WO2017018717A1 - Composition d'hydrogel d'agent de remplissage dermique - Google Patents

Composition d'hydrogel d'agent de remplissage dermique Download PDF

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WO2017018717A1
WO2017018717A1 PCT/KR2016/007945 KR2016007945W WO2017018717A1 WO 2017018717 A1 WO2017018717 A1 WO 2017018717A1 KR 2016007945 W KR2016007945 W KR 2016007945W WO 2017018717 A1 WO2017018717 A1 WO 2017018717A1
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hyaluronic acid
hydrogel
filler
alginic acid
acid
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PCT/KR2016/007945
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English (en)
Korean (ko)
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이근용
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한양대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/446—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with other specific inorganic fillers other than those covered by A61L27/443 or A61L27/46
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels

Definitions

  • the present invention relates to a hydrogel composition for dermal filler that is easy to control mechanical properties, mechanical properties and durability is improved.
  • botulinum toxin eg, the brand name “Botox”
  • Dermal fillers are agents that are injected into a patient, for example to improve facial lines and reduce wrinkles, and unlike Botox, which uses the principle of paralyzing facial muscles that cause wrinkles, Use the principle.
  • hyaluronic acid-based compounds that are biocompatible materials have been frequently used.
  • Hyaluronic acid also known as hyaluronan, is a non-sulfated glycosaminoglycan that is widely distributed in binding, epithelial and nervous tissues throughout the human body.
  • Hyaluronic acid is abundant in many different layers of skin and includes, for example, the ability to ensure good hydration, to assist the organization of the extracellular matrix, and to act as a filler; And complex functions such as those involved in tissue regeneration mechanisms.
  • the amount of hyaluronic acid, collagen, elastin, and other matrix polymers present in the skin decreases. For example, repeated exposure to ultraviolet light, for example, from the sun allows dermal cells to reduce their production of hyaluronan as well as to increase their rate of degradation.
  • Injectable dermal fillers are successfully used for skin aging. Dermal fillers can replace lost endogenous matrix polymers or treat / promote the function of existing matrix polymers to treat these skin conditions.
  • the present inventors earnestly researched to develop a hydrogel composition for dermal filler that is easy to control mechanical properties, and improved mechanical properties and durability.
  • a hydrogel for dermal filler having improved mechanical properties and durability can be prepared as compared to a conventional hyaluronic acid hydrogel. It became.
  • an object of the present invention is to provide a hydrogel composition for dermal filler.
  • Still another object of the present invention is to provide a method for preparing a dermal filler hydrogel.
  • the present invention to solve the above problems,
  • an ion crosslinkable hyaluronic acid hydrogel comprising hyaluronic acid, alginic acid bound to the hyaluronic acid, and an ion crosslinking agent.
  • the ion crosslinking agent provides a hydrogel composition for dermal filler that is a divalent cation or a salt thereof.
  • step (b) reacting the reaction product of step (a) with a second reaction material, hyaluronic acid or alginic acid, to obtain an alginic acid-linked hyaluronic acid reformer linked through a linker;
  • composition for reinforcing mechanical properties of a chemical crosslinked hyaluronic acid filler comprising an ion crosslinkable hyaluronic acid hydrogel comprising:
  • the present invention provides a hydrogel composition for dermal filler with improved persistence and mechanical properties (G ′).
  • the present invention provides a method for producing a hydrogel for dermal filler.
  • the present invention provides a composition for reinforcing mechanical properties of chemical cross-linked hyaluronic acid filler.
  • composition of the present invention can be applied to parts of the body where high mechanical properties are required.
  • Hyaluronic acid was first modified with ethylenediamine (NH2-HA). NH2-HA was then reacted with alginic acid (AL) through the carbodiimide chemistry.
  • FIG. 2A shows an image of a hyaluronic acid (HA) or alginic acid-grafted-hyaluronic acid (HGA) solution after mixing, in the presence (+) or absence (-) condition of calcium ions.
  • HA hyaluronic acid
  • HGA alginic acid-grafted-hyaluronic acid
  • FIG. 2B shows an image of HGA after injection through a syringe needle.
  • FIG. 2C shows the change in storage modulus (G ′, filled representation) and loss modulus (G ′′, empty representation) of HGA1 (circle) and HA (square) solutions in the presence of calcium ions.
  • 2D shows the change in storage modulus dependent on hyaluronic acid content.
  • 3A shows the change in dry weight of both hyaluronic acid / alginic acid mixed gels or alginic acid-grafted-hyaluronic acid gels after treatment with hyaluronidase at 37 ° C. for 2 weeks.
  • 3B shows a cross-sectional SEM image of the alginic acid / hyaluronic acid mixed gel.
  • 4A shows the 1 H-NMR spectra of hyaluronic acid, alginic acid and alginic acid-grafted hyaluronic acid.
  • 4B shows the results of DMMB analysis for quantitative graft efficacy of alginic acid-grafted hyaluronic acid.
  • Figures 5a-5b shows the results of comparison of hydrogel properties according to the molecular weight of hyaluronic acid used in the ion-crosslinked alginic acid-linked hyaluronic acid reformer.
  • Figure 5a shows the properties of the hydrogel when using an alginic acid-linked hyaluronic acid reformer containing a hyaluronic acid of 1000 kD.
  • Figure 5b shows the properties of the hydrogel when using an alginic acid-linked hyaluronic acid reformer containing a hyaluronic acid of 1000 kD.
  • 5A and 5B show the concentration of the alginic acid-binding hyaluronic acid modifier.
  • FIGS. 6a-6c show the results of physical property changes when the ion-crosslinked alginic acid-bonded hyaluronic acid hydrogel is mixed with a conventional chemical crosslinked hyaluronic acid filler.
  • Figure 6a shows the physical properties when the existing filler cugel Aqua S (BNC Korea) and alginic acid-linked hyaluronic acid hydrogel containing hyaluronic acid having an average molecular weight of 1000 kD at 50:50.
  • FIG. 6B shows the physical properties of the existing filler cugel Aqua S and an alginic acid-linked hyaluronic acid hydrogel including hyaluronic acid having an average molecular weight of 1500 kD at 50:50.
  • Figure 6c shows the physical properties when the mixture of the existing filler cugel S (BNC Korea) and alginic acid-linked hyaluronic acid hydrogel containing hyaluronic acid having an average molecular weight of 1500 kD at 50:50.
  • 6A to 6C show the types and concentrations of hydrogels used.
  • Figure 7 shows the cytotoxicity test results according to the mixing weight ratio when the ion-crosslinked alginic acid-linked hyaluronic acid hydrogel is mixed with the conventional chemical cross-linked hyaluronic acid filler.
  • Figure 8 shows the results of the change in physical properties (elastic coefficient) according to the mixing weight ratio when the ion-crosslinked alginic acid-linked hyaluronic acid hydrogel is mixed with the conventional chemically cross-linked hyaluronic acid filler.
  • the present invention includes a hyaluronic acid, an alginic acid bonded to the hyaluronic acid, and an ion crosslinkable hyaluronic acid hydrogel comprising an ion crosslinking agent, wherein the ion crosslinking agent is a divalent cation or a salt thereof It provides a hydrogel composition for dermal filler, characterized in that.
  • the present inventors earnestly researched to develop a hydrogel composition for dermal filler that is easy to control mechanical properties, and improved mechanical properties and durability.
  • a hydrogel for dermal filler having improved mechanical properties and durability can be prepared as compared with a conventional hyaluronic acid hydrogel.
  • the term "dermal filler” is used for improving or treating wrinkles, depressions, scars, etc. formed on the skin of a subject, and specifically, for improving wrinkles such as forehead, brow, and nasolabial folds, It may mean a biocompatible material for injection into the dermis for the purpose of raising the nose, increasing the skin area volume, improving the scar area, and the like.
  • the term "chemical crosslinking hyaluronic acid filler” may refer to a conventional hyaluronic acid filler prepared using a chemical crosslinking agent, and may be any hyaluronic acid filler prepared by adding a chemical crosslinking agent.
  • Commercially available filler products Restylane, Irete, Cutegel, Juviderm Perfectha, Teosial, Rofilan, Elevess For example, Elavie (Elavie), Glytone (Glytone), Princess (Princess) and the like.
  • the hydrogel composition for dermal fillers according to the present invention may further include a chemically cross-linked hyaluronic acid filler, through which the mechanical properties of the dermal filler can be significantly improved.
  • the weight ratio of the ion crosslinkable hyaluronic acid hydrogel and the chemical crosslinked hyaluronic acid filler may be 1: 1-9, more specifically 1: 1.
  • hyaluronic acid into which alginic acid is introduced is crosslinked by a divalent cation to form a hydrogel.
  • a divalent cation for example as specifically Ca 2 +, Ba 2 +, Cu 2 +, Fe 2 2, such as +, and Mg 2 +, causing cross-linking by the interaction with a cation is introduced into hyaluronic acid, alginate, a hydrogel To form.
  • the hydrogel may be adjusted to have higher mechanical properties (G ′) than the hydrogel formed from a simple mixture in which hyaluronic acid and alginic acid are not bound.
  • the divalent cation of the present invention may be calcium, barium or magnesium ions.
  • the calcium, barium or magnesium ions of the present invention may be provided in the form of sulfate or chloride salts with respect to the alginic acid bound hyaluronic acid.
  • divalent cations can be introduced by adding calcium chloride, calcium sulfate, barium chloride, magnesium chloride, magnesium sulfate, and the like.
  • the composition of the present invention may have a mechanical property (G ′) of 10-3000 Pa. More specifically, it may have a mechanical property of 20-3000 pa, even more specifically 500-3000 pa, even more specifically 500-2500 pa, even more specifically 1000-2500 pa, even more specifically 1500-2500 pa. .
  • the composition of the present invention may have a mechanical property of 1500-2500 pa, the composition may be used as a dermal filler composition for skeletal reinforcement.
  • the composition may be used as a dermal filler composition for skeletal reinforcement.
  • the term "skeletal reinforcement” may refer to a use for applying a deformation to a facial contour including a nose, a cheekbone, and a jawline, and in the case of the skeletal reinforcement dermal filler, a filler having a purpose of removing wrinkles is higher. May require mechanical properties.
  • the term mechanical property (G ′) may mean shear storage modulus or elastic modulus, and specifically, the sugar storage modulus is a shear test that transmits a force pushing the sample. It may mean the force required to apply a constant deformation to the sample, the elastic modulus may refer to the force required to apply a constant deformation to the sample by shear test by the force to pull the sample.
  • the alginic acid of the present invention may have a molecular weight of 10000-1000000 g / mol
  • the hyaluronic acid of the present invention may have a molecular weight of 10000-2500000 g / mol.
  • the alginic acid of the present invention may have a molecular weight of 20000 to 500000 g / mol, more specifically, may be used 50000 to 500000 g / mol, and more specifically 100000 to 300000 g / mol. have.
  • the hyaluronic acid of the present invention may be used having a molecular weight of 100000 to 2500000 g / mole, more specifically 300000 to 2500000 g / mol can be used, and more specifically 500000 to 1500000 g / mol can be used. .
  • the weight ratio of hyaluronic acid and alginic acid of the present invention may be 4: 1 to 1: 2.
  • the weight ratio of the hyaluronic acid and the alginic acid described above may mean a ratio of the total weight of each of the hyaluronic acid and the alginic acid included in the hydrogel composition.
  • the combined composition ratio of hyaluronic acid and alginic acid included in the hydrogel composition of the present invention is one of the factors that can control the mechanical properties (G ') of the hyaluronic acid gel, to achieve the required physical properties according to the site and purpose of use of the dermal filler
  • the weight ratio of the hyaluronic acid and alginic acid in the composition ratio can be suitably used in the range of 4: 1 to 1: 2. More specifically, hyaluronic acid and alginic acid may be used in a weight ratio of 2: 1 to 1: 1.5, and even more specifically, 2: 1 to 1: 1.
  • the divalent cation of the present invention can be used by adjusting the amount in a desired range in order to achieve the desired mechanical properties in the preparation of the hydrogel.
  • an appropriate amount may be selected and mixed in consideration of solubility and the like.
  • the divalent cation salt of the invention may be 5 mM to 100 mM calcium sulfate.
  • the divalent cation salt of the invention may be 1 mM to 50 mM calcium chloride.
  • the coupling of hyaluronic acid and alginic acid of the present invention may be a covalent bond.
  • the covalent bond of the present invention may be made through a linker covalent-formable with the carboxyl group of alginic acid and the carboxyl group of hyaluronic acid.
  • the covalent bond of the present invention may be a direct bond between hyaluronic acid and alginic acid, or may be a bond through a linker as described above.
  • one molecule forms a bond with both hyaluronic acid and alginic acid, thereby binding the hyaluronic acid and alginic acid.
  • the linker of the present invention may use any linker known in the art including at least two functional group moieties capable of covalent bond formation with a carboxyl group included in hyaluronic acid and a carboxyl group included in alginic acid. .
  • the linker of the invention is diamine, divinylsulfone, 1,4-butanediol, diglycidyl ether (BDDE) and glutaraldehyde, carbodiimide, hydroxysuccine It is selected from the group consisting of imide, imido ester, maleimide, haloacetyl, disulfide, hydroazide, alkoxyamine. More specifically, for example, diamine may be used as the linker of the present invention.
  • the composition of the present invention may further comprise an analgesic agent.
  • the hydrogel composition for dermal filler of the present invention is injected into the dermis via topical injection, and may cause short-term pain at the injected site.
  • an analgesic may be additionally injected, but by using a hydrogel composition including the analgesic, the same effect may be achieved by only one injection.
  • the analgesic agent of the present invention lidocaine, mepivacaine, bupivacaine, procaine, chloroprocaine, ethidocaine, prilocaine diclonin, hexylcaine, procaine, cocaine, Ketamine, morphine, pramoxin, propofol, phenol, naloxone, meperidine, butorpanol, pentazosin, morphine-6-glucuronide, codeine, dihydrocodeine, diamorphine, dextrosepropoxyphene, fetidine , Fentanyl, alfentanil, alphaprodine, buprenorphine, dextromoramide, diphenoxylate, dipiphanone, heroin (diacetylmorphine), hydrocodone (dihydrocodeinone), hydromolphone (die Hydromorphinone), levopanol, meptazinol, methadone, methopone (methyl
  • composition of the present invention may be prepared as a pharmaceutical composition.
  • compositions of the present invention may be prepared by mixing with one or more additional pharmaceutically acceptable aqueous carriers or excipients.
  • Topical pharmaceutical or cosmetic pharmaceutical compositions including, for example, pharmaceutically acceptable carriers or diluents, such as buffered saline, as well as dermatologically or pharmaceutically acceptable carriers, vehicles or mediums. It may include other components typically used in.
  • dermatologically or pharmaceutically acceptable may mean that the composition or ingredient described above does not cause excessive toxicity, incompatibility, allergic reactions, etc. upon skin contact.
  • compositions of the present invention may optionally include other pharmaceutically acceptable ingredients, including but not limited to buffers, preservatives, isotonicity modifiers, salts, antioxidants, osmolality modifiers, emulsifiers, wetting agents and the like.
  • Pharmaceutically acceptable buffers are buffers that can be used to prepare the hydrogel compositions disclosed herein provided that the formulations obtained are pharmaceutically acceptable.
  • Non-limiting examples of pharmaceutically acceptable buffers may include acetate buffers, borate buffers, citrate buffers, neutral buffered saline, phosphate buffers and phosphate buffered saline. Any concentration of pharmaceutically acceptable buffer can be useful for formulating the hydrogel compositions disclosed herein, provided that the effective amount of active ingredient can be calculated in consideration of this effective concentration of buffer.
  • Physiologically acceptable buffer concentrations range, for example, from about 0.1 mM to about 900 mM, but are not limited thereto.
  • the pH of the pharmaceutically acceptable buffer can be adjusted provided that the agent obtained is pharmaceutically acceptable.
  • acids or bases may be used to adjust the pH of the hydrogel composition.
  • Any buffered pH level may be useful for formulating pharmaceutical compositions, provided that a therapeutically effective amount of active matrix polymer ingredient can be calculated in consideration of this effective pH level.
  • Physiologically acceptable pH is, for example, in the range of about pH 5.0 to about pH 8.5, but not limited thereto.
  • the pH of the hydrogel compositions disclosed herein can be about 5.0 to about 8.0, or about 6.5 to about 7.5, about 7.0 to about 7.4, or about 7.1 to about 7.3.
  • Pharmaceutically acceptable preservatives may include sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene.
  • Pharmaceutically acceptable preservatives include benzalkonium chloride, chlorobutanol, thimerosal, phenylmercury acetate, phenylmercury nitrate, stabilized oxychloro composition, such as PURITE® (California) Allergan Inc., Irvine, Ltd.) and chelating agents, such as DTPA or DTPA-bisamide, calcium DTPA, and CaNaDTPA-bisamide.
  • compositions disclosed herein include, for example, sodium chloride and potassium chloride; And salts such as glycerine, but are not limited to these.
  • the composition may be provided as a salt and may be formed by a number of acids, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous or other protic solvents than the corresponding free base forms. It is understood that these and other materials known in the pharmaceutical arts can be included in the hydrogel compositions disclosed herein.
  • composition may further comprise any ingredient conventionally used for cosmetic purposes, such as improvement of skin aging, such as wrinkles, or in the field of treatment of neuromuscular related diseases.
  • the composition of the present invention is an injectable composition, which may be used in the form of being administered to the skin region of a subject using an injection device having a fine needle.
  • injection device having a fine needle may refer to an injection device having a needle of, for example, about 21 gauge to about 32 gauge.
  • the route of administration of the hydrogel composition to a subject may typically be determined based on the cosmetic and / or clinical effects desired by the subject and / or operator, and the body part or region in which the composition is administered. have.
  • the compositions disclosed herein may be administered by any means known to those of skill in the art, including needled syringes, pistols (eg, hand-compressed pistols), catheters by topical or direct surgical implantation. It is not limited to this.
  • the hydrogel compositions disclosed herein can be administered into a skin region, such as, for example, the dermal or subcutaneous region.
  • the suitable dosage of the hydrogel composition of the present invention can be determined in various ways by factors such as the mode of administration, the subject's age, weight, sex, time of administration and site of administration. On the other hand, the dosage of the hydrogel composition of the present invention may be preferably 0.001-100 mg / kg (body weight) per serving.
  • the present invention provides a method for preparing a hydrogel composition for dermal filler comprising the following steps:
  • step (b) reacting the reaction product of step (a) with a second reaction material, hyaluronic acid or alginic acid, to obtain an alginic acid-linked hyaluronic acid reformer linked through a linker;
  • Step (a) of the present invention is a step of covalently linking a linker to a carboxyl group of alginic acid or hyaluronic acid by reacting the linker with alginic acid or hyaluronic acid, and the linker used is the same as the linker which may be included in the hydrogel composition.
  • first reactant may refer to a substance that first reacts with a linker in hyaluronic acid or alginic acid to form a bond.
  • the linker of the present invention may be used in an amount capable of binding to all of the carboxyl groups included in the first reactant.
  • Step (b) of the present invention is a step of coupling the hyaluronic acid and alginic acid through the linker by reacting a linker-bound hyaluronic acid or alginic acid with a second reactant.
  • second reactant may refer to a compound other than the compound selected as the first reactant among hyaluronic acid or alginic acid.
  • alginic acid-linked hyaluronic acid modifier may refer to hyaluronic acid in a state in which alginic acid is bound.
  • Step (c) of the present invention is a step of adding a divalent cation or a salt thereof to the resulting alginic acid-linked hyaluronic acid reformer to cause a crosslinking reaction to gel.
  • the crosslinking reaction occurs by the interaction of the divalent cation added with alginic acid, thereby obtaining a hydrogel.
  • the present invention may further comprise the step of mixing the obtained crosslinked hyaluronic acid hydrogel with a chemical cross-linked hyaluronic acid filler;
  • the mechanical properties of the dermal filler can be significantly improved.
  • the mixed weight ratio of the ion crosslinkable hyaluronic acid hydrogel and the chemical crosslinked hyaluronic acid filler may be 1: 1-9, and more specifically 1: 1.
  • the divalent cation of the present invention may be calcium, barium or magnesium ions.
  • the calcium, barium or magnesium ions of the present invention can be used in the form of sulfate or chloride salts with respect to alginic acid bound hyaluronic acid. Specifically, for example, it may be used in the form of calcium chloride, calcium sulfate, barium chloride, magnesium chloride, or magnesium sulfate compound.
  • the present invention further comprises the step of adding an analgesic agent to the hydrogel of the present invention after the step (c).
  • Method for producing a hydrogel composition for dermal filler relates to a method for producing a "hydrogel composition for dermal filler" which is another embodiment of the present invention, the overlapping content is used, and excessive In order to avoid complexity, description of duplicate contents will be omitted.
  • the present invention provides a composition for reinforcing mechanical properties of a chemical cross-linked hyaluronic acid filler comprising an ion-crosslinkable hyaluronic acid hydrogel comprising:
  • the weight ratio of the ion crosslinkable hyaluronic acid hydrogel and the chemical crosslinking-hyaluronic acid filler may be 1: 1-9, more specifically 1: 1.
  • the hyaluronic acid of the present invention may have a molecular weight of 500000 g / mol to 2500000 g / mol.
  • the divalent cation of the present invention may be calcium, barium or magnesium ions.
  • the divalent cations of the invention may be added in the form of chloride salts.
  • the chloride salt of the invention may be calcium chloride.
  • composition for reinforcing the mechanical properties of the chemical cross-linked hyaluronic acid filler according to the present invention uses the hydrogel composition for dermal filler, which is one embodiment of the present invention described above, and the overlapping content is omitted so as to avoid excessive complexity of the present specification. do.
  • hyaluronic acid (molecular weight 600000-850000; Lifecore) was reacted with ethylenediamine (Sigma-Aldrich).
  • Synthesis of NH2-hyaluronic acid was synthesized by a conventionally known method using an EDC / NHS reaction. In order to suppress the crosslinking between hyaluronic acid chains during the reaction, an excess of 10 times molar ratio of ethylenediamine was added. And it reacted for 20 hours at room temperature. The solution was dialyzed and treated with activated charcoal, filtered through a 0.22 ⁇ m filter for sterilization and lyophilized. Alginic acid was combined with NH 2 -hyaluronic acid via a carbodiimide chemistry according to a conventionally known method (see FIG. 1).
  • Hyaluronic acid, alginic acid and alginic acid-binding hyaluronic acid modifiers were analyzed by 1 H NMR spectroscopy (Bruker assemble 500 MHz) at 70 ° C. Samples were dissolved in D 2 O at 3 mg / ml.
  • DMMB analysis was performed to quantify the alginic acid / hyaluronic acid content in the alginic acid-binding hyaluronic acid reformer. That is, 16 mg of DMMB was dissolved in 25 ml of ethanol, filtered with filter paper, and 100 ml of 1 M guanidine hydrochloride containing 0.17 M of sodium formate and 1 ml of formic acid was mixed with the filtered DMMB. . The solution was mixed with deionized water to a total volume of 500 ml. Each sample was diluted with deionized water and 0.1% by weight solution was obtained. 1 ml of DMMB solution was added to 100 ⁇ l of each sample and mixed vigorously for 30 minutes.
  • the cultured samples were centrifuged at 12000 g for 10 minutes to precipitate the complex. The supernatant was removed and dried at room temperature for 30 minutes. The pellet was dissolved in 1 ml of decomplexation solution. A decomplexed solution was prepared with 50 mM sodium acetate buffer (pH 6.8) containing 10% 1-propanol and 4 M guanidine hydrochloride. After 30 minutes of mixing, 100 ⁇ l of each sample was transferred to a 96-well plate. Absorbance was measured at 656 nm using a spectrophotometer (SpectraMax M2; Molecular Devices).
  • Gel discs were obtained using a punch (10 mm diameter), fed into a PBS solution containing hyaluronidase in a concentration range of 0 to 100 ⁇ g / ml and incubated at 37 ° C. for 24 hours. Gel discs were frozen and lyophilized before measuring dry weight.
  • Viscoelastic properties of ion-crosslinked alginic acid-linked hyaluronic acid gels were measured using a rotary rheometer with a cone-and-plate (20 mm diameter plate, 4 ° cone angle) coneand-plate fixture (Bohlin Gemini 150). . A 150 ⁇ m gap opening was set at the top of the cone and plate, and the operating temperature was set constant at 37 ⁇ 0.1 ° C.
  • Alginic acid-binding hyaluronic acid modifiers were dissolved in PBS.
  • the 2 wt% polymer solution was mixed with a CaSO 4 slurry in an amount dependent on the alginic acid content of the polymer solution (0.42 g of CaSO 4 per 1 g of alginic acid could be mixed).
  • Mechanical properties of ion-crosslinked hydrogels were measured using a rotary rheometer (Bohlin Gemini 150). Measurement was made at 0.5 Hz and the temperature was kept constant at 37 ⁇ 0.1 ° C.
  • Example 7 Ion crosslinking Alginic acid-linked hyaluronic acid On the reformer Comparison of Hydrogel Properties by Hyaluronic Acid Molecular Weight
  • Alginic acid-bound hyaluronic acid modifiers synthesized using hyaluronic acid having a molecular weight of 1000000 g / mol or 1500000 g / mol were dissolved in PBS. 2 wt% of the polymer solution was mixed with a CaSO 4 slurry in an amount depending on the alginic acid content of the polymer solution (0.42 g of CaSO 4 per 1 g of alginic acid could be mixed).
  • Mechanical properties of ion-crosslinked hydrogels were measured using a rotary rheometer (Bohlin Gemini 150). Measurement was made at 0.5 Hz and the temperature was kept constant at 37 ⁇ 0.1 ° C.
  • Example 8 Chemical crosslinking Hyaluronic acid With filler Ion crosslinking Changes in Physical Properties of Alginate-Linked Hyaluronic Acid Modifiers
  • the mixed weight ratios of Humidics' elavies and ion-crosslinkable alginic acid-bonded hyaluronic acid hydrogels were respectively set to 9: 1.
  • the elastic modulus was measured after mixing at 7: 3 and 5: 5.
  • DDC / F containing ATug5 mouse chondrogenic cell line (ECACC) 10ug / ml human transferring (Sigma-Aldrich), 3 ⁇ 10 -8 M sodium selenite (Sigma-Aldrich), 5% FBS, 1% antibiotics Cells were cultured using -12 culture. The cells were cultured at 37 ° C. and 5% CO 2 , and 5000 cells were cultured in each well in a Transwell 0.4um polyester membrane 12-well plate (Costar).
  • HGA Alginic acid-linked hyaluronic acid
  • HGA hyaluronic acid
  • HA hyaluronic acid
  • NH 2 -HA ethylenediamine
  • HA-g-AL carbodiimide
  • HGA1, HGA2, and HGA4 weight ratios of hyaluronic acid and alginic acid
  • HGA Alginic acid-bound hyaluronic acid
  • DMMB dimethylmethylene blue
  • Ion crosslinked alginic acid-linked hyaluronic acid hydrogels can be injected through a cylinder with a 23-G needle (see FIG. 2B). This is also attractive in terms of enabling minimally invasive delivery of drugs and / or cells to the body.
  • the viscoelastic and gelling behavior of the alginic acid-bound hyaluronic acid gels were investigated using a rotary rheometer (see FIGS. 2C-2G).
  • the storage modulus (G ') was higher than the loss modulus (G ") at various frequencies (Fig. 2c), indicating that the mixture builds a hydrogel structure through ion crosslinking.
  • the hyaluronic acid solution does not form a hydrogel against calcium addition, and then examines the effect of the alginic acid content on the mechanical properties of the ion-crosslinked alginic acid-linked hyaluronic acid gel (see FIG. 2D).
  • the weight ratios between hyaluronic acid and alginic acid varied.
  • the mechanical properties of the gel changed significantly from 2.5 ⁇ 0.2 kPa to 0.075 ⁇ 0.001 kPa.
  • the change in the elasticity of the gel changes with calcium concentration (see Fig. 2e)
  • the G 'value of the alginic acid-bound hyaluronic acid gel is 0.3 ⁇ 0.1.
  • the kPa increased from 2.7 ⁇ 0.6 kPa, but no further increase was observed when the calcium sulfate concentration increased to 60 mM.
  • the gelation time of the alginic acid-linked hyaluronic acid gel was significantly affected by the alginic acid content in the gel.
  • the shear modulus of the alginic acid-linked hyaluronic acid gel depends on the alginic acid molecular weight (see Fig. 2g), as the alginic acid molecular weight increases from 50000 g / mol to 250000 g / mol.
  • the G 'of the ion crosslinked gel also increased from 0.04 ⁇ 0.003 to 2.4 ⁇ 0.2 kPa
  • Alginate-linked hyaluronic acid gels made with 50000 g / mol or 100000 g / mol alginic acid showed poor mechanical properties, even calcium Even in the presence of ions, it is very important to control the mechanical properties of the polymer scaffold in relation to cellular behavior (eg adhesion, proliferation and migration) [39-41].
  • mechanical properties are also important for the regulation of stem cell differentiation, which has an immediate effect on tissue regeneration [41–44].
  • the mechanical properties of alginic acid-binding hyaluronic acid gels which can be very attractive for use in tissue engineering, are readily controllable through control of polymer composition and calcium ion concentration.
  • Alginic acid-binding hyaluronic acid hydrogels were treated with hyaluronidase to test stability for in vitro enzymatic reactions. A slight decrease in gel weight was observed on the two week incubation time. Gel weight was dependent on enzyme concentration (see FIG. 3A). The hydrogels maintained their initial weight of approximately 80% after treatment with 100 ⁇ g / ml hyaluronidase for 2 weeks compared to control conditions without enzymes. Alginic acid-binding hyaluronic acid gels maintained a well packed porous structure after enzymatic treatment (see FIG. 3C).
  • gels prepared from a simple mixture of hyaluronic acid and alginic acid showed a significant reduction in gel weight during culturing even in the presence of 1 ⁇ g / ml hyaluronidase. Regardless of the hyaluronidase content, a weight loss of approximately 40% was observed. The weight loss is similar to that expected based on the hyaluronic acid content on the gel.
  • Hyaluronic acid / alginic acid mixed gel cross-sectional images after treatment with hyaluronidase showed significant gel loss by enzymatic digestion (FIG. 3B).
  • Hyaluronic acid was cleaved into small oligosaccharide fragments by the action of hyaluronidase [45, 46]. However, hyaluronic acid was rapidly degraded in the absence of crosslinking or chemical modification by endogenous hyaluronidase in vitro [47]. During the development of new ECMs from delivered cells, fast gel degradation can be detrimental to the regeneration of many types of tissues, as the gels must provide a 3-D structural environment and maintain their integrity as scaffolds. Hyaluronic acid / alginic acid mixed hydrogels do not form fully interpenetrating polymer network structures, since only alginic acid in the mixture participates in gel formation when calcium ions are added.
  • Hyaluronic acid in the mixed gels was mostly degraded entirely by the action of hyaluronidase (see Figure 3).
  • the degradation of hyaluronic acid in the alginic acid-binding hyaluronic acid was limited by the covalent conjugation of the alginic acid chain leading to slow gel degradation by hyaluronidase. This may make these gels more suitable than mixed gels for in vivo applications.
  • the G 'value of the alginic acid-linked hyaluronic acid gel increased from 480 Pa to 600 Pa. All samples tested produced hydrogels at a constant alginic acid / calcium concentration ratio. This means that at the same alginic acid molecular weight and calcium ion concentration, the hyaluronic acid molecular weight has an important effect on the physical properties of the alginic acid-linked hyaluronic acid gel. In addition, as the concentration of the alginic acid-bound hyaluronic acid reformer decreased, the G 'value also decreased.
  • FIG. 8 shows the results of changes in the physical properties (elastic coefficient) according to the mixing weight ratio when the ion-crosslinkable alginic acid-bonded hyaluronic acid hydrogel is mixed with the conventional chemically crosslinked hyaluronic acid filler. It was confirmed that the modulus of elasticity of the ella crosslinked alginic acid-bonded hyaluronic acid hydrogel mixture tended to increase as the content of the ionic crosslinked alginic acid-bonded hyaluronic acid hydrogel increased. When the alginic acid-bonded hyaluronic acid hydrogel was mixed at a ratio of 5: 5, it was confirmed that the physical properties (elastic coefficient) increased by about 35% than when the elavie was used alone (FIG. 8).
  • Figure 7 shows the cytotoxicity test results according to the mixing weight ratio when the ion-crosslinked alginic acid-linked hyaluronic acid hydrogel is mixed with the conventional chemical cross-linked hyaluronic acid filler.
  • a filler having excellent mechanical properties without using a biotoxic chemical crosslinking agent, which can replace a commercially available hyaluronic acid filler, and as a filler to a body part requiring high mechanical properties. It can be usefully applied.

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Abstract

La présente invention concerne : une composition d'hydrogel d'agent de remplissage dermique facilitant le réglage de propriétés mécaniques et ayant une durabilité et des propriétés mécaniques améliorées ; et son procédé de préparation. La composition de la présente invention est utilisée pour être appliquée, comme agent de remplissage, sur des parties de corps nécessitant des propriétés mécaniques élevées et, si le procédé de la présente invention est utilisé, les propriétés mécaniques d'un hydrogel d'agent de remplissage dermique, à préparer, peuvent être facilement réglées.
PCT/KR2016/007945 2015-07-24 2016-07-21 Composition d'hydrogel d'agent de remplissage dermique WO2017018717A1 (fr)

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CN114450040A (zh) * 2019-09-26 2022-05-06 株式会社再生生物链 组织再生用水凝胶组合物以及利用其制备的支撑体
CN114177352A (zh) * 2021-12-22 2022-03-15 西安德诺海思医疗科技有限公司 一种梯度降解皮肤填充剂及其制备方法

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