2300 kDa.

3. The method according to claim 1, characterized in that the solvent is water for injection.

4. The method according to paragraph 1, characterized in that the sterile filter has a pore size of 0.2 µm.

5. The method according to claim 1, characterized in that the non-cross-linked hyaluronic acid has a molecular weight in the range from 700 to 1200 kDa.

6. The method of claim 1, further comprising mixing an anesthetic agent after step c) mixing the hyaluronic acid and the pH modifier.

7. The method according to claim 6, characterized in that the anesthetic agent is lidocaine or its salt.

8. The method according to claim 6, characterized in that the anesthetic agent is contained in a concentration of 0.3% (w / v) relative to the total volume of the final filler.

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