JP5165281B2 - Two-reactor type water-containing medical gel-forming agent and hyaluronic acid gel obtained therefrom - Google Patents

Description

  The present invention relates to a two-component reaction type hyaluronic acid gel applied as a medical material such as a therapeutic material for joint diseases such as osteoarthritis and rheumatoid arthritis, a tissue regeneration scaffold, and a carrier for DDS, and a method for producing the same. In particular, it is composed of a liquid containing the first reaction component and a liquid containing the second reaction component, and by mixing the first reaction component and the second reaction component, they are reacted with each other and cured in a gel form. The present invention relates to a medical material that is decomposed and fluidized and decomposed and metabolized by enzymes in a living body after a certain period of time.

  Hyaluronic acid is a linear high-molecular polysaccharide composed of disaccharide units in which D-glucuronic acid and N-acetyl-D-glucosamine are alternately linked by β-1,4 bonds and β-1,3 bonds. It is known that this hyaluronic acid is distributed in the connective tissue of mammals and is present at the tip of chickens, and exhibits biocompatibility that does not show any harmful effects even when injected into a living body. Previously it was extracted from the chicken crest and animal umbilical cord, but recently some products have been purified from streptococcal cultures. Hyaluronic acid is highly soluble in water, both in its acid form and in its salt form (sodium and potassium salts). However, although hyaluronic acid has high water retention, it is susceptible to enzymatic action in the living body, so its half-life in the body is very short, about 1 to 3 days. When used as a medical material in vivo, It has been pointed out that the residence time is short. For this reason, attempts have been made to extend the residence time in the body by various methods such as chemical modification, chemical cross-linking, and the like. As typical examples of these, a crosslinked hyaluronic acid gel using a difunctional crosslinking agent such as divinyl sulfone, bisepoxides and formaldehyde has been reported (Japanese Patent Laid-Open No. 7-97401). In addition, a bifunctional reagent such as divinyl sulfone, bisepoxide, formaldehyde or the like can be used as a crosslinking agent, and the resulting highly swellable crosslinked hyaluronic acid gel can be exemplified (US Pat. No. 4,582,865). (See the specification, Japanese Patent Publication No. 6-37575, Japanese Patent Laid-Open No. 7-97401, Japanese Patent Laid-Open No. 60-130601). Further, a method for chemically modifying hyaluronic acid utilizing the feature that tetrabutylammonium salt of hyaluronic acid is dissolved in an organic solvent such as dimethyl sulfoxide is disclosed (Japanese Patent Laid-Open No. 3-105003). In addition, as a method that does not use a chemical reagent that forms a covalent bond, hyaluronic acid and a polymer compound having an amino group or imino group are ion-complexed with a carboxyl group of hyaluronic acid and an amino group or imino group of the polymer compound. A method for preparing a hyaluronic acid polymer composite by binding as a body is disclosed (see JP-A-6-73103). However, in such a conventional chemical modification method, the crosslinked hyaluronic acid fine particles must be used in a state of being dispersed in a solvent such as physiological saline. Is difficult, and the body retention period is not so long. Furthermore, it cannot be used in situ by in-situ injection at the required site using a syringe or spray device. However, the two-component reaction type hyaluronic acid gel of the present application can be injected in situ at a target site using a two-component mixing device or a spray device, and its use is simple.

  In recent years, there is an interest in regenerative medicine based on the development of the tissue engineering field. In this tissue engineering, a scaffold for tissue regeneration that helps the proliferation of various cells is important. Cultured skin, cultured cartilage and jawbone regeneration are known as applications to tissue engineering using hyaluronic acid gel. Examples thereof include water-insoluble medical materials comprising an ionic complex of hyaluronic acid and a polymer compound having an amino group or imino group (JP-A-6-73103), hyaluronic acid and a crosslinking agent di (or bis) hydrazine, A water-insoluble biocompatible material (Japanese Patent Laid-Open No. 9-59303) by a condensation reaction with di (or bis) hydrazide, and a poorly water-soluble hyaluronic acid gel to a tissue regeneration base material of cartilage tissue or tooth peripheral tissue Application (Japanese Patent Laid-Open No. 2003-10308) has been proposed. However, since these hyaluronic acid gels are used after seeding various cells after forming the gel in vitro, they cannot be used in situ because they cannot be injected with a syringe.

On the other hand, as an effective means for treating various arthropathy such as osteoarthritis and rheumatoid arthritis, a method of injecting an aqueous solution of hyaluronic acid into a diseased joint site has been adopted. “Alz (trade name)” (manufactured by Seikagaku Corporation, average molecular weight 900,000), “Hyalgan (trade name)” (Fidia, average molecular weight <500,000), “Svenir (trade name)” (Aventis Pharma / Chugai Pharmaceutical Co./Electrochemical Co., Ltd., average molecular weight 1.9 million) is known. In addition, “Synvisc (trade name)” (manufactured by Genzyme) has been developed in which hyaluronic acid is chemically cross-linked and polymerized to improve viscoelasticity. This cross-linked hyaluronic acid gel is a hyaluronic acid gel obtained by chemically cross-linking hyaluronic acid with a cross-linking agent divinylsulfone, and is a high run (US Pat. No. 4,713,448). In addition, single and mixed gels based on hyaluronic acid are described in US Pat. Nos. 4,582,865 and 4,605,691. The treatment using these joint treatment preparations requires injection to the patient's joint directly at a frequency of 3 to 5 times per week, which is extremely painful for the patient, and reduction thereof is desired.
International Publication WO2006 / 080523 JP 2003-10308 A

  The conventional reforming method has been particularly desired to be improved because it is difficult to store in a target place for a long period of time and the in-vivo storage period is short. Therefore, the present inventors provide a two-component reaction type hyaluronic acid gel that can be used in situ and solves these problems.

  Use high-toxicity low-molecular chemical cross-linking agents to maximize the benefits of biocompatibility inherent in hyaluronic acid itself, and to provide medical materials with superior safety to medical care. In addition, a means for providing a hyaluronic acid gel without forming a complex with a cationic polymer compound has not been developed so far. The inventors of the present application have found for the first time that a water-insoluble hyaluronic acid gel comprising hyaluronic acid alone is produced by a simple method without using a low-molecular cross-linking agent or the like. As a result of intensive studies on the possibility of application to therapeutic materials, carriers for DDS, cartilage tissues, and tissue regeneration scaffolds for tissue regeneration, the present invention was completed by finding its usefulness.

  The two-reactant type hydrous gel-forming agent for medical use of the present invention has a first reaction solution comprising an aqueous solution of an aldehyded hyaluronic acid having a weight average molecular weight of 1,000 to 3,000,000 and a weight average molecular weight of 1,000 to 3,000,000. It comprises a second reaction solution comprising an aqueous solution of an amino group-containing hyaluronic acid, and when the first and second reactants are mixed, the pH is 5.0 to 8.0. To do.

  The two-reactant type water-containing gel-forming agent for medical use of the present invention is used by applying it to the affected area in the form of an aqueous solution before or after mixing the two liquids, or in an uncured sol state. It may be applied to the affected area after being molded into a sheet-like, film-like, crushed, sponge-like, lump-like, fiber-like, or tube-like hydrated gel. It can also be used as a carrier for DDS. In this case, physiologically active substances include cell growth factors such as insulin, hepatocyte growth factor, transforming growth factor, nerve growth factor, epidermal growth factor, and platelet-derived growth. Factors, insulin-like growth factor, fibroblast growth factor, colony stimulating factor, erythropoietin, transferrin, interleukin, interferon and the like can be included. In addition, antibiotics, proteins, oligosaccharides or nucleic acids, gynecological disease treatment agents such as intrauterine drugs, intravaginal drugs and the like can be contained alone or in combination with other physiologically active substances.

  When the medical hydrogel of the present invention is used as a scaffold for cell tissue regeneration, the tissues to be regenerated include articular cartilage, costal cartilage, tracheal cartilage, pharyngeal cartilage, skull, alveolar bone, periodontal tissue, cementum, periodontal ligament, A tendon, a ligament, etc. can be mentioned. That is, almost any living tissue can be regenerated. At that time, chondrocytes, stem cells, bone marrow cells, osteoblasts, ES cells, etc. are seeded, the cells are cultured on a scaffold, proliferated, and then filled into the biological tissue defect part to regenerate the defective tissue. It can be used to promote

  Hyaluronic acid can be stored in the gel for a long period of time and applied as a medical material, for example, joint disease treatment material, adhesion prevention material, hemostatic agent, wound dressing material, pressure ulcer treatment material, artificial skin, biological tissue It can be suitably applied to a scaffold for regeneration, an auxiliary material for ophthalmic surgery, a carrier for drug delivery (DDS), and the like. Further, it can be expected to be applied to medical materials for surgical operation, for example, digestive organs, genitourinary mucosa and oral mucosa, or epithelial swelling of skin tissue in cosmetic surgery.

  Aldehydated hyaluronic acid forming the first reaction liquid is one in which hyaluronic acid is oxidized to introduce an aldehyde group, and has a weight average molecular weight in the range of 1,000 to 2,000,000. Hyaluronic acid includes hyaluronic acid and hyaluronate salts such as sodium hyaluronate (sodium salt), potassium hyaluronate, magnesium hyaluronate and calcium hyaluronate, and these may be used in combination. The aldehyde group can be introduced by a general periodate oxidation method, and preferably 0.01 to 1.0 aldehyde group, more preferably 0.02 to 0.0. Eight, more preferably 0.05 to 0.5 aldehyde groups are introduced.

  The hyaluronic acid used to obtain the aldehyded hyaluronic acid has a weight average molecular weight of preferably 2000 to 10 million, more preferably 2000 to 2 million. For example, cosmetic use and pharmaceutical use commercially available from Kibun Food Chemifa Co., Ltd. can be used. The optimum molecular weight of aldehyde-modified hyaluronic acid varies depending on the specific application, and the period until fluidization can be adjusted by selecting a specific molecular weight or molecular weight distribution. When the molecular weight of the aldehyded hyaluronic acid is excessively large, the sol-formation is excessively delayed. Moreover, when the molecular weight of aldehyde-ized hyaluronic acid is too small, the time which maintains a gelled state becomes too short.

The weight average molecular weight and molecular weight distribution of hyaluronic acid can be easily determined by a general aqueous GPC (gel filtration chromatography; formally size exclusion chromatography (SEC)) measurement. Specifically, a GPC column composed of a water-soluble polymer cross-linked product (TOSOH TSK gel G3000PW and G5000PW, TSK guard column PWH) is heated to 40 ° C., and a buffer solution (10 mM KH ₂ PO ₄ +10 mM K ₂ HPO ₄ ). Can be obtained by measurement using as an eluent.

  Aldehydated hyaluronic acid (first reaction solution) can be used after being introduced with aldehyde groups by periodate oxidation and then dissolved in distilled water via freeze-drying. In some cases, the powder can be dissolved in distilled water after being powdered by spray drying at a relatively low temperature while blowing an inert gas such as nitrogen under reduced pressure. Furthermore, aldehyde-modified hyaluronic acid (first reaction solution) can be freeze-dried and used in a powder state.

  The aminated hyaluronic acid constituting the second reactant has a weight average molecular weight of 2000 to 10 million, preferably 2000 to 2 million.

  As the aminated hyaluronic acid (second reaction liquid), the carboxyl group of hyaluronic acid is activated with carbodiimide, reacted with dihydrazine adipate, and then freeze-dried and dissolved in distilled water. In some cases, the powder can be dissolved in distilled water after being powdered by spray drying at a relatively low temperature while blowing an inert gas such as nitrogen under reduced pressure. Furthermore, aminated hyaluronic acid (second reaction solution) can be freeze-dried and used in a powder state. Both the first reactant and the second reactant may be in the form of powder. In this case, for example, after the powders are mixed with each other, the moisture in the living body is sucked and gelled. Alternatively, the first reactant powder and the second reactant powder may be mixed and gelled by adding water. The first reactant powder and the second reactant powder are uniformly mixed. Water may be added to the mixed powder mixed in to form a gel. Storage stability can be improved by making one or both of the first reactant and the second reactant into a powder form.

  If the molecular weight of the aminated hyaluronic acid is too high or contains too high a molecular weight compartment, the period until fluidization becomes too long.

  The molar ratio of aldehyde group / amino group in a state where the first reaction solution and the second reaction solution are mixed is 0.1 or more and less than 5, preferably 0.2 to 4.0, more preferably 1.0 to. 3.5. When the aldehyde group / amino group molar ratio is smaller than these ranges, the resulting gel decomposes too quickly, and when it is larger, rapid gelation cannot be achieved.

  When using the 2-reactant type hyaluronic acid gel of the present invention, mixing and application of the first reaction solution and the second reaction solution can be performed by various methods. For example, mixing can be performed by applying one of the first and second adhesive stock solutions to the surface of the adherend and then applying the other. Further, after the first reaction liquid and the second reaction liquid are mixed in the mixing chamber of the coating apparatus, spraying may be performed by ejecting from the nozzle, or the liquid may be fed from the slit of the applicator and applied. May be performed.

  When the first reaction solution and the second reaction solution are mixed, a Schiff bond is formed between the aldehyde group of the aldehyded hyaluronic acid and the amino group of the aminated hyaluronic acid, and this serves as a crosslinking point to form a network structure. A hydrogel having is formed. As a result, curing occurs from 2 to 600 seconds, preferably from 3 to 500 seconds, more preferably from 5 to 300 seconds after mixing. The preferred time from mixing to curing varies somewhat depending on the application.

  The hydrated gel-like cured adhesive layer or the hydrated gel-like resin produced by such a curing reaction changes to a liquid state by self-decomposition after the design liquefaction period. That is, even if it is in a water-containing state without undergoing enzymatic decomposition or the like in vivo, it is naturally decomposed and converted into a liquid state (flowable sol state). Therefore, in a living body, when a predetermined period is elapsed, it can be quickly absorbed or excreted and disappear. The design degradation period is arbitrarily set within a range of several hours to 4 months, usually within a range of 1 day to 1 month, particularly within a range of 2 days to 2 weeks.

  In contrast, in the case of an existing biodegradable resin that is decomposed and absorbed only by enzymatic decomposition in the living body, the decomposition period varies greatly, and it is rapidly decomposed after a necessary adhesion holding period. It was difficult.

  The decomposition period by self-decomposition can be arbitrarily adjusted and set by selecting or adjusting the molecular weight or distribution of aldehyded hyaluronic acid and / or aminated hyaluronic acid, and adjusting the pH when mixing two liquids. it can. In other words, the period of decomposition and absorption can be arbitrarily designed by adjusting the configuration of the two-component adhesive.

  The mechanism of autolysis without enzymatic degradation is not clear, but when the aldehyde group of aldehyde-modified hyaluronic acid binds to an amino group to form a Schiff base, the glucoside bond adjacent to the Schiff base is degraded in a hydrous state. We think that it became easy to receive.

  In addition, as the amino group-containing polymer compound that forms the second reaction solution, collagen, gelatin, albumin, chitosan, or multi-branched polyethylene glycol having an amino group introduced at the end thereof may be used instead of aminated hyaluronic acid. You can also. Here, in order to increase the reaction rate of gelation by mixing aqueous solutions of the first and second reactants or by mixing powder-liquid, more amino groups are added to the polymer chain such as collagen, gelatin, albumin and chitosan. Can also be introduced. Furthermore, as the multi-branched polyethylene glycol, one having a branch number (the number of functional groups in the starting material to which ethylene oxide is added) of 2 to 32 and a weight average molecular weight of 2,000 to 200,000 is used. it can.

  The hyaluronic acid gel obtained from the two-component reactive hydrogel of the present invention is used as a medical material as a joint disease treatment material, an adhesion prevention material, a hemostatic agent, a wound dressing material, a pressure ulcer treatment material, artificial skin, and biological tissue regeneration. The present invention can be suitably applied to scaffolds for ophthalmic use, ophthalmic surgery aids, drug delivery (DDS) carriers, and the like. It can also be applied to medical materials for surgery, for example, skin, digestive organs, genitourinary mucosa and oral mucosa, and epithelial swelling in cosmetic surgery. EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

  2 g of sodium hyaluronate having a molecular weight of 100,000 (Kibun Food Chemifa, FCH-SU, lot number: 5108) was dissolved in 200 ml of distilled water. Next, 0.2 g of sodium periodate (molecular weight: 213.89) was added and reacted at 50 ° C. with stirring for 3 hours. The solution after the reaction was dialyzed against distilled water for 24 hours (using a dialysis membrane having a molecular weight cut off of 14,000) and then freeze-dried to obtain 1.89 g of aldehyde-modified hyaluronic acid.

  1 g of sodium hyaluronate having a molecular weight of 100,000 (Kibun Food Chemifa, FCH-SU, lot number: 5108) was dissolved in 100 ml of distilled water. Next, 9.44 g of adipic acid dihydrazide (molecular weight 174.2) was added to adjust the pH of the solution to 4.75. To this, 2.08 g of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (molecular weight 191.7) was added to keep the pH of the solution at 4.75, and the reaction was conducted while stirring at 25 ° C. for 2 hours. I let you. The solution after the reaction was dialyzed against distilled water for 24 hours (using a dialysis membrane having a molecular weight cut off of 14,000) and then freeze-dried to obtain 0.700 g of aminated hyaluronic acid.

  Aldehydated hyaluronic acid obtained in Example 1 was dissolved in distilled water to prepare a 10% by weight aqueous solution, and aminated hyaluronic acid obtained in Example 3 was dissolved in distilled water to give 1% by weight. An aqueous solution of was prepared. 1 ml of each of these was added to a glass test tube having a diameter of 15 mm and mixed with a vortex mixer.

  Aldehydated hyaluronic acid obtained in Example 1 was dissolved in distilled water to prepare a 5 wt% aqueous solution, and aminated hyaluronic acid obtained in Example 3 was dissolved in distilled water to give 0.5%. A weight percent aqueous solution was prepared. 1 ml of each of these was added to a glass test tube having a diameter of 15 mm and mixed with a vortex mixer.

  Aldehydated hyaluronic acid obtained in Example 1 was dissolved in distilled water to prepare a 2.5% by weight aqueous solution, and aminated hyaluronic acid obtained in Example 3 was dissolved in distilled water to obtain 0%. A 25 wt% aqueous solution was prepared. 1 ml of each of these was added to a glass test tube having a diameter of 15 mm and mixed with a vortex mixer.

  Aldehydated hyaluronic acid obtained in Example 1 was dissolved in distilled water to prepare a 1.25 wt% aqueous solution, and aminated hyaluronic acid obtained in Example 3 was dissolved in distilled water to obtain 0%. A 125 wt% aqueous solution was prepared. 1 ml of each of these was added to a glass test tube having a diameter of 15 mm and mixed with a vortex mixer.

The gelation time when the two liquids were mixed in Examples 4 to 7 and the time until the shape changed from gel to sol at 25 ° C. were examined visually. The results are shown in Table 1.

  From Table 1, when the concentration of the two liquids is high, the gelation time tends to be short and the solation time tends to be long. That is, when the concentration is high, the crosslinking density increases, so the time until sol formation is also delayed. In contrast to the conventional hyaluronic acid cross-linking, the morphology could not be maintained for more than one month, whereas in the present invention the gel state could be maintained for more than 28 days.

As a scaffold for biological tissue regeneration, cells (about 2 × 10 ⁸ cells) collected from the bone marrow of a 12-week-old rabbit were placed in the aminated hyaluronic acid aqueous solution prepared in Example 2, and the aldehyde-modified hyaluronic acid of Example 1 was used. The aqueous solution was injected into the defect cartilage on the femoral side of the knee joint of a female Japanese white rabbit 5 months old using a mixing device. As a result, significant regeneration of cartilage and other bones was observed after 12 weeks.

Bone marrow-derived mesenchymal stem cells (approximately 2 × 10 ⁸ cells) are placed in an aminated hyaluronic acid aqueous solution in the same manner as in Example 8, and the aldehyde-treated hyaluronic acid aqueous solution is mixed with the beagle canine alveolar bone defect using a mixing device. Transplanted. As a result, remarkable alveolar bone, cementum and periodontal ligament regeneration were observed after 3 months.

  As a simple substance for DDS, danazol for treating endometriosis is suspended in the aminated hyaluronic acid aqueous solution prepared in Example 2, and the aldehyde-containing hyaluronic acid aqueous solution of Example 1 is extruded using a mixing device, whereby danazol-containing cyclic A ring-shaped hyaluronic acid gel preparation was prepared. An in vitro sustained release test of a drug (danazol) contained in the obtained gynecological disease treatment agent was conducted in 3 L of 0.14 mol / L phosphate buffer (pH 7.4) containing 10 units / ml of bovine testicular hyaluronidase. The mixture was stirred at 37 ° C. for several tens of days, and the amount of danazol released into the liquid every day was measured by a liquid chromatography method. As a result, the release was stably maintained at about 200 μg / day.

  In order to confirm the efficacy of the two-component reactive hyaluronic acid gel as an intra-articular treatment, 5 Japanese white rabbits (weight approximately 3 kg) were anesthetized and the aminated hyaluronic acid prepared in Example 2 was applied to the right knee joint. The acid aqueous solution and the aldehyde-treated hyaluronic acid aqueous solution of Example 1 were injected using a mixing device. Four weeks later, all the cases were sacrificed and the knee joint portion into which the hyaluronic acid aqueous solution was injected was observed. As a result, hyaluronic acid gel remained in all knee joints.

  As described above, the two-component reactive hyaluronic acid gel of the present invention is excellent in biocompatibility and safety, and can be injected in situ at a target site using a two-component mixing device or a spray device. As a medical material, joint materials, anti-adhesion materials, hemostatic agents, wound dressings, pressure ulcer treatment materials, artificial skin, biological tissue regeneration scaffolds, ophthalmic surgical aids, and drug delivery ( It can be suitably applied to a carrier for DDS). It can also be applied to medical materials for surgery, for example, skin, digestive organs, genitourinary mucosa and oral mucosa, and epithelial swelling in cosmetic surgery.

Claims (9)

A first reactant comprising an aldehyde-treated hyaluronic acid having a weight average molecular weight of 1,000 to 3,000,000 and a second reactant comprising an amino group-containing hyaluronic acid having a weight average molecular weight of 1,000 to 3,000,000. 1 reactant and 2 reactant type medical hydrogel forming agent, characterized in that the second reactant is capable of forming a hydrogel by reacting in the presence of water.   The aldehyde-modified hyaluronic acid is obtained by oxidizing hyaluronic acid having a weight average molecular weight of 1000 to 3 million with periodic acid or periodate to introduce an aldehyde group. A two-agent type water-containing gel-forming agent for medical use. The amino group-containing hyaluronic acid is obtained by introducing an amino group into hyaluronic acid having a weight average molecular weight of 1,000 to 3,000,000 using adipic acid dihydrazide or other dihydrazide compounds. The two-reagent-type medical hydrogel forming agent as described. A first reactant made of aldehyded hyaluronic acid having a weight average molecular weight of 1,000 to 3,000,000 and a second reactant made of aminated hyaluronic acid having a weight average molecular weight of 1,000 to 3,000,000 are reacted in the presence of water. A water-containing gel-like medical resin obtained by the treatment, and if stored in a water-containing state, after passing through a settable gel state holding period, it is changed into a sol state by self-decomposition. Resin. The two-reagent-type medical hydrogel according to any one of claims 1 to 3, wherein the first and second reactants are sterilized by irradiation with an electron beam of 10 to 50 KGy. Forming agent. The first reactant and the second reactant are both aqueous solutions, the first reactant is powder and the second reactant is liquid, or both the first reactant and second reactant are powder (powder- The two-reactant type hydrous gel-forming agent for medical use according to any one of claims 1 to 3 and 5 . The two-reactant type hydrous gel-forming agent for medical use according to any one of claims 1 to 3, which is in the form of a mixed powder obtained by mixing a powder of the first reactant and a powder of the second reactant . The aldehyde-modified hyaluronic acid has 0.01 to 1.0 aldehyde groups introduced per repeating unit, and the molar ratio of aldehyde groups / amino groups is 0.1 or more and less than 5. And a two-reactant type hydrous gel-forming agent for medical use according to any one of 5 to 7 . It is obtained from the medical hydrous gel-forming agent according to any one of claims 1 to 3 and 5 to 8, and is used for joint disease treatment material, adhesion prevention material, hemostatic agent, wound dressing material, pressure ulcer treatment material, artificial skin, and biological tissue regeneration. A hydrogel used as a scaffold, an ophthalmic surgical aid, or a drug delivery (DDS) carrier.