WO2017131298A1 - Combinaison d'acides hyaluroniques réticulés et son procédé de préparation - Google Patents

Combinaison d'acides hyaluroniques réticulés et son procédé de préparation Download PDF

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Publication number
WO2017131298A1
WO2017131298A1 PCT/KR2016/005819 KR2016005819W WO2017131298A1 WO 2017131298 A1 WO2017131298 A1 WO 2017131298A1 KR 2016005819 W KR2016005819 W KR 2016005819W WO 2017131298 A1 WO2017131298 A1 WO 2017131298A1
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Prior art keywords
cross
hyaluronic acid
linked
linked hyaluronic
combination
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PCT/KR2016/005819
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English (en)
Inventor
Sung Chul Choi
Hyun Il Kim
Ki Young Yang
Hyo Seung Park
Back Ho LEE
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Hanmi Pharm. Co., Ltd.
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Priority claimed from KR1020160012519A external-priority patent/KR20170090965A/ko
Application filed by Hanmi Pharm. Co., Ltd. filed Critical Hanmi Pharm. Co., Ltd.
Priority to JP2018538690A priority Critical patent/JP6821689B2/ja
Priority to US15/322,326 priority patent/US20180215840A1/en
Priority to CN201680080495.0A priority patent/CN108602898A/zh
Publication of WO2017131298A1 publication Critical patent/WO2017131298A1/fr
Priority to US16/448,180 priority patent/US11180576B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates

Definitions

  • the present disclosure relates to a preparation method of a cross-linked hyaluronic acid and a combination of cross-linked hyaluronic acids having a viscoelasticity appropriate for use in the living body, comprising the cross-linked hyaluronic acid prepared using the same method.
  • Hyaluronic acids are a biopolymer material including linearly linked repeating units consisting of N-acetyl-D-glucosamine and D-glucuronic acid, and are also known to be prevalent in animal placenta, vitreous humour, synovial fluid, rooster combs, and the like. Hyaluronic acids are also known to be produced via fermentation by microorganisms of the Streptococcus spp. (for example, Streptococcus equi , or Streptococcus zooepidemicus ) or the Staphylococcus spp.
  • Synvisc-One ® a cross-linked hyaluronic acid injection, which provide its effect lasting up to 6 months with one injection, are commercially available in the U.S.
  • Synvisc-One ® includes a cross-linked hyaluronic acid obtained by extracting hyaluronic acids from rooster combs with a formalin-containing aqueous solution, having a low viscoelasticity due to the light cross-linking of proteins connected to hyaluronic acids with formalin (US 4,713,448).
  • the lightly cross-linked hyaluronic acid is combined with its further cross-linked hyaluronic acid having increased viscoelasticity prepared by further cross-linking the lightly cross-linked hyaluronic acid using divinyl sulfone (DVS) as a cross-linking agent, thereby preparing a combination of cross-linked hyaluronic acids (Synvisc-One ® having appropriate viscoelasticity for applying to the joint cavity in the human body.
  • DVDS divinyl sulfone
  • hyaluronic acids of animal origin such as from rooster combs may have a quality control problem due to animal-origin viruses and inconsistent quality of source material.
  • the present disclosure provides a method of preparing a cross-linked hyaluronic acid having a low viscoelasticity.
  • the present disclosure provides a combination of cross-linked hyaluronic acids having an appropriate viscoelasticity applicable to the human body.
  • the present disclosure provides a biocompatible material including the combination of cross-linked hyaluronic acids.
  • the present disclosure provides a method of preparing the combination of cross-linked hyaluronic acids having an appropriate viscoelasticity applicable to the living body.
  • a method of preparing a cross-linked hyaluronic acid having an elasticity of about 50 Pa to about 200 Pa and a viscosity of about 20 Pa to about 100 Pa including cross-linking hyaluronic acid with an epoxy-based cross-linking agent having at least two epoxy functional groups in an ethanol-containing aqueous alkaline solution.
  • a method of preparing a cross-linked hyaluronic acid having an elasticity of about 400 Pa to about 800 Pa and a viscosity of about 40 Pa to about 100 Pa including further cross-linking the cross-linked hyaluronic acid prepared using the above-described method with an epoxy-based cross-linking agent having at least two epoxy functional groups in an aqueous alkaline solution.
  • a combination of cross-linked hyaluronic acids including a cross-linked hyaluronic acid having an elasticity of about 50 to about 200 Pa and a viscosity of about 20 to about 100 Pa, and a cross-linked hyaluronic acid having an elasticity of about 400 to about 800 Pa and a viscosity of about 40 to about 100 Pa.
  • a combination of cross-linked hyaluronic acids including: a cross-linked hyaluronic acid having an elasticity of about 50 to about 200 Pa and a viscosity of about 20 to about 100 Pa prepared using a method including cross-linking hyaluronic acid with an epoxy-based cross-linking agent having at least two epoxy functional groups in an ethanol-containing aqueous alkaline solution; and
  • a cross-linked hyaluronic acid having an elasticity of about 400 to about 800 Pa and a viscosity of about 40 to about 100 Pa prepared using a method including cross-linking the cross-linked hyaluronic acid having an elasticity of about 50 to about 200 Pa and a viscosity of about 20 to about 100 Pa with an epoxy-based cross-linking agent having at least two epoxy functional groups in an aqueous alkaline solution.
  • a biocompatible material including any of the above-described combinations of cross-linked hyaluronic acids.
  • a method of preparing a combination of cross-linked hyaluronic acids including combining the cross-linked hyaluronic acid having an elasticity of about 50 to about 200 Pa and a viscosity of about 20 to about 100 Pa with the cross-linked hyaluronic acid having an elasticity of about 400 to about 800 Pa and a viscosity of about 40 to about 100 Pa, wherein the combining ratio is adjusted to meet a target viscoelasticity of the combination of cross-linked hyaluronic acids to be prepared.
  • ethanol is added to the aqueous alkaline solution, so that the prepared cross-linked hyaluronic acid may have a low viscoelasticity.
  • the viscoelasticity of the cross-linked hyaluronic acid may be adjusted depending on the reaction conditions.
  • a combination of cross-linked hyaluronic acids having a desired viscoelasticity may be prepared by combining the cross-linked hyaluronic acid having a low viscoelasticity with the cross-linked hyaluronic acid having a high viscoelasticity obtained through further cross-linking of the low-viscoelasticity cross-linked hyaluronic acid. Therefore, a biocompatible combination of cross-linked hyaluronic acids having a viscoelasticity appropriate for use in the human body, and in particular, having a similar viscoelasticity similar to the human synovial fluid may be prepared for effective use in osteoarthritis treatment.
  • FIG. 1 is a flowchart illustrating a method of preparing a combination of cross-linked hyaluronic acids, according to an embodiment of the present disclosure.
  • FIG. 2 is a graph of particle size distribution as a result of measuring particle sizes of a combination of cross-linked hyaluronic acids prepared with the primary cross-linked product and secondary cross-linked product of high-molecular weight (about 2.5 MDa) hyaluronic acids in a mixed ratio of about 9:1 by weight, according to an embodiment of the present disclosure.
  • FIG. 3 is a graph of cell survival ratio illustrating the results of the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium) assay on the combination of cross-linked hyaluronic acids prepared with the primary cross-linked product and secondary cross-linked product of high-molecular weight hyaluronic acids in a mixed ratio of about 9:1 by weight, according to an embodiment of the present disclosure, and positive (Teflon) and negative (Latex) control groups.
  • MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium
  • the inventors of the present disclosure attempted to prepare lightly cross-linked hyaluronic acids from purified source material of hyaluronic acids originating from microorganisms, but failed due to low protein content of the purified hyaluronic acids of microorganism origin.
  • the inventors found that lightly cross-linked hyaluronic acids having a novel range of low viscoelasticity are obtained by introducing ethanol to an aqueous alkaline solution where a conventional hyaluronic acid cross-linking reaction with a multifunctional epoxy-based cross-linking agent takes place.
  • cross-linked hyaluronic acids having an increased viscoelasticity may be obtained by a secondary cross-linking reaction of the lightly cross-linked hyaluronic acids having a low viscoelasticity with a multifunctional epoxy-based cross-linking agent.
  • An aspect of the present disclosure provides a cross-linked hyaluronic acid having an elasticity of 50 to 200 Pa and a viscosity of 20 to 100 Pa.
  • cross-linked hyaluronic acids are not limited only to those cross-linked hyaluronic acids obtained by a specific cross-linking method.
  • the cross-linked hyaluronic acid may be prepared by a method including a step of cross-linking hyaluronic acids with an epoxy-based cross-linking agent having at least two epoxy functional groups in an ethanol-containing aqueous alkaline solution. This will be described later in more detail.
  • the cross-linked hyaluronic acid having an elasticity of 50 to 200 Pa and a viscosity of 20 to 100 Pa may be used as any biocompatible material using hyaluronic acids, for example, selected from the group consisting of arthritis treatment implants, wrinkle fillers, cosmetic fillers, and drug carriers.
  • the cross-linked hyaluronic acid may be used as a more appropriate biocompatible material in a combination with a cross-linked hyaluronic acid having an increased viscoelasticity that may be prepared via a further cross-linking reaction, if required.
  • Another aspect of the present disclosure provides a method of preparing a cross-linked hyaluronic acid having an elasticity of 50 to 200 Pa and a viscosity of 20 to 100 Pa, the method including cross-linking hyaluronic acids with an epoxy-based cross-linking agent having at least two epoxy functional groups in an ethanol-containing aqueous alkaline.
  • Another aspect of the present disclosure provides a method of preparing a cross-linked hyaluronic acid having an elasticity of 400 to 800 Pa and a viscosity of 40 to 100 Pa, the method including further cross-linking the cross-linked hyaluronic acid prepared using the above-described method with an epoxy-based cross-linking agent having at least two epoxy functional groups in an aqueous alkaline solution.
  • the cross-linked hyaluronic acid having an elasticity of 50 to 200 Pa and a viscosity of 20 to 100 Pa, prepared using the above-describe method is also referred to as a "primary cross-linked product" and the cross-linked hyaluronic acid having an elasticity of 400 to 800 Pa and a viscosity of 40 to 100 Pa, prepared by further cross-linking the primary cross-linked product, is also referred to as a "secondary cross-linked product".
  • the primary cross-linked product may be in the form of hyaluronic acid gel, and the secondary cross-linked product may be in the form of hyaluronic acid particles.
  • hyaluronic acid may be construed as any of hyaluronic acid, a salt of hyaluronic acid, or any mixtures thereof.
  • the salt of hyaluronic acid may be any biocompatible salt form, for example, selected from the group consisting of sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, tetrabutylammonium hyaluronate, and any combinations thereof.
  • the salt of hyaluronic acid may be sodium hyaluronate.
  • the hyaluronic acid may have a molecular weight of about 100,000 to about 6,000,000.
  • a viscoelasticity of the cross-linked hyaluronic acid may vary depending on the molecular weight of the hyaluronic acid.
  • the hyaluronic acid may be sodium hyaluronate having a molecular weight of about 100,000 to about 6,000,000.
  • the hyaluronic acid may include any hyaluronic acids known in the art to which the present disclosure pertains.
  • the hyaluronic acid may be obtained from any sources.
  • the hyaluronic acid may be obtained, for examples, from animal sources (for example, animal placenta, rooster combs, or the like) or any microorganisms that may produce hyaluronic acids through fermentation (for example, microorganisms of the Staphylococcus spp . , the Streptococcus spp . , or the like).
  • the hyaluronic acid may be hyaluronic acid of microorganism origin, for example, of the Staphylococcus spp. microorganism origin.
  • Hyaluronic acids of microorganism origin may be free of the problems with animal-origin hyaluronic acids, such as virus or inconsistent quality of source material, and thus may be advantageous in view of quality control in drug preparation.
  • the epoxy-based cross-linking agent having at least two epoxy functional groups may be any epoxy-based cross-linking agent having at least two epoxy functional groups known in the art, for example, selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylpropane polyglycidyl ether, 1,2-(bis(2,3-epoxypropoxy)ethylene, pentaerythritol polyglycidyl ether,
  • the epoxy-based cross-linking agent having at least two epoxy functional groups may be BDDE, EGDGE, 1,6-hexanediol diglycidyl ether, or any combinations thereof.
  • the amount of the epoxy-based cross-linking agent that may be reacted with the hyaluronic acid or cross-linked hyaluronic acid may be in a range of about 10 ⁇ l/g to about 100 ⁇ l/g, and in some embodiments, about 20 ⁇ l/g to about 100 ⁇ l/g with respect to the hyaluronic acid or cross-linked hyaluronic acid.
  • the amount of the epoxy-based cross-linking agent that reacted with the hyaluronic acid or cross-linked hyaluronic acid is less than these ranges, gel formation may not occur in preparation of the primary cross-linked product or cross-linked particles may not be obtained in preparation of the secondary cross-linked product.
  • a primary cross-linked product may be obtained in the form of particles, not an intended gel having a low viscoelasticity.
  • the ethanol-containing aqueous alkaline solution may be an aqueous alkaline solution containing about 5 to 13 %w/w of ethanol.
  • the viscosity of the cross-linked hyaluronic acid may be controlled with the concentration of the ethanol. When the concentration of the ethanol is within this range, sodium hyaluronate may be stably mixed with the ethanol, not extracted by the ethanol, so that a cross-linking reaction may take place.
  • the aqueous alkaline solution may be an aqueous alkaline solution of about pH 9 to 13.
  • the aqueous alkaline solution may be any aqueous alkaline solution known to be available in preparation of cross-linked hyaluronic acids.
  • the aqueous alkaline solution may be an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution, or ammonia water.
  • the aqueous alkaline solution may be an aqueous sodium hydroxide solution.
  • the ethanol-containing aqueous alkaline solution may be an about 0.7 to about 1.3 %w/w aqueous sodium hydroxide solution containing about 5 to about 13 %w/w of ethanol.
  • hyaluronic acid gel was formed only when hyaluronic acids reacted with an epoxy-based cross-linking agent having at least two epoxy functional groups in an aqueous alkaline solution containing ethanol, but not in an aqueous alkaline solution containing other alcohols, for example, methanol or isopropanol. It was also found that the viscoelasticity of the formed hyaluronic acid gel varies depending on the reaction time. In other words, it was found that hyaluronic acid hydrogel may be formed when the aqueous alkaline solution contains especially ethanol among organic solvents. It was also found that the reaction time may be appropriately varied to form hyaluronic acid gel having a desired viscoelasticity.
  • the cross-linking reaction may be performed in the presence of ethanol at a temperature range higher than room temperature for rapid cross-linking reaction. This rapid cross-linking reaction may induce light cross-linking.
  • the temperature range higher than room temperature may be from about 40 to about 60°C.
  • the primary cross-linked product may be a cross-linked hyaluronic acid in hydrogel form.
  • the secondary cross-linked product obtained through further cross-linking of the primary cross-linked product may be a cross-linked hyaluronic acid in particle form.
  • Another aspect of the present disclosure provides a combination of cross-linked hyaluronic acids including a low-viscoelasticity cross-linked hyaluronic acid having an elasticity of 50 to 200 Pa and a viscosity of 20 to about 100 Pa, and a high-viscoelasticity cross-linked hyaluronic acids having an elasticity of 400 to 800 Pa and a viscosity of 40 to 100 Pa.
  • the low-viscoelasticity cross-linked hyaluronic acid and the high-viscoelasticity cross-linked hyaluronic acid are not limited to cross-linked hyaluronic acids prepared using a specific cross-linking method.
  • the low-viscoelasticity cross-linked hyaluronic acid and the high-viscoelasticity cross-linked hyaluronic acid may be the primary cross-linked product of hyaluronic acids and the secondary cross-linked product of hyaluronic acids, respectively, prepared using the above-described methods of preparing cross-linked hyaluronic acids.
  • a ratio of combination of the low-viscoelasticity cross-linked hyaluronic acid and the high-viscoelasticity cross-linked hyaluronic acid may be adjusted depending on a desired viscoelasticity of the combination of cross-linked hyaluronic acids.
  • Another aspect of the present disclosure provides a combination of cross-linked hyaluronic acids including a primary cross-linked product of hyaluronic acids having an elasticity of about 50 to about 200 Pa and a viscosity of about 20 to about 100 Pa, and a secondary cross-linked product of hyaluronic acids having an elasticity of about 400 to about 800 Pa and a viscosity of about 40 to about 100 Pa.
  • a ratio of combination of the primary cross-linked product and the secondary cross-linked product may be adjusted depending on a desired viscoelasticity of the combination of cross-linked hyaluronic acids.
  • Another aspect of the present disclosure provides a biocompatible material including a combination of cross-linked hyaluronic acids according to any of the above-described embodiments.
  • the biocompatible material may be a biocompatible material using hyaluronic acids, for example, selected from the group consisting of arthritis treatment implants, wrinkle fillers, cosmetic fillers, and drug carriers.
  • the biocompatible material may have a target viscoelasticity that may vary depending on its use.
  • a ratio of combination of the low-viscoelasticity cross-linked hyaluronic acid and the high-viscoelasticity cross-linked hyaluronic acid in the combination of cross-linked hyaluronic acids, or a ratio of combination of the primary cross-linked product of hyaluronic acids and the secondary cross-linked product of hyaluronic acids in the combination of cross-linked hyaluronic acids may be adjusted depending on a target viscoelasticity of the combination of cross-linked hyaluronic acids.
  • the combination of cross-linked hyaluronic acids may be a combination of cross-linked hyaluronic acids having an elasticity of about 100 to about 150 Pa and a viscosity of about 20 to about 60 Pa, which may result in a viscoelasticity appropriate for use as a synovial fluid supplement in the human body. It is known that a viscoelasticity similar to that of human synovial fluid may be obtained at an elasticity of about 100 to about 150 Pa and a viscosity of about 20 to about 60 Pa ( Balazs , E. A ., "The physical properties of synovial fluid and the special role of hyaluronic acid," Disorders of the Knee 2 (1974): 61-74).
  • the ratio of combination of the low-viscoelasticity cross-linked hyaluronic acid and the high-viscoelasticity cross-linked hyaluronic acid, or the ratio of combination of the primary cross-linked product and the secondary cross-linked product may be varied according to the molecular weight of the hyaluronic acids.
  • a high-molecular weight (about 2.5 MDa) hyaluronic acid and a low-molecular weight (about 0.9 MDa) hyaluronic acid were each subjected to a cross-linking reaction to obtain primary cross-linked products.
  • the resulting primary cross-linked products were each subjected to a further cross-linking reaction to obtain a secondary cross-linked product.
  • the primary cross-linked product and the secondary cross-linked product were combined in different ratios to prepare combinations of cross-linked hyaluronic acids, followed by a viscoelasticity measurement.
  • the primary cross-linked products were found to have a different viscoelasticity depending on whether a high-molecular weight hyaluronic acid or a low-molecular weight hyaluronic acid was used as a start material.
  • the secondary cross-linked products were found to have a significantly increased viscoelasticity, compared to that of their primary cross-linked product, irrespective of the molecular weight of the start material.
  • the combination of cross-linked hyaluronic acids also each had a different viscoelasticity depending on the ratio of combination of the primary cross-linked product and the secondary cross-linked product ( refer to Experimental Example 2).
  • the ratio of combination of the primary cross-linked product and the secondary cross-linked product may vary depending on the molecular weight of hyaluronic acid source used as the start material.
  • a combination ratio of about 9:1 by weight of the primary cross-linked product and the secondary cross-linked product thereof for the case of a high-molecular weight (about 2.5 MDa) hyaluronic acid source being used as the start material, or a combination ratio of 8:2 by weight of the primary cross-linked product and the secondary cross-linked product thereof for the case of a low-molecular weight (about 0.9 MDa) hyaluronic acid source being used as the start material may obtain a combination of cross-linked hyaluronic acids having a viscoelasticity range (at an elasticity of about 100 to about 150 Pa and a viscosity of about 20 to 60 Pa) appropriate for use as arthritis treatment implants ( refer to Experimental Example 2).
  • the combination of cross-linked hyaluronic acids may include the primary cross-linked product of hyaluronic acids having an elasticity of 50 to 200 Pa and a viscosity of 20 to 100 Pa and the secondary cross-linked product of hyaluronic acids having an elasticity of 400 to 800 Pa and a viscosity of 40 to 100 Pa in a weight ratio of about 8:2 to about 9:1.
  • the combination of cross-linked hyaluronic acids may be in the form of particles, obtained by particlization, having an average particle size of about 500 to about 750 ⁇ m.
  • the particlization may be performed by a general pulverization process after combination of the primary cross-linked product and the secondary cross-linked product.
  • FIG. 1 is a flowchart illustrating a method of preparing a combination of cross-linked hyaluronic acids, according to an embodiment of the present disclosure. This preparation method now will be described below.
  • step (a) is preparing a primary cross-linked product of hyaluronic acids in hydrogel form.
  • sodium hyaluronate also abbreviated to "HA”
  • HA hyaluronate
  • step (a1) is mixing a sodium hyaluronate-containing buffer with an ethanol-containing aqueous sodium hydroxide solution.
  • a viscoelasticity of a resulting hyaluronic acid hydrogel may vary depending on a mixing ratio of ethanol and sodium hydroxide in step (a1).
  • Step (a2) is mixing sodium hyaluronate with a cross-linking agent.
  • the amount of the cross-linking agent may be about 10 ⁇ l to about 100 ⁇ l per unit gram of sodium hyaluronate. When the amount of the cross-linking agent is less than this range, a cross-linked hyaluronic acid having a desired viscoelasticity may not be obtained.
  • Step (a3) is a process of cross-linking reaction of sodium hyaluronate with a cross-linking agent in the ethanol-containing aqueous alkaline solution.
  • rapid cross-linking reaction may be performed due to a reaction temperature higher than room temperature and the presence of ethanol in a reactant, so that a light cross-linking may be induced.
  • This reaction step may be performed in an about 40 to about 60°C oven for about 4 to about 6 hours.
  • the resulting cross-linked hyaluronic acids in hydrogel form may be subjected to dialysis in a buffer (step (a4)), followed by a neutralization reaction with distilled water (step (a5)) and then precipitation of hyaluronic acid as hydrogel may be induced with about 95% ethanol (step (a6)) thereby obtaining a cross-linked hyaluronic acid in powder form.
  • Step (b) is preparing a secondary cross-linked product of hyaluronic acids in the form of particles through further cross-linking of the primary cross-linked product of hyaluronic acids obtained in step (a).
  • Step (b1) is adding the cross-linked sodium hyaluronate (HA) obtained in step (a6), to an about 0.7 to about 1.3 %w/w aqueous sodium hydroxide solution and mixing them together.
  • Step (b2) is adding a cross-linking agent to the resulting mixture from step (b1) and mixing together. In this step, about 10 to about 100 ⁇ l of the cross-linking agent may be added per one gram of the cross-linked HA obtained in step (a).
  • Step (b3) is a cross-linking reaction process of HA with the cross-linking agent. This reaction may be performed in an about 40 to about 60°C oven for about 8 to 10 hours. This cross-linking reaction in step (b3) may be performed for a time longer than step (a3) to induce generation of cross-linked hyaluronic acids in the form of particles.
  • the obtained cross-linked hyaluronic acids in the form of particles may be subjected to dialysis in a buffer (step (b4)), followed by a neutralization reaction with distilled water (step (b5)) and then precipitation of hyaluronic acid as particles may be induced with about 95% ethanol (step (b6)) thereby obtaining cross-linked hyaluronic acids in powder form.
  • Step (c) is mixing the primary cross-linked product of sodium hyaluronate in hydrogel form obtained in step (a) with the secondary cross-linked product of sodium hyaluronate in particle form obtained in step (b) in a variety of ratios, and preparing a final product via pulverization, filling, and sterilization.
  • Step (c1) is mixing the primary cross-linked product of sodium hyaluronate in hydrogel form and the secondary cross-linked product of sodium hyaluronate in particle form in a ratio of about 9:1 to about 8:2 and dissolving the mixture in a buffer to prepare a combination of cross-linked hyaluronic acids.
  • Step (c2) is pulverizing the combination of cross-linked hyaluronic acids into particles having a particle size of about 500 to about 750 ⁇ m, followed by filling a syringe with the combination of cross-linked hyaluronic acids (step (c3)) and sterilizing the combination of cross-linked hyaluronic acids-filled syringe (step (c4)) thereby obtaining a final combination of cross-linked hyaluronic acids having a target viscoelasticity.
  • the biocompatible material may be a synovial fluid supplement including a combination of the cross-linked hyaluronic acid having an elasticity of 50 to 200 Pa and a viscosity of 20 to 100 Pa and the cross-linked hyaluronic acid having an elasticity of 400 to 800 Pa and a viscosity of 40 to 100 Pa, the combination in particle form having an average particle size of about 500 to about 750 ⁇ m and having an elasticity of 100 to 150 Pa and a viscosity of 20 to 60 Pa.
  • the biocompatible material may be a synovial fluid supplement including a combination of the primary cross-linked product of hyaluronic acids having an elasticity of 50 to 200 Pa and a viscosity of 20 to 100 Pa and the secondary cross-linked product of hyaluronic acids having an elasticity of 400 to 800 Pa and a viscosity of 40 to 100 Pa, the combination in particle form having an average particle size of about 500 to about 750 ⁇ m and an elasticity of 100 to 150 Pa and a viscosity of 20 to 60 Pa.
  • the synovial fluid supplement may have a viscoelasticity similar to that of the human's real synovial fluid, and may be effectively used as a synovial fluid supplement.
  • the synovial fluid supplement may include both the primary cross-linked product in hydrogel form and the secondary cross-linked product in particle form to thus provide lubricating and separating functions for joints, and may serve a very similar function like the human synovial fluid. Due to the inclusion of both the primary cross-linked product in hydrogel form and the secondary cross-linked product in particle form, the biocompatible material may be structurally stable, not easily decomposable in the human body, and have an effect as a synovial fluid supplement lasting for about 6 months or longer with one injection.
  • Another aspect of the present disclosure provides a method of preparing a combination of cross-linked hyaluronic acids according to any of the above-described embodiments, the method including combining the cross-linked hyaluronic acid having an elasticity of about 50 to about 200 Pa and a viscosity of about 20 to about 100 Pa and the cross-linked hyaluronic acid having an elasticity of about 400 to about 800 Pa and a viscosity of about 40 to about 100 Pa, wherein the combining ratio is adjusted to meet a target viscoelasticity of the combination of cross-linked hyaluronic acids to be prepared.
  • a combination of cross-linked hyaluronic acid having a desired viscoelasticity may be obtained by adjustment of the combining ratio of the cross-linked hyaluronic acids having an elasticity of about 50 to about 200 Pa and a viscosity of about 20 to about 100 Pa and the cross-linked hyaluronic acid having an elasticity of about 400 to about 800 Pa and a viscosity of about 40 to about 100 Pa.
  • the combining ratio of the primary cross-linked product of hyaluronic acids and the secondary cross-linked product of hyaluronic acids may be varied. Even when preparing a combination of cross-linked hyaluronic acids having the same viscoelasticity, the combining ratio of the primary cross-linked product of hyaluronic acids and the secondary cross-linked product of hyaluronic acids may also be varied depending on the molecular weight of hyaluronic acid source and the preparation conditions of the primary cross-linked product and the secondary cross-linked product (reaction temperature, reaction time, and the like).
  • the cross-linked hyaluronic acids of Preparation Example 2 using the ethanol-containing aqueous alkaline solution were found to have a significantly low viscoelasticity, compared to the cross-linked hyaluronic acids of Preparation Example 1 using the organic solvent-free aqueous alkaline solution. This indicates that adding ethanol to an aqueous alkaline solution for a cross-linking reaction may suppress the cross-linking reaction, and consequentially result in cross-linked hyaluronic acids having a reduced viscoelasticity.
  • Example 1 Preparation of primary cross-linked product of hyaluronic acids in hydrogel form
  • the preparation processes of Examples 1 to 3 were separately performed using each of a high-molecular weight sodium hyaluronate (having a molecular weight of 2.5 Mda) and a low-molecular weight sodium hyaluronate (having a molecular weight of 0.9 Mda).
  • the primary cross-linked product of hyaluronic acids obtained in Example 1 was subjected to a secondary cross-linking reaction.
  • the primary cross-linked product of hyaluronic acids in powder form obtained in Example 1 was mixed with a 1 %w/w sodium hydroxide aqueous solution in a weight ratio of about 1:5 and then completely dissolved.
  • 50 ⁇ l of BDDE with respect to 1 g of the primary cross-linked product was added to the resulting reaction mixture and then mixed together. After the mixing, a cross-linking reaction was performed at a reaction temperature of about 40°C for about 8 hours.
  • the resulting secondary cross-linked product of hyaluronic acids in particle form was subjected to dialysis in a PBS buffer for about 12 to 24 hours, followed by washing with distilled water to remove BDDE.
  • the resulting neutralized hyaluronic acids in particle form were subjected to extraction with a 95% ethanol aqueous solution to thereby yield a secondary cross-linked product of hyaluronic acids in powder form.
  • the primary cross-linked product of hyaluronic acids in hydrogel form obtained in Example 1 and the secondary cross-linked product of hyaluronic acids in particle form obtained in Example 2 were mixed together in a weight ratio of about 9:1 or about 8:2 in a PBS buffer to prepare a 2 %w/w of mixed solution.
  • the mixed solution was pulverized by passing the mixed solution through a 500 ⁇ m-mesh sieve with physical force.
  • the pulverized mixture was filled into a syringe, followed by sterilization at about 121°C for about 20 minutes.
  • the primary cross-linked product of hyaluronic acids in hydrogel form obtained in Example 1 and the secondary cross-linked product of hyaluronic acids in particle form obtained in Example 2 were mixed together in a weight ratio of about 10:0, 9:1, 8:2, and 0:10 in a PBS buffer to prepare 2 %w/w of mixed solutions.
  • the mixed solutions were each pulverized by passing them through a 500 ⁇ m-mesh sieve with physical force.
  • the viscoelasticity of each of the mixed solutions was measured using a rotational rheometer (Kinexus Pro Rheometer, available from Malvern, Worchestershire, UK). The results are shown in Table 3.
  • the combination of cross-linked hyaluronic acids obtained with the primary cross-linked product and secondary cross-linked product from the high-molecular weight hyaluronic acid in a mixed ratio of about 9:1 by weight had an elasticity of about 114.40 Pa and a viscosity of about 45.19 Pa.
  • the combination of cross-linked hyaluronic acids obtained with the primary cross-linked product and secondary cross-linked product from the low-molecular weight hyaluronic acids in a mixed ratio of about 8:2 by weight had an elasticity of 106.1 Pa and a viscosity of 45.71 Pa. Accordingly, these two combinations of cross-linked hyaluronic acids were found to have a similar viscoelasticity to the human synovial fluid.
  • the viscoelasticities of the cross-linked hyaluronic acid prepared under the same conditions varied depending on the molecular weight of the starting hyaluronic acid source. Even prepared under the same cross-linking conditions, the cross-linked hyaluronic acid from the starting high-molecular weight hyaluronic acid source had a higher viscoelasticity than those from the starting low-molecular weight hyaluronic acid source. Therefore, to prepare a combination of cross-linked hyaluronic acids having a desired viscoelasticity, the combination ratio of the primary cross-linked product and the secondary cross-linked product may vary depending on the molecular weight of a starting hyaluronic acid source.
  • the combination of cross-linked hyaluronic acids was found to have an average particle size of about 606 ⁇ m (D 10 : 235.8 ⁇ m, D 50 : 553.5 ⁇ m, and D 90 : 1009 ⁇ m).
  • Cytotoxicity of the combination of cross-linked hyaluronic acids obtained with the primary cross-linked product and secondary cross-linked product from the high-molecular weight hyaluronic acids in a mixed ratio of about 9:1 by weight was evaluated by the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium) assay.
  • the cell survival ratios of Teflon and Latex were 100% and 37%, respectively.
  • the combination of cross-linked hyaluronic acids had a cell survival ratio of about 89.93%, which is above 80%, the lower limit of cytotoxicity. Therefore, the combination of cross-linked hyaluronic acids as an embodiment of the present disclosure was found to have high biocompatibility.

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Abstract

La présente invention concerne un procédé de préparation d'un acide hyaluronique réticulé ayant une élasticité d'environ 50 à environ 200 Pa et une viscosité d'environ 20 à environ 100 Pa, le procédé comprenant la réticulation de l'acide hyaluronique avec un agent de réticulation à base d'époxy comportant au moins deux groupes fonctionnels époxy dans une solution alcaline aqueuse contenant de l'éthanol ; un procédé de préparation d'un acide hyaluronique réticulé ayant une élasticité d'environ 400 à environ 800 Pa et une viscosité d'environ 40 à environ 100 Pa, le procédé comprenant la réticulation de l'acide hyaluronique réticulé ayant une élasticité d'environ 50 à environ 200 Pa et une viscosité d'environ 20 à environ 100 Pa avec un agent de réticulation à base d'époxy comportant au moins deux groupes fonctionnels époxy dans une solution alcaline aqueuse ; et une combinaison d'acides hyaluroniques réticulés comprenant l'acide hyaluronique réticulé ayant une élasticité d'environ 50 à environ 200 Pa et une viscosité d'environ 20 à environ 100 Pa et l'acide hyaluronique réticulé ayant une élasticité d'environ 400 à environ 800 Pa et une viscosité comprise entre environ 40 et environ 100 Pa.
PCT/KR2016/005819 2016-01-29 2016-06-01 Combinaison d'acides hyaluroniques réticulés et son procédé de préparation WO2017131298A1 (fr)

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JP2018538690A JP6821689B2 (ja) 2016-01-29 2016-06-01 複合ヒアルロン酸架橋物及びその製造方法
US15/322,326 US20180215840A1 (en) 2016-01-29 2016-06-01 Combination of cross-linked hyaluronic acids and method of preparing the same
CN201680080495.0A CN108602898A (zh) 2016-01-29 2016-06-01 交联透明质酸的组合及其制备方法
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WO2023100219A1 (fr) * 2021-11-30 2023-06-08 Kewpie Corporation Poudre d'acide hyaluronique

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