WO2012026468A1 - 架橋ヒアルロン酸組成物及び自己架橋ヒアルロン酸粒子 - Google Patents
架橋ヒアルロン酸組成物及び自己架橋ヒアルロン酸粒子 Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- 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
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
- C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/87—Application Devices; Containers; Packaging
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/91—Injection
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T428/00—Stock material or miscellaneous articles
- Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
- Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
Definitions
- the present invention relates to a crosslinked hyaluronic acid composition, self-crosslinked hyaluronic acid particles used therefor, an injection containing the crosslinked hyaluronic acid composition, and a prefilled syringe preparation containing the same.
- Hyaluronic acid is a linear polymer polysaccharide in which ⁇ -DN-acetylglucosamine and ⁇ -D-glucuronic acid are alternately bound. Since hyaluronic acid exhibits excellent biocompatibility and viscoelasticity, it has been developed into the medical field. As one of them, Patent Document 1 discloses the use of crosslinked hyaluronic acid as a viscosity replenisher for knee osteoarthritis.
- an object of the present invention is to provide a crosslinked hyaluronic acid composition capable of obtaining a sufficient therapeutic effect for osteoarthritis of the knee even when the administration frequency is reduced as compared with the conventional art, and self-crosslinked hyaluronic acid particles used therefor Is to provide.
- the present invention relates to a crosslinked hyaluronic acid composition containing self-crosslinked hyaluronic acid particles having an equilibrium swelling ratio of 3 to 10 times, and an aqueous solvent, and the self-crosslinked hyaluronic acid with respect to the total volume of the crosslinked hyaluronic acid composition Providing a crosslinked hyaluronic acid composition wherein the dry weight of the particles is 3-8 w / v%.
- a high concentration for example, 3 to 8 w / v% by dry weight
- the viscosity increased rapidly and it was very difficult to inject from the syringe (syringe) into the affected area.
- the molecular weight and concentration of hyaluronic acid or cross-linked hyaluronic acid must be reduced, and a sufficient therapeutic effect for knee osteoarthritis can be obtained with a small number of administrations. I could not.
- the viscosity does not increase rapidly even at a high concentration. Therefore, the crosslinked hyaluronic acid composition was applied to an injection. In some cases, a sufficient therapeutic effect for osteoarthritis of the knee can be obtained even with a small number of administrations.
- the self-crosslinked hyaluronic acid particles preferably have an average volume particle size of 10 to 100 ⁇ m.
- the viscosity does not increase rapidly even at high concentrations, so the crosslinked hyaluronic acid composition is applied to injections. In this case, a sufficient therapeutic effect for knee osteoarthritis can be obtained even with a small number of administrations.
- the self-crosslinked hyaluronic acid particles have a self-crosslinked esterification degree of 0.05 to 0.50 mol%. It is preferable.
- Self-crosslinked hyaluronic acid particles are made of self-crosslinked hyaluronic acid crosslinked by an ester bond that is easily hydrolyzed under in vivo conditions. When the ester bond is hydrolyzed, molecular hyaluronic acid is generated.
- the molecular weight of hyaluronic acid produced by hydrolysis (defined as primary molecular weight and represented by viscosity average molecular weight) is preferably 800,000 or more in view of its therapeutic effect.
- the self-crosslinked hyaluronic acid particles have an ethyl ester amount of 0.05 mol% or less and a self-crosslinked esterification degree of 0.05 to 0.50 mol%. Is preferred.
- self-crosslinked hyaluronic acid particles having an ethyl ester amount, a degree of self-crosslinking esterification, and an equilibrium swelling ratio within the above predetermined range the viscosity does not increase rapidly even at a high concentration. When applied to an injection, a sufficient therapeutic effect on knee osteoarthritis can be obtained even with a small number of administrations.
- the self-crosslinked hyaluronic acid particles are crosslinked by ester bonds that are easily hydrolyzed under in vivo conditions.
- the molecular weight of hyaluronic acid produced by hydrolysis (defined as primary molecular weight and represented by viscosity average molecular weight) is preferably 800,000 or more in view of its therapeutic effect.
- Self-crosslinking hyaluronic acid particles having an average volume particle size of 10 to 100 ⁇ m, an equilibrium swelling ratio of 3 to 10 times, a primary molecular weight of 800,000 or more, and a self-crosslinking esterification degree of 0.05 to 0.50 mol% are used.
- self-crosslinked hyaluronic acid particles having an ethyl ester amount of 0.05 mol% or less, a degree of self-crosslinking esterification of 0.05 to 0.50 mol%, an equilibrium swelling ratio of 3 to 10 times, and a primary molecular weight of 800,000 or more By using it, the effect of reducing the number of administrations and treating knee osteoarthritis becomes more prominent.
- the entire amount of crosslinked hyaluronic acid can be dissolved within 30 days.
- High molecular weight hyaluronic acid can be produced in this manner, thereby obtaining a high therapeutic effect.
- the viscosity measured at 25 ⁇ 2 ° C. and a shear rate of 50 S ⁇ 1 can be reduced to 300 mPa ⁇ s or less by the rotational viscosity measuring method using a cone plate, so that injection as an injection becomes easy.
- the present invention provides an injection containing the above-mentioned crosslinked hyaluronic acid composition.
- conventional hyaluronic acid-based injections as described above, when the concentration of hyaluronic acid is increased to 3 to 8 w / v%, the viscosity rapidly increases and it is very difficult to inject from the syringe (syringe) into the affected area. Met.
- the injection according to the present invention uses a crosslinked hyaluronic acid composition containing self-crosslinked hyaluronic acid particles having the above-mentioned predetermined physical properties, sufficient osteoarthritis of the knee can be achieved even with a small number of administrations. A therapeutic effect can be obtained.
- the injection used is provided.
- the concentration of hyaluronic acid is high, the viscosity increases and it is difficult to inject from the syringe into the affected area. Therefore, an amount of hyaluronic acid that produces a sufficient therapeutic effect could not be administered at a time.
- the injection according to the present invention uses a crosslinked hyaluronic acid composition containing self-crosslinked hyaluronic acid particles having the above-described physical properties, it is 1.25 mg / kg body weight or more per time or 75 mg or more per time. It can be used so that self-crosslinking hyaluronic acid is administered, and even with a single administration, a sufficient therapeutic effect on knee osteoarthritis can be obtained.
- the present invention provides a prefilled syringe preparation containing the above injection.
- a prefilled syringe preparation containing a conventional hyaluronic acid-based injection has a very high viscosity when hyaluronic acid is used at a high concentration (for example, 3 to 8 w / v%) as described above. It was difficult to inject.
- the prefilled syringe preparation according to the present invention contains a crosslinked hyaluronic acid composition containing self-crosslinked hyaluronic acid particles having the above-mentioned predetermined physical properties as an injection inside the syringe, so that the number of administrations is small. However, a sufficient therapeutic effect for knee osteoarthritis can be obtained.
- the present invention also provides self-crosslinked hyaluronic acid particles having an average volume particle size of 10 to 100 ⁇ m and an equilibrium swelling ratio of 3 to 10 times.
- the self-crosslinked hyaluronic acid particles having the predetermined physical properties described above when the crosslinked hyaluronic acid composition containing the particles is applied to an injection, the viscosity rapidly increases even at a high concentration of 3 to 8 w / v%. Therefore, even with a small number of administrations, a sufficient therapeutic effect for osteoarthritis of the knee can be obtained.
- the self-crosslinked hyaluronic acid particles of the present invention having an average volume particle size and an equilibrium swelling ratio within the above predetermined range have a primary molecular weight of 800,000 or more and a degree of self-crosslinking esterification of 0.05 to 0.50 mol%. It is preferable. By using such self-crosslinked hyaluronic acid particles, the number of administrations is reduced and the therapeutic effect of knee osteoarthritis becomes more remarkable.
- the present invention also provides self-crosslinked hyaluronic acid particles having an ethyl ester amount of 0.05 mol% or less, a degree of self-crosslinking esterification of 0.05 to 0.50 mol%, and an equilibrium swelling ratio of 3 to 10 times.
- the self-crosslinked hyaluronic acid particles having the predetermined physical properties described above when the crosslinked hyaluronic acid composition containing the particles is applied to an injection, the viscosity rapidly increases even at a high concentration of 3 to 8 w / v%. Therefore, even with a small number of administrations, a sufficient therapeutic effect for osteoarthritis of the knee can be obtained.
- the primary molecular weight of the self-crosslinked hyaluronic acid particles of the present invention in which the amount of ethyl ester, the degree of self-crosslinking esterification, and the equilibrium swelling ratio are within the predetermined ranges are preferably 800,000 or more.
- a crosslinked hyaluronic acid composition capable of obtaining a sufficient therapeutic effect for osteoarthritis of the knee even when the number of administrations is less than before, and self-crosslinked hyaluronic acid particles used therefor can do.
- the crosslinked hyaluronic acid composition according to the first embodiment of the present invention contains self-crosslinked hyaluronic acid particles having an average volume particle size of 10 to 100 ⁇ m and an equilibrium swelling ratio of 3 to 10 times.
- the crosslinked hyaluronic acid composition according to the second embodiment of the present invention has an ethyl ester amount of 0.05 mol% or less, a self-crosslinked esterification degree of 0.05 to 0.50 mol%, and an equilibrium swelling. It contains self-crosslinked hyaluronic acid particles having a magnification of 3 to 10 times.
- the self-crosslinking hyaluronic acid particles are particles obtained by granulating self-crosslinking hyaluronic acid.
- self-crosslinked hyaluronic acid refers to hyaluronic acid having a crosslinked structure, and tertiary carboxyl groups in the hyaluronic acid molecule are self-esterified with the hydroxyl groups of the same and / or different hyaluronic acid molecules.
- the original network structure is formed, and those using a chemical cross-linking agent or a chemical modifier are not included.
- the degree of self-crosslinking esterification in self-crosslinked hyaluronic acid particles is expressed in mol% as the ratio of the integral value of the chemical shift peak derived from the crosslinked ester to the integral value of the chemical shift peak derived from the main chain structure of hyaluronic acid. It is a thing.
- the degree of self-crosslinking esterification in the self-crosslinking hyaluronic acid particles according to the first embodiment of the present invention is preferably 0.05 to 0.50 mol%. More preferably, it is 0.08 to 0.30 mol%.
- the degree of self-crosslinking esterification in the self-crosslinking hyaluronic acid particles according to the second embodiment of the present invention is 0.05 to 0.50 mol%. Preferably, it is 0.08 to 0.30 mol%.
- the amount of ethyl ester in the self-crosslinked hyaluronic acid particles according to the first embodiment of the present invention is preferably 0.05 mol% or less.
- the amount of ethyl ester in the self-crosslinked hyaluronic acid particles according to the second embodiment of the present invention is 0.05 mol% or less.
- the amount of ethyl ester is the content of ethyl ester contained in the self-crosslinked hyaluronic acid particles, and it is considered that ethanol contained in the hyaluronic acid raw material as a residual solvent was ethyl esterified.
- the ethanol contained in the hyaluronic acid raw material is removed by freezing and vacuum drying, 80 ° C. ventilation, acetone washing or room temperature ventilation for 2 to 3 days.
- the amount of ethyl ester in the self-crosslinked hyaluronic acid particles can be 0.05 mol% or less.
- the content of ethanol contained in the hyaluronic acid raw material can be measured by extraction with acetonitrile using a gas chromatography / mass spectrometer (GC-MS).
- the amount of ethyl ester in the self-crosslinked hyaluronic acid particles is more preferably 0.03 mol% or less.
- the amount of ethyl ester can be measured by NMR similarly to the degree of self-crosslinking esterification.
- hyaluronic acid can be used regardless of its origin, whether it is extracted from animal tissue or manufactured by fermentation.
- the strain used in the fermentation method is a microorganism having the ability to produce hyaluronic acid such as Streptococcus genus isolated from the natural world, or Streptococcus equii FM-100 described in JP-A No. 63-123392 (90-year-old Microtechnical Laboratories No. 9027). No.) and Streptococcus equii FM-300 described in Japanese Patent Application Laid-open No. Hei 2-23489 are desirable. Those cultured and purified using the above mutant strains are used.
- Hyaluronic acid is also used in a concept including its alkali salts, such as sodium, potassium and lithium salts.
- Self-crosslinking hyaluronic acid is obtained by coexisting hyaluronic acid, water having a hyaluronic acid concentration of 5% by mass or more, and a carboxyl group of hyaluronic acid in an equimolar amount or more and maintaining the coexistence state at a low temperature. It is done.
- the acid coexisting with hyaluronic acid is not particularly limited, and any known acid can be used, but it is preferably an acid stronger than hyaluronic acid, and more preferably an inorganic acid. More preferred are hydrochloric acid, nitric acid, and sulfuric acid. Among these, nitric acid that is excellent in handling and the like is particularly preferred.
- the amount of the coexisting acid is not particularly limited, but can be an amount of the acid component equimolar or more with the carboxyl group of hyaluronic acid.
- the acid coexisting with hyaluronic acid is preferably retained in such an amount that hyaluronic acid is contained in an amount of 15% by mass or more, preferably 20% by mass or more and 40% by mass or less.
- This holding can be performed at a temperature between -30 ° C and 25 ° C for any period between 1 hour and 20 days. Particularly preferably, it can be carried out at a temperature between ⁇ 25 ° C. and ⁇ 5 ° C. for 1 day to 15 days.
- Mixing with the coexisting acid with the hyaluronic acid allows the coexisting acid to be kneaded so that the hyaluronic acid is 15% by mass or more, preferably 20% by mass or more of the whole, and the coexisting acid can be made uniform.
- the acid coexisting with hyaluronic acid is 15% by mass or more, preferably 20% by mass or more.
- the acidic aqueous solution of hyaluronic acid prepared at a low concentration can be concentrated so that hyaluronic acid is contained at 15% by mass or more, preferably 20% by mass or more of the whole.
- the average volume particle size of the self-crosslinking hyaluronic acid particles according to the first embodiment of the present invention is 10 to 100 ⁇ m, preferably 20 to 80 ⁇ m, more preferably 40 to 70 ⁇ m.
- the average volume particle size of the self-crosslinked hyaluronic acid particles according to the second embodiment of the present invention is preferably 10 to 100 ⁇ m, more preferably 20 to 80 ⁇ m, and further preferably 40 to 70 ⁇ m.
- the average volume particle diameter is equivalent to the diameter of a circle having the same area as the particle image projected when the particles are photographed, for example Calculate as diameter.
- the average volume particle diameter is equivalent to the diameter of a circle having the same area as the particle image projected when the particles are photographed, for example Calculate as diameter.
- the total volume of 10,000 particles can be employed.
- a particle size / shape distribution measuring instrument PITA-1 (trade name, manufactured by Seishin Enterprise) can be used.
- Self-crosslinked hyaluronic acid particles can be produced by making a mixed liquid of self-crosslinked hyaluronic acid and an aqueous solvent into a fine particle by passing through a slit while applying a shearing force at less than 50 ° C.
- the self-crosslinked hyaluronic acid can be pulverized using a high-speed rotating device that passes through a slit while applying a shearing force to form fine particles, so that the average volume particle size can be reduced to 10 to 100 ⁇ m.
- FIG. 1 is a schematic configuration diagram of a high-speed rotating device used for producing self-crosslinked hyaluronic acid particles according to the present invention.
- the high-speed rotating device 10 includes a rotor 1 and a screen 2, and the rotor 1 and the screen 2 rotate in opposite directions, and the self-crosslinking hyaluronic acid passes through the slits 3 of the screen 2 to be atomized as self-crosslinking hyaluronic acid particles. To do.
- a large shearing force can be obtained, and atomized self-crosslinked hyaluronic acid particles can be obtained.
- the self-crosslinking hyaluronic acid when the self-crosslinking hyaluronic acid is atomized by the high-speed rotating device 10, the main chain of hyaluronic acid constituting the fine particles can be efficiently crushed without being randomly cut. However, it is presumed that self-crosslinked hyaluronic acid particles having low viscosity can be obtained.
- Creamics double motion (trade name, manufactured by M Technique Co., Ltd.) is preferable.
- This device is composed of a rotor that rotates at high speed and a screen that surrounds the rotor. A large velocity gradient in the vicinity of the rotor surface that rotates at high speed causes the self-crosslinked hyaluronic acid to pass through the liquid passage holes (slits) of the screen. Large particles are sheared and atomized.
- the degree of atomization by the high-speed rotating device is specified as shown in Table 1 according to the rotation speed of the rotor and the screen and the processing time.
- the ratio of the rotational speed of the screen to the rotational speed of the rotor is preferably 50 to 100%, particularly 90%.
- the high-pressure type crusher for example, there is Nanomizer (trade name, manufactured by Nanomizer Co., Ltd.), but the high-pressure and high-speed sample tends to become high temperature, and the physical property deterioration degree of self-crosslinked hyaluronic acid is large. It is not preferable to use for the atomization treatment for the self-crosslinked hyaluronic acid particles according to the present invention. Even in a high-speed rotating device, for example, T.W. K. An apparatus that cannot atomize particles to an average volume particle size of 200 ⁇ m or less, such as homomic (trade name, manufactured by Primix Co., Ltd.), is used for atomization treatment for self-crosslinked hyaluronic acid particles according to the present invention. It is not preferable.
- physical property deterioration means that the physical properties such as 60 ° C solubility half-life and primary molecular weight of self-crosslinked hyaluronic acid before crushing are inferior to the initial values before crushing due to particle formation (atomization) such as crushing. Say. It is preferable that the physical properties of the self-crosslinked hyaluronic acid particles by the atomization treatment are not deteriorated as much as possible.
- the 60 ° C. solubility half-life is the time until the gel fraction, which is 100% at the start, reaches 50% when self-crosslinked hyaluronic acid is heated under the conditions of 60 ° C. and pH 7.4.
- the initial value retention is 80%, This means that 20% of physical property deterioration has occurred due to particle formation.
- the primary molecular weight of the self-crosslinked hyaluronic acid particles of the present invention is the molecular weight of hyaluronic acid produced by hydrolysis of the ester bond of the self-crosslinked hyaluronic acid particles, preferably 800,000 or more, and more than 800,000 to 3 million It is more preferable to be within the range.
- a material having a primary molecular weight within the above-mentioned range can be preferably used from a high molecular weight to a low molecular weight obtained by hydrolysis.
- the above primary molecular weight is expressed as a viscosity average molecular weight.
- a differential refractometer is used as a detector for GPC, and the retention time at the peak top of the molecular weight distribution. It can be calculated from For the calculation of the viscosity average molecular weight from the retention time, a calibration curve created using the retention time at the peak top of the molecular weight distribution of hyaluronic acid with a known viscosity average molecular weight is used.
- the viscosity average molecular weight of hyaluronic acid used for preparing a calibration curve is obtained by dissolving hyaluronic acid with a 0.2M sodium chloride solution, measuring the flow-down time at 30 ° C. using an Ubbelohde viscometer, and calculating from the obtained reduced viscosity.
- the equilibrium swelling ratio of the self-crosslinked hyaluronic acid particles was determined by the volume of the self-crosslinked hyaluronic acid particles when the aqueous solvent (buffer solution) of the crosslinked hyaluronic acid composition was removed by filtration, and the self-crosslinked hyaluronic acid particles were further dried. Expressed by the magnification of
- the equilibrium swelling ratio is a ratio of the wet weight of the self-crosslinked hyaluronic acid particles when the aqueous solvent (buffer solution) of the crosslinked hyaluronic acid composition is removed by filtration to the weight of the self-crosslinked hyaluronic acid particles when further dried.
- (Qw) and density it can be calculated by the following equation (2).
- Equilibrium swelling ratio 1 + ( ⁇ / ⁇ 0 ) ⁇ (Qw ⁇ 1) (2) ( ⁇ : density of self-crosslinking hyaluronic acid particles, ⁇ 0 : density of aqueous solvent (buffer))
- the equilibrium swelling ratio is affected by the salt concentration, pH, temperature, swelling time, etc. of the solvent.
- 10 mM phosphate buffered saline (pH 6.0) and the NaCl concentration is 0.9 wt. % Can be used and can be measured after swelling at 5 ° C. for 1 day and reaching an equilibrium swollen state.
- the method for removing the solvent of the crosslinked hyaluronic acid composition by filtration is not particularly limited, and for example, a centrifugal filtration method using a centrifugal filter unit, a vacuum filtration method using a membrane filter, or the like can be appropriately used.
- the equilibrium swelling ratio of the self-crosslinked hyaluronic acid particles of the present invention is 3 to 10 times, preferably 4 to 8 times.
- the equilibrium swelling ratio of the self-crosslinked hyaluronic acid particles is in the above range, there is no inconvenience that the self-crosslinked hyaluronic acid particles cannot be discharged from the syringe due to excessive swelling when contained in the crosslinked hyaluronic acid composition. It becomes possible to inject.
- the dry weight of the self-crosslinked hyaluronic acid particles with respect to the total volume of the crosslinked hyaluronic acid composition is 3 to 8 w / v%.
- 3 w / v% indicates the concentration of self-crosslinked hyaluronic acid particles in the crosslinked hyaluronic acid composition.
- the dry weight of the self-crosslinked hyaluronic acid particles with respect to the total volume of the crosslinked hyaluronic acid composition is preferably 3 to 7 w / v%.
- the concentration of self-crosslinked hyaluronic acid particles can be quantified as follows, for example. First, the crosslinked hyaluronic acid suspension is diluted with distilled water, a sodium hydroxide solution is added, and the mixture is allowed to stand at room temperature to hydrolyze the ester crosslinking of the self-crosslinked hyaluronic acid and dissolve the self-crosslinked hyaluronic acid. . Next, hydrochloric acid is added to the solution for neutralization, and then the glucuronic acid concentration is quantified by the carbazole sulfate method. Using this glucuronic acid concentration and a known concentration of hyaluronic acid as a standard substance, the concentration of self-crosslinked hyaluronic acid particles can be calculated.
- crosslinked hyaluronic acid composition of the present invention when immersed in 10 mM phosphate buffered saline at pH 7.0 ⁇ 0.5 and temperature 36.0 ⁇ 2.0 ° C., the entire amount of self-crosslinked hyaluronic acid dissolves within 30 days.
- Hyaluronic acid having a viscosity average molecular weight of 800,000 or more is produced.
- Such hyaluronic acid production conditions are typically immersion in 10 mM phosphate buffered saline at pH 7.4 and temperature of 37.0 ° C.
- the aqueous solvent contained in the crosslinked hyaluronic acid composition is used in the crosslinked hyaluronic acid composition so that the dry weight of the self-crosslinked hyaluronic acid particles is 3 to 8 w / v% with respect to the total volume of the crosslinked hyaluronic acid composition. It is a solvent contained in The aqueous solvent may be an aqueous solution having a physiologically acceptable aqueous medium. Physiologically acceptable means that when the joint therapeutic agent is injected into the joint cavity, the aqueous medium itself does not cause undesirable effects or side effects, such as tissue swelling or shrinkage, inflammation, and the like.
- Physiologically acceptable aqueous media are usually inorganic salts such as alkali or alkaline earth metal chlorides, sulfates, phosphates or bicarbonates such as sodium chloride, sodium sulfate, magnesium chloride, and the corresponding potassium.
- inorganic salts such as alkali or alkaline earth metal chlorides, sulfates, phosphates or bicarbonates such as sodium chloride, sodium sulfate, magnesium chloride, and the corresponding potassium.
- organic acid salts such as calcium salt, sodium lactate, sodium acetate, or neutral organic substances such as glucose, mannose, polyhydric alcohol, such as glycerin, mannitol, etc. It is an aqueous solution.
- the preparation may be prepared according to a pharmacologically known method by using an appropriate excipient; an isotonic agent; an antiseptic; an emulsifier; a dispersant; a stabilizer; a solubilizing agent; Antioxidants; low molecular weight (less than about 10 residues) polypeptide (eg, polyarginine or tripeptide); protein (eg, serum albumin, gelatin, or immunoglobulin); hydrophilic polymer (eg, polyvinylpyrrolidone); Amino acids (eg, glycine, glutamic acid, aspartic acid, or arginine); chelating agents (eg, EDTA); counter ions (eg, sodium); and / or non-ionic surfactants (eg, polysorbates, poloxamers), etc.
- a pharmaceutical additive, hyaluronic acid, etc. can be mixed suitably and can be prepared. Such substances that enhance isotonicity and chemical stability are non-toxic
- the number of days to reach the gel fraction can be used.
- the number of days to reach the gel fraction is the number of days until the gel fraction is lowered to the reference value when the crosslinked hyaluronic acid composition is allowed to stand under certain measurement conditions and gradually released.
- a water-based solvent is added to self-crosslinked hyaluronic acid, and a crosslinked hyaluronic acid suspension adjusted to a certain concentration is heated in a certain environment to obtain a predetermined gel fraction (for example, 97% gel fraction or 95% gel fraction).
- the time required to reach the gel fraction can be determined as the number of days reaching the gel fraction.
- the equilibrium sedimentation concentration is the concentration of hyaluronic acid contained in the precipitate when the crosslinked hyaluronic acid suspension is allowed to stand and the self-crosslinked hyaluronic acid particles are completely precipitated.
- the equilibrium sedimentation concentration can be regarded as the upper limit of the hyaluronic acid concentration. That is, a crosslinked hyaluronic acid suspension having a hyaluronic acid concentration equal to the equilibrium sedimentation concentration indicates that the entire suspension is precipitated and has no supernatant, and the hyaluronic acid concentration cannot be increased any further.
- the viscosity of the crosslinked hyaluronic acid composition is preferably 300 mPa ⁇ s or less at 25 ⁇ 2 ° C. and a shear rate of 50 S ⁇ 1 .
- the viscosity of the crosslinked hyaluronic acid composition is 300 mPa ⁇ s or less, it can be easily injected when injected into a living body using an injector as an injection, and the burden on the patient is reduced.
- the viscosity of the crosslinked hyaluronic acid composition can be measured, for example, by using a rotational viscosity measurement method.
- the discharge pressure of the crosslinked hyaluronic acid composition was as follows: a syringe having a diameter of 0.45 cm with a 23 G needle having an inner diameter of 0.40 mm and a needle length of 25 mm, and a temperature of 25 ° C. and an injection speed of 50 mm / min. It is preferably 8N or less, more preferably 0.2 to 0.8N, still more preferably 0.2 to 0.6N.
- the cross-linked hyaluronic acid composition having a discharge pressure in the above range can be easily injected when injected into a living body using an injector (syringe) as an injection, thereby reducing the burden on the patient.
- the discharge pressure of the crosslinked hyaluronic acid composition should be measured with an extrusion pressure measuring device by filling the syringe with the crosslinked hyaluronic acid composition, attaching an injection needle, and pushing out the syringe (syringe) at a predetermined speed. Can do.
- an extrusion pressure measuring device a static compression testing device used in a general material test can be used.
- the injection of the present invention contains the above-mentioned crosslinked hyaluronic acid composition. Since the injection using the present crosslinked hyaluronic acid composition has a low viscosity and a low discharge pressure, it is easy to inject when injected into a living body.
- the injection of the present invention is preferably used so that 1.25 mg / kg body weight or more per administration, or 75 mg or more of the self-crosslinking hyaluronic acid per administration is administered.
- Conventionally used hyaluronic acid joint preparations have a hyaluronic acid concentration of about 1 w / v% and the dose per administration is about 25 mg.
- the injection according to the present invention has the above-mentioned prescribed physical properties. Since the cross-linked hyaluronic acid composition containing self-cross-linked hyaluronic acid particles is used, the concentration can be 3 w / v% or higher, so that 75 mg or more of self-cross-linked hyaluronic acid is administered at a time. Can be done. Further, when the average human body weight is 60 kg, self-crosslinking hyaluronic acid of 1.25 mg / kg body weight or more can be administered at a time.
- the injection is preferably used so that 1.7 mg / kg body weight or more per administration, or 100 mg or more of the self-crosslinking hyaluronic acid per administration is administered, more preferably 2.0 mg per administration. / Kg body weight or more, or 120 mg or more of the self-crosslinking hyaluronic acid per administration.
- the upper limit is 4.2 mg / kg body weight or less per dose, or 250 mg or less per dose of the self-crosslinked hyaluronic acid, more preferably 3.3 mg / kg per dose. It is used so that the above self-crosslinked hyaluronic acid is administered in an amount of not more than kg body weight or not more than 200 mg per one time.
- the prefilled syringe preparation of the present invention contains the above-mentioned injection in the syringe barrel, and the injection using the present crosslinked hyaluronic acid composition as described above has a low viscosity and discharge pressure.
- Cross-linked hyaluronic acid can be easily injected into the living body.
- the crosslinked hyaluronic acid composition of the present invention can be administered to a living body by any appropriate administration route. It is preferably done parenterally and is preferably prepared as an injection.
- the crosslinked hyaluronic acid composition according to the present invention is administered to a rabbit joint as an injection, it is confirmed that hyaluronic acid having a viscosity average molecular weight of 800,000 or more is generated in the living body. It is considered that the crosslinking point is cut by the pH and temperature in the body, and hyaluronic acid is generated in the joint.
- the administration of the crosslinked hyaluronic acid composition of the present invention can be carried out parenterally, intramuscularly, subcutaneously, etc., and particularly preferably directly into a tissue such as a joint cavity.
- the crosslinked hyaluronic acid composition of the present invention can be a drug that is contained in an amount effective to achieve the intended purpose of hyaluronic acid, and the “therapeutically effective amount” or “pharmacologically effective amount” An amount of a drug that is well recognized by those skilled in the art and effective to produce a pharmacological result. The determination of a therapeutically effective dose is well known to those skilled in the art.
- the therapeutically effective amount refers to the amount of a drug that reduces the disease state by administration.
- the therapeutic effect and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
- the dose is preferably within a range of circulating concentrations that include the LD50 with little or no toxicity. This dose will vary within this range depending on the mode of administration used, the sensitivity of the patient, and the route of administration.
- the dose of the complex is appropriately selected depending on the age and other patient conditions, the type of disease, the type of complex to be used, and the like.
- the self-crosslinked hyaluronic acid particles and crosslinked hyaluronic acid composition of the present invention can be used without particular limitation in the field where general biodegradable medical materials and hyaluronic acid are used in addition to knee osteoarthritis. it can.
- biopharmaceutical products or pharmaceuticals such as pharmacologically active substance carriers, wound dressings, tissue-replacement-type biological tissue repair agents, anti-adhesion agents, hemostatic agents, artificial extracellular matrix, medical devices / medical devices used for diagnosis / treatment, etc.
- the use to a composition is mentioned.
- Example 1 (first embodiment)
- 22.5 g of sodium hyaluronate powder with a viscosity average molecular weight of 2,200,000 (water content 10%) was put into a frozen nitric acid and kneaded for 1 hour at -10 ° C. and 100 rpm until uniform rubbery (sodium hyaluronate) 20.8% by mass).
- the mixture of hyaluronic acid and nitric acid was put in a freezer set at ⁇ 20 ° C. Ten days later, 1 L of pure water at 5 ° C. was added, and the exchange of pure water every other hour was repeated twice. Furthermore, it is put into 1 L of 50 mM phosphate buffer at 5 ° C., and the 50 mM phosphate buffer is exchanged every 1 hour 5 times, and neutralization washing is performed until nitric acid is completely removed, and self-crosslinking hyaluronic acid is obtained.
- the particle size of the obtained self-crosslinked hyaluronic acid particles was quantified using a particle size / shape distribution measuring instrument PITA-1 (manufactured by Seishin Enterprise). As pretreatment, self-crosslinked hyaluronic acid was stained with methylene blue (stain concentration: 1 w / v%, staining time: 1 minute or more). As the measurement conditions for PITA-1, the carrier liquid was distilled water, the number of measured particles was 10,000, and the objective lens was 4 times. As a result, the average volume particle size of the obtained self-crosslinked hyaluronic acid particles was 65 ⁇ m.
- Example 2 Self-crosslinked hyaluronic acid was synthesized in the same manner as in Example 1, and the resulting self-crosslinked hyaluronic acid was made into particles in the same manner as in Example 1 except that the treatment time was 30 minutes. As a result, the average volume particle size of the obtained self-crosslinked hyaluronic acid particles was 52 ⁇ m.
- Example 3 Self-crosslinked hyaluronic acid was synthesized in the same manner as in Example 1, and the resulting self-crosslinked hyaluronic acid was made into particles in the same manner as in Example 1 except that the treatment time was 120 minutes. As a result, the average volume particle size of the obtained self-crosslinked hyaluronic acid particles was 41 ⁇ m.
- Example 1 The self-crosslinked hyaluronic acid particles obtained in Example 1 were crushed using a high-pressure crusher (product name: Nanomizer, manufactured by Nanomizer Co., Ltd.). A crushing generator having a diameter of 100 ⁇ m was attached to the crushing section of the apparatus, and it was treated three times at 200 MPa while being cooled so that the self-crosslinked hyaluronic acid particles immediately became below room temperature. The particle size of the self-crosslinked hyaluronic acid particles was measured using a laser diffraction / scattering particle size distribution meter (product name: SALD-7000, manufactured by Shimadzu Corporation).
- SALD-7000 laser diffraction / scattering particle size distribution meter
- the refractive index of the sample was 1.300, and 10 mM phosphate buffered saline was used as the dispersion medium.
- the average volume particle size of the obtained self-crosslinked hyaluronic acid particles was 5 ⁇ m, but the yield was very low and not practical.
- the particle size of the self-crosslinked hyaluronic acid particles was quantified using a particle size / shape distribution measuring instrument PITA-1 (manufactured by Seishin Enterprise), but could not be measured due to the presence of large particles. Therefore, when classification was performed using a sieve having an opening of 0.2 mm, 90% or more remained on the sieve on a weight basis, so the average volume particle size was 200 ⁇ m or more.
- ⁇ Viscosity average molecular weight> A phosphate buffer component was added to physiological saline at a concentration of 10 mM to prepare phosphate buffered saline having a pH of 7.4.
- the self-crosslinked hyaluronic acid particles of Examples 1 to 3 and Comparative Examples 1 to 2 are added to 100 ml of this phosphate buffered physiological saline, and then 37.0 for 30 days until the self-crosslinked hyaluronic acid is completely dissolved. Immersion at °C.
- the viscosity average molecular weight of hyaluronic acid eluted in phosphate-buffered physiological saline was measured by injecting 0.1 ml into the GPC apparatus after filtering the supernatant with a 0.2 ⁇ m membrane filter.
- the viscosity average molecular weight of hyaluronic acid was calculated from the retention time at the peak top of the molecular weight distribution using a differential refractometer as a detector of the GPC apparatus.
- the GPC apparatus uses one SB806HQ manufactured by Showa Denko KK as the GPC column and RI-71S manufactured by Shodex as the differential refractive index detector, 0.2 M aqueous solution of solvent sodium nitrate, measuring temperature 40 ° C., flow rate 0. Measured at 3 ml / min.
- a calibration curve prepared using the retention time at the peak top of the molecular weight distribution of hyaluronic acid with a known viscosity average molecular weight was used.
- Table 2 shows the measurement results of Examples 1 to 3 and Comparative Examples 1 and 2.
- the self-crosslinked hyaluronic acid particles of Example 1 had a solubility half-life after treatment of 25 hours, a viscosity average molecular weight of 1,700,000, a retention rate of 100% from before the treatment, and deterioration of physical properties due to particle formation. There wasn't.
- the self-crosslinked hyaluronic acid particles of Examples 2 and 3 hardly deteriorated in physical properties due to the formation of particles.
- the self-crosslinked hyaluronic acid particles of Comparative Example 1 have a solubility half-life after particle formation of 18 hours, a viscosity average molecular weight of 1.3 million, and retention ratios of 69% and 76% from before particle formation. The physical properties were deteriorated by the conversion, and the yield was very low and not practical.
- Example 4 The self-crosslinked hyaluronic acid particles obtained in Example 1 were put into 10 mM phosphate buffered saline at 5 ° C. and pH 7.0, and 10 mM phosphate buffered saline was exchanged every other hour twice. Repeated. The dry weight (concentration) of the self-crosslinked hyaluronic acid particles with respect to the total volume of the crosslinked hyaluronic acid composition was adjusted as follows so as to be 6 w / v%.
- the concentration of the self-crosslinking hyaluronic acid was determined by diluting 50 mg of the crosslinked hyaluronic acid composition with 1.55 ml of distilled water, adding 0.2 ml of 1N sodium hydroxide solution thereto, and allowing to stand at room temperature for 30 minutes.
- the self-crosslinked hyaluronic acid ester bridge was hydrolyzed to dissolve the self-crosslinked hyaluronic acid.
- the concentration of self-crosslinked hyaluronic acid was calculated by a carbazole sulfate method using a known concentration of hyaluronic acid (viscosity average molecular weight 1.9 million) as a standard substance. Based on this quantitative result, the concentration of the self-crosslinked hyaluronic acid particles was adjusted to 6 w / v% to obtain a crosslinked hyaluronic acid composition.
- Example 5 Example 4 except that the dry weight of the self-crosslinked hyaluronic acid particles relative to the total volume of the crosslinked hyaluronic acid composition was adjusted to 10 wt% phosphate buffered saline at pH 7.0 so that the dry weight was 3 w / v%. In the same manner, a crosslinked hyaluronic acid composition was prepared.
- Example 3 A high-speed rotating device (product name: Creamix Single Motion, manufactured by MTechnic Co., Ltd.) was used to make self-crosslinked hyaluronic acid particles, and the rotor of the device was rotated 10,000 rpm in the forward direction so that the crosslinked hyaluronic acid was 30 ° C.
- Self-crosslinked hyaluronic acid particles having an average volume particle size of 300 ⁇ m were prepared in the same manner as in Example 1 except that the particles were atomized for 6 minutes while being cooled to below. Further, a crosslinked hyaluronic acid composition having a concentration of self-crosslinked hyaluronic acid particles of 6 w / v% was prepared in the same manner as in Example 4.
- Example 4 A high-speed rotating device (product name: Creamix Single Motion, manufactured by MTechnic Co., Ltd.) is used to granulate self-crosslinked hyaluronic acid, and the rotor of the device is rotated at 20,000 rpm in the forward direction to obtain 30 crosslinked hyaluronic acid.
- Self-crosslinked hyaluronic acid particles having an average volume particle size of 153 ⁇ m were prepared in the same manner as in Example 1 except that the particles were atomized for 4 minutes while cooling to less than 0 ° C.
- a crosslinked hyaluronic acid composition having a concentration of self-crosslinked hyaluronic acid particles of 6 w / v% was prepared in the same manner as in Example 4.
- Example 5 A high-speed rotating device (product name: Creamix Single Motion, manufactured by MTechnic Co., Ltd.) was used to granulate self-crosslinked hyaluronic acid, and the rotor of the device was rotated in the forward direction at 20,000 rpm. The mixture was atomized for 20 minutes without cooling to prepare self-crosslinked hyaluronic acid particles having an average volume particle size of 100 ⁇ m. At this time, the temperature of the self-crosslinking hyaluronic acid rose to 85 ° C. Further, a crosslinked hyaluronic acid composition having a concentration of self-crosslinked hyaluronic acid particles of 6 w / v% was prepared in the same manner as in Example 4.
- Hyaluronic acid joint preparation “Smony” (trade name, manufactured by Chugai Pharmaceutical Co., Ltd.) (Viscosity average molecular weight 2 million, hyaluronic acid concentration 1 w / v%)
- Hyaluronic acid joint preparation “Alz” (trade name, manufactured by Seikagaku Corporation) (Viscosity average molecular weight 800,000, hyaluronic acid concentration 1 w / v%)
- Hyaluronic acid joint preparation “Synvisc” (trade name, manufactured by Genzyme Corporation) (Hyaluronic acid concentration 0.8 w / v%)
- Hyaluronic acid joint formulation “Durolane” (trade name, manufactured by Q-MED) (Hyaluronic acid concentration 2.0 w / v%)
- MCR300 (trade name, manufactured by Physica) was used as a rheometer which is a viscosity measuring device.
- Viscosity comparison of the crosslinked hyaluronic acid compositions of Examples 4 and 5 and Comparative Example 3 and Reference Examples 1 to 5 was performed. Table 3 shows the measurement results of the viscosity.
- the crosslinked hyaluronic acid composition of Example 4 contains 6 w / v% of self-crosslinked hyaluronic acid particles at a high concentration, but the viscosity average molecular weight of Reference Example 1 is 800,000, 1 w / v. Compared to v% hyaluronic acid, the viscosity was 1/6 or less.
- ⁇ Measurement of Discharge Pressure of Crosslinked Hyaluronic Acid Composition 1 ml of a crosslinked hyaluronic acid composition is packed in a syringe Terumo syringe SS-01T (trade name, manufactured by Terumo) having an inner diameter of 0.45 cm, and an injection needle 23G (manufactured by Terumo) having an inner diameter of 0.40 mm and a needle length of 25 mm is attached. It was.
- the extrusion pressure measuring instrument EZ-TEST (trade name, manufactured by Shimadzu Corporation) under the discharge conditions of a temperature of 25 ° C. and a speed of 50 mm / min, the crosslinked hyaluronic acid compositions of Example 4, Comparative Example 3 and Comparative Example 4
- the pressure applied to the syringes of Reference Examples 1 to 5 was measured. Table 4 shows the measurement results.
- the crosslinked hyaluronic acid composition of Example 4 can keep the discharge pressure low even though it contains 6 w / v% and hyaluronic acid six times as high as that of Reference Example 1. It was.
- the crosslinked hyaluronic acid composition of Example 4 keeps the discharge pressure low despite containing 6 w / v% and hyaluronic acid six times as high as that of Reference Example 1. It was suggested that a thin needle could be used, which could reduce patient pain during injection.
- the viscosity average molecular weight of hyaluronic acid was calculated from the retention time at the peak top of the molecular weight distribution using a differential refractometer as a detector of the GPC apparatus.
- the GPC apparatus uses one SB806HQ manufactured by Showa Denko KK as the GPC column and RI-71S manufactured by Shodex as the differential refractive index detector, 0.2 M aqueous solution of solvent sodium nitrate, measuring temperature 40 ° C., flow rate 0. Measured at 3 ml / min.
- a calibration curve prepared using the retention time at the peak top of the molecular weight distribution of hyaluronic acid with a known viscosity average molecular weight was used.
- the viscosity average molecular weight of hyaluronic acid used for preparing a calibration curve is determined by dissolving the hyaluronic acid with a 0.2M sodium chloride solution and using a Ubbelohde viscometer, the flow time of the 0.2M sodium chloride solution at 30 ° C.
- a phosphate buffer component was added to physiological saline at a concentration of 10 mM to prepare phosphate buffered saline having a pH of 7.4.
- phosphate buffered saline having a pH of 7.4.
- 0.5 ml of the samples of Examples 4 and 5 and Comparative Example 5 were added to 100 ml of this phosphate buffered physiological saline, and immersed at 37.0 ° C. for 30 days until the self-crosslinking hyaluronic acid was completely dissolved. did.
- the viscosity average molecular weight of hyaluronic acid eluted in phosphate buffered physiological saline was measured in the same manner as the measurement of the viscosity average molecular weight of the crosslinked hyaluronic acid.
- mice 7 days after injection, the mice were euthanized under anesthesia, the knees were cut out, and the joint fluid was collected with a high viscosity pipette.
- the joint fluid was diluted exactly 100 times with distilled water and centrifuged at 15,000 rpm for 10 minutes at 4 ° C. The supernatant was filtered through a 0.2 ⁇ m membrane filter, and then 0.1 ml was injected into the GPC apparatus to measure the viscosity average molecular weight. Table 7 shows the measurement results.
- Comparative Example 5 and Reference Examples 1 and 3 had a joint fluid volume of 30 ⁇ l, a joint fluid viscosity average molecular weight of 1.7 million or more, and a hyaluronic acid concentration of 0.3% by mass, as in the case of no sample administration. .
- the synovial fluid amounts of Examples 4 and 5 were 200 and 300 ⁇ l, the viscosity average molecular weight of the synovial fluid was 1.7 million or more, and the hyaluronic acid concentration was 0.6% by weight.
- needle clogging occurred and injection into the joint was difficult.
- the increase in the synovial fluid and the increase in the hyaluronic acid concentration in the synovial fluid of Examples 4 and 5 are considered to be derived from the crosslinked hyaluronic acid composition of the present invention, and if the crosslinked hyaluronic acid composition of the present invention is used as an injection, It was found that hyaluronic acid having a viscosity average molecular weight of 1.7 million or more was retained in the joint fluid even 7 days after the injection.
- Animals are individually housed in bracket-type metal wire mesh cages (350W x 500D x 350H mm) mounted on a movable rack, temperature 20 ⁇ 3 ° C, humidity 50 ⁇ 20%, ventilation rate 12-18 times / hour
- the animals were reared in an environment with an illumination time of 8:00 to 20:00 (12 hours light, 12 hours dark).
- the feed is given by a stainless steel feeder and the experimental animal solid feed RC4 (produced by Oriental Yeast Co., Ltd.) is given as a restricted feed of 150 g / day, and the drinking water is freely supplied by a polypropylene water bottle (made by a stainless steel pipe). Gave to.
- the individual identification of the animal is identified by entering the individual identification number with magic ink on the auricle, the card with the sex and individual identification number written in the cage before the grouping, the test number, administration group, sex, A card with the animal number, the date of surgery, the date of administration, the date of examination, and the individual identification number was attached.
- the meniscus partial excision operation was performed on the day after the grouping date, and the day of partial meniscal excision was defined as day 0 after the operation.
- a meniscus partially excised osteoarthritis model was prepared using animals of 14 to 15 weeks of age with reference to the methods described in References 1 to 3.
- Reference 1 has 32 KBL: JW rabbits (13 weeks old, female), and under ketamine and xylazine anesthesia, the lateral collateral ligament and seed bone of the left knee joint The ligament was dissected, the meniscus was partially excised from 3.0 to 4.0 mm, and a 26G syringe needle was used, 5 times in the knee joint at a rate of 2 times per week for each of the 8 birds in groups A and B.
- a molecular weight HA solution is injected, physiological saline is injected into 8 control group C birds, and loxonin is orally administered daily to groups C and D to evaluate pain suppression effect and cartilage degeneration prevention effect.
- Reference 2 also prepares 72 New Zealand white rabbits (2 to 3 kg in weight), and under anesthesia, the left knee joint ligament is dissected, the meniscus is partially excised 3 to 4 mm, and a week is placed in the knee joint.
- 48 birds in group A were injected with a 1-0.01% HA solution having a molecular weight of 1.9 million, and 12 birds in group B were injected with 1-0. It is described that a 01% HA solution is injected, physiological saline is injected into 12 birds of Group C, and after sacrifice, the knee joint is collected and evaluated for drug efficacy.
- Reference 7 Shinsuke Miyazaki, Kiyoshi Nagano, Keitaro Suzuki, Sachiko Goto, Toshijiro Yamaguchi, Atsushi Namiki: Effects of sodium hyaluronate on rabbit fixed joints, Orthopedic Science, Vol. 11, 125-127 (1984)
- Reference 8 T.W. Kawano, H .; Miura, T .; Mawatari, T .; Moro-Oka, Y.M. Nakanishi, H .; Higashi and Y.H.
- the external meniscus was exposed by excising the tendon at the starting part of the popliteal muscle, and the central part of the meniscus was excised over 3.0 to 4.0 mm. Thereafter, the subcutaneous muscle layer and the skin were each sutured, and about 0.2 ml of ampicillin (product name: Vicillin sol-15%, manufactured by Meiji Seika Co., Ltd.) was injected into the thigh muscle.
- ampicillin product name: Vicillin sol-15%, manufactured by Meiji Seika Co., Ltd.
- ⁇ Method for measuring pain suppression effect For the measurement of the weight distribution of both hind paws, an analgesic evaluation device for small animals, Incapacitance Tester (manufactured by Linton Instrument, UK) was used. This device accurately detects the weight distribution between the right and left legs of the animal placed in the main body container in grams using a dual-channel sensor pad installed on the bottom of the container. Averaged over the set time. The main body container used was for rabbits. The measurement setting time was 5 seconds when the animal was stationary.
- the left rear foot weight distribution ratio (%) was calculated from the left and right weights (loads) according to the following formula (4).
- Left rear foot weight distribution ratio (%) ⁇ left load (g) / (right load (g) + left load (g)) ⁇ 100 ⁇ (4)
- the average value of the left hind paw weight distribution ratio (%) calculated three times was defined as the left hind paw weight distribution ratio (%) per measurement. The result is shown in FIG.
- the above five test samples were placed in a constant temperature environment tester (manufactured by ESPEC) set at 60 ° C. and heated for a predetermined time.
- the heating time was different for the five test samples. That is, one test sample was taken out at each time point 0, 2, 4, 6, and 10 hours after the start of heating.
- the cross-linked hyaluronic acid composition (0.4 ml) was centrifuged at 5 ° C., 2,000 rpm for 30 minutes using a centrifugal filter unit (0.45 micrometer pore size, manufactured by Millipore) to remove the solvent.
- the filter unit was dried for 20 hours to obtain the weight of the self-crosslinked hyaluronic acid from which the solvent had been removed and the weight of the dried self-crosslinked hyaluronic acid, and the equilibrium swelling ratio was calculated.
- the solvent was 10 mM phosphate buffered saline (pH 6.0), the NaCl concentration was 0.9 wt%, the sample was swollen at 5 ° C. for 1 day, and measured after reaching an equilibrium swollen state.
- the degree of crosslinking (degree of self-crosslinking esterification) in self-crosslinked hyaluronic acid was determined by the chemical shift intensity (peak area value) by proton nuclear magnetic resonance (NMR). For the measurement, it was necessary to hydrolyze the hyaluronic acid structure in advance to reduce the molecular weight, so the hydrolysis treatment with a hyaluronic acid hydrolase that selectively hydrolyzes only the main chain structure of self-crosslinked hyaluronic acid was performed. went.
- Hyaluronic acid hydrolyzing enzyme uses Hyaluronidize form tests type V, lyophilized powder, activity:> 1,500 units / mg solid (manufactured by Sigma-Aldrich), cation exchange column G50 Purified using Healthcare.
- the above enzyme is dissolved in 10 mM, pH 5.0 acetate buffer so as to have a concentration of 0.1 g / mL, and 0.1 mL of this solution is passed through the above column equilibrated with the same buffer, and NaCl is added.
- 0.8 mL of an enzyme solution of a purified fraction eluted at a concentration of 0.05 to 0.15 mol / L was obtained.
- the cross-linked hyaluronic acid was adjusted to a concentration of 3 wt% in 10 mL of pH 5.0 acetate buffered saline, 0.2 mL of the purified enzyme solution was added, and the mixture was shaken at 37 ° C. and 160 rpm.
- the reaction was performed for 24 hours under the conditions, and then 0.2 mL of the enzyme solution was added and the reaction was further performed for 24 hours under the same conditions.
- the solution after the reaction was frozen at ⁇ 30 ° C., then freeze-dried for 18 hours, and used as an NMR measurement sample.
- the crosslinked hyaluronic acid composition having a reduced degree of crosslinking by increasing the heating time also has a 60 ° C. solubility half-life and a quadratic function correlation.
- the equilibrium swelling ratio increased as the cross-linked hyaluronic acid composition with a lower degree of cross-linking, indicating a correlation similar to the Flory-Lehner equation.
- the gel fraction arrival days became shorter as the heating time increased.
- the heating time to reach a gel fraction of 97% was shortened to 0.7 days
- the heating time to reach a gel fraction of 95% was shortened to 1.1 days.
- the smaller the half-life and the degree of crosslinking the shorter the gel fraction arrival days, indicating that these physical property values correlate with storage stability (FIGS. 8 and 9).
- Comparative Example 6 has a low average sedimentation concentration of 5.2% by weight, indicating that the concentration cannot be increased as compared with Examples 6-9.
- Example 10 (Second Embodiment) ⁇ Pretreatment of ethanol removal from hyaluronic acid raw material> Sodium hyaluronate powder having a viscosity average molecular weight of 2,200,000 was put in a container equipped with a ventilation filter, sucked with a pump at room temperature, and aerated for 3 days to remove ethanol.
- the mixture of hyaluronic acid and nitric acid was put in a freezer set at ⁇ 20 ° C. Ten days later, 1 L of pure water at 5 ° C. was added, and the exchange of pure water every other hour was repeated twice. Furthermore, it is put into 1 L of 50 mM phosphate buffer at 5 ° C., and the 50 mM phosphate buffer is exchanged every 1 hour 5 times, and neutralization washing is performed until nitric acid is completely removed, and self-crosslinking hyaluronic acid is obtained.
- Example 11 Self-crosslinked hyaluronic acid particles were obtained in the same manner as in Example 10 using a hyaluronic acid raw material adjusted to contain 1000 ppm of ethanol.
- the degree of self-crosslinking esterification (degree of cross-linking) in self-cross-linking hyaluronic acid was determined by the chemical shift intensity (peak area value) by proton nuclear magnetic resonance (NMR). For the measurement, it was necessary to hydrolyze the hyaluronic acid structure in advance to reduce the molecular weight, so the hydrolysis treatment with a hyaluronic acid hydrolase that selectively hydrolyzes only the main chain structure of self-crosslinked hyaluronic acid was performed. went.
- Hyaluronic acid hydrolyzing enzyme uses Hyaluronidize form tests type V, lyophilized powder, activity:> 1,500 units / mg solid (manufactured by Sigma-Aldrich), cation exchange column G50 Purified using Healthcare.
- the above enzyme is dissolved in 10 mM, pH 5.0 acetate buffer so as to have a concentration of 0.1 g / mL, and 0.1 mL of this solution is passed through the above column equilibrated with the same buffer, and NaCl is added.
- 0.8 mL of an enzyme solution of a purified fraction eluted at a concentration of 0.05 to 0.15 mol / L was obtained.
- the cross-linked hyaluronic acid was adjusted to a concentration of 3 wt% in 10 mL of pH 5.0 acetate buffered saline, 0.2 mL of the purified enzyme solution was added, and the mixture was shaken at 37 ° C. and 160 rpm.
- the reaction was performed for 24 hours under the conditions, and then 0.2 mL of the enzyme solution was added and the reaction was further performed for 24 hours under the same conditions.
- the solution after the reaction was frozen at ⁇ 30 ° C., then freeze-dried for 18 hours, and used as an NMR measurement sample.
- ⁇ Viscosity average molecular weight> A phosphate buffer component was added to physiological saline at a concentration of 10 mM to prepare phosphate buffered saline having a pH of 7.4.
- the self-crosslinked hyaluronic acid particles of Examples 10 to 11 and Comparative Examples 7 to 8 are added to 100 ml of this phosphate buffered physiological saline, and 37.0 for 30 days until the self-crosslinked hyaluronic acid is completely dissolved. Immersion at °C.
- the viscosity average molecular weight of hyaluronic acid eluted in phosphate-buffered physiological saline was measured by injecting 0.1 ml into the GPC apparatus after filtering the supernatant with a 0.2 ⁇ m membrane filter.
- the viscosity average molecular weight of hyaluronic acid was calculated from the retention time at the peak top of the molecular weight distribution using a differential refractometer as a detector of the GPC apparatus.
- the GPC apparatus uses one SB806HQ manufactured by Showa Denko KK as the GPC column and RI-71S manufactured by Shodex as the differential refractive index detector, 0.2 M aqueous solution of solvent sodium nitrate, measuring temperature 40 ° C., flow rate 0. Measured at 3 ml / min.
- a calibration curve prepared using the retention time at the peak top of the molecular weight distribution of hyaluronic acid with a known viscosity average molecular weight was used.
- Table 12 shows the measurement results of Examples 10 to 11 and Comparative Examples 7 to 8.
- the self-crosslinked hyaluronic acid particles of Examples 10 to 11 had a small ethanol content, a small amount of ethyl ester, and no decrease in solubility half-life and primary molecular weight.
- the self-crosslinked hyaluronic acid particles of Comparative Examples 7 to 8 having a large ethanol content and ethyl ester amount have lower solubility half-life and primary molecular weight values than those of Examples 10 to 11, and physical properties are deteriorated. It was. Further, since the degree of self-crosslinking esterification in Comparative Examples 7 to 8 was lower than that in Examples 10 to 11, it was confirmed that ethyl esterification inhibited self-esterification.
- ⁇ Average volume particle size> The particle size of the self-crosslinked hyaluronic acid particles obtained in Example 10 was quantified using a particle size / shape distribution measuring instrument PITA-1 (manufactured by Seishin Enterprise). As pretreatment, self-crosslinked hyaluronic acid was stained with methylene blue (stain concentration: 1 w / v%, staining time: 1 minute or more). As the measurement conditions for PITA-1, the carrier liquid was distilled water, the number of measured particles was 10,000, and the objective lens was 4 times. As a result, the average volume particle size of the obtained self-crosslinked hyaluronic acid particles was 65 ⁇ m.
- Example 12 ⁇ Preparation of crosslinked hyaluronic acid composition> (Example 12)
- the self-crosslinked hyaluronic acid particles obtained in Example 10 were put into 10 mM phosphate buffered saline at 5 ° C. and pH 7.0, and the 10 mM phosphate buffered saline was changed every other hour twice. Repeated.
- the dry weight (concentration) of the self-crosslinked hyaluronic acid particles with respect to the total volume of the crosslinked hyaluronic acid composition was adjusted as follows so as to be 6 w / v%.
- the concentration of the self-crosslinking hyaluronic acid was determined by diluting 50 mg of the crosslinked hyaluronic acid composition with 1.55 ml of distilled water, adding 0.2 ml of 1N sodium hydroxide solution thereto, and allowing to stand at room temperature for 30 minutes.
- the self-crosslinked hyaluronic acid ester bridge was hydrolyzed to dissolve the self-crosslinked hyaluronic acid.
- the concentration of self-crosslinked hyaluronic acid was calculated by a carbazole sulfate method using a known concentration of hyaluronic acid (viscosity average molecular weight 1.9 million) as a standard substance. Based on this quantitative result, the concentration of the self-crosslinked hyaluronic acid particles was adjusted to 6 w / v% to obtain a crosslinked hyaluronic acid composition.
- Example 13 Example 12 except that the dry weight of the self-crosslinked hyaluronic acid particles with respect to the total volume of the crosslinked hyaluronic acid composition was adjusted to 10 wt% phosphate buffered saline at pH 7.0 so that the dry weight was 3 w / v%. In the same manner, a crosslinked hyaluronic acid composition was prepared.
- Example 9 A high-speed rotating device (product name: Creamix Single Motion, manufactured by MTechnic Co., Ltd.) is used to granulate self-crosslinked hyaluronic acid, and the rotor of the device is rotated at 10,000 rpm in the forward direction to obtain 30 crosslinked hyaluronic acid.
- Self-crosslinked hyaluronic acid particles having an average volume particle size of 300 ⁇ m were prepared in the same manner as in Example 10 except that the particles were atomized for 6 minutes while cooling to less than 0 ° C. Further, a crosslinked hyaluronic acid composition having a self-crosslinked hyaluronic acid particle concentration of 6 w / v% was prepared in the same manner as in Example 12.
- Example 10 A high-speed rotating device (product name: Creamix Single Motion, manufactured by MTechnic Co., Ltd.) is used to granulate self-crosslinked hyaluronic acid, and the rotor of the device is rotated at 20,000 rpm in the forward direction to obtain 30 crosslinked hyaluronic acid.
- Self-crosslinked hyaluronic acid particles having an average volume particle size of 153 ⁇ m were prepared in the same manner as in Example 10 except that the particles were atomized for 4 minutes while cooling to less than 0 ° C. Further, a crosslinked hyaluronic acid composition having a self-crosslinked hyaluronic acid particle concentration of 6 w / v% was prepared in the same manner as in Example 12.
- Example 11 A high-speed rotating device (product name: Creamix Single Motion, manufactured by MTechnic Co., Ltd.) was used to granulate self-crosslinked hyaluronic acid, and the rotor of the device was rotated in the forward direction at 20,000 rpm. The mixture was atomized for 20 minutes without cooling to prepare self-crosslinked hyaluronic acid particles having an average volume particle size of 100 ⁇ m. At this time, the temperature of the self-crosslinking hyaluronic acid rose to 85 ° C. Further, a crosslinked hyaluronic acid composition having a self-crosslinked hyaluronic acid particle concentration of 6 w / v% was prepared in the same manner as in Example 12.
- Hyaluronic acid joint preparation “Smony” (trade name, manufactured by Chugai Pharmaceutical Co., Ltd.) (Viscosity average molecular weight 2 million, hyaluronic acid concentration 1 w / v%)
- Hyaluronic acid joint preparation “Alz” (trade name, manufactured by Seikagaku Corporation) (Viscosity average molecular weight 800,000, hyaluronic acid concentration 1 w / v%)
- Hyaluronic acid joint preparation “Synvisc” (trade name, manufactured by Genzyme Corporation) (Hyaluronic acid concentration 0.8 w / v%)
- Hyaluronic acid joint formulation “Durolane” (trade name, manufactured by Q-MED) (Hyaluronic acid concentration 2.0 w / v%)
- MCR300 (trade name, manufactured by Physica) was used as a rheometer which is a viscosity measuring device.
- Viscosity comparison of the crosslinked hyaluronic acid compositions of Examples 12 to 13 and Comparative Example 9 and Reference Examples 1 to 5 was performed. Table 13 shows the measurement results of the viscosity.
- the crosslinked hyaluronic acid composition of Example 12 contained 6 w / v% and a high concentration of self-crosslinked hyaluronic acid particles, but the viscosity average molecular weight of Reference Example 1 was 800,000, 1 w / v. Compared to v% hyaluronic acid, the viscosity was 1/6 or less.
- ⁇ Measurement of Discharge Pressure of Crosslinked Hyaluronic Acid Composition 1 ml of a crosslinked hyaluronic acid composition is packed in a syringe Terumo syringe SS-01T (trade name, manufactured by Terumo) having an inner diameter of 0.45 cm, and an injection needle 23G (manufactured by Terumo) having an inner diameter of 0.40 mm and a needle length of 25 mm is attached. It was.
- the extrusion pressure measuring device EZ-TEST (trade name, manufactured by Shimadzu Corporation) under the discharge conditions of a temperature of 25 ° C. and a speed of 50 mm / min, the crosslinked hyaluronic acid compositions of Example 12 and Comparative Examples 9 to 10, The pressure applied to the syringes of Reference Examples 1 to 5 was measured. Table 14 shows the measurement results.
- Example 14 As shown in Table 14, in particular, the crosslinked hyaluronic acid composition of Example 12 was able to keep the discharge pressure low despite containing 6 w / v% and hyaluronic acid 6 times as high as that of Reference Example 1. It was.
- the crosslinked hyaluronic acid composition of Example 12 keeps the discharge pressure low even though it contains 6 w / v% and hyaluronic acid six times as high as that of Reference Example 1. It was suggested that a thin needle could be used, which could reduce patient pain during injection.
- mice 7 days after injection, the mice were euthanized under anesthesia, the knees were cut out, and the joint fluid was collected with a high viscosity pipette.
- the joint fluid was diluted exactly 100 times with distilled water and centrifuged at 4 ° C., 15,000 rpm for 10 minutes. The supernatant was filtered through a 0.2 ⁇ m membrane filter, and then 0.1 ml was injected into the GPC apparatus to measure the viscosity average molecular weight. Table 17 shows the measurement results.
- Comparative Example 11 and Reference Examples 1 and 3 had a joint fluid volume of 30 ⁇ l, a joint fluid viscosity average molecular weight of 1.7 million or more, and a hyaluronic acid concentration of 0.3% by mass, as in the case of no sample administration. .
- the synovial fluid amounts of Examples 12 and 13 were 200 and 300 ⁇ l, the viscosity average molecular weight of the synovial fluid was 1.7 million or more, and the hyaluronic acid concentration was 0.6% by weight.
- needle clogging occurred and injection into the joint was difficult.
- the increase in the synovial fluid and the increase in the hyaluronic acid concentration in the synovial fluid of Examples 12 and 13 are considered to be derived from the crosslinked hyaluronic acid composition of the present invention, and if the crosslinked hyaluronic acid composition of the present invention is used as an injection, It was found that hyaluronic acid having a viscosity average molecular weight of 1.7 million or more was retained in the joint fluid even 7 days after the injection.
- Animals are individually housed in bracket-type metal wire mesh cages (350W x 500D x 350H mm) mounted on a movable rack, temperature 20 ⁇ 3 ° C, humidity 50 ⁇ 20%, ventilation rate 12-18 times / hour
- the animals were reared in an environment with an illumination time of 8:00 to 20:00 (12 hours light, 12 hours dark).
- the feed is a stainless steel feeder and the experimental animal chow RC4 (Oriental Yeast Co., Ltd.) is given as a limited feed of 150 g / day, and the drinking water is freely supplied with tap water by a polypropylene water bottle (made of a stainless steel pipe). Gave.
- the individual identification of the animal is identified by entering the individual identification number with magic ink on the auricle, the card with the sex and individual identification number written in the cage before the grouping, the test number, administration group, sex, A card with the animal number, the date of surgery, the date of administration, the date of examination, and the individual identification number was attached.
- the meniscus partial excision operation was performed on the day after the grouping date, and the day of partial meniscal excision was defined as day 0 after the operation.
- a meniscus partially excised osteoarthritis model was prepared using animals of 14 to 15 weeks of age with reference to the methods described in References 1 to 3 above.
- the external meniscus was exposed by excising the tendon at the starting part of the popliteal muscle, and the central part of the meniscus was excised over 3.0 to 4.0 mm. Thereafter, the subcutaneous muscle layer and the skin were each sutured, and about 0.2 ml of ampicillin (product name: Vicillin sol-15%, manufactured by Meiji Seika Co., Ltd.) was injected into the thigh muscle.
- ampicillin product name: Vicillin sol-15%, manufactured by Meiji Seika Co., Ltd.
- Table 18 shows four groups injecting the injections of Examples 12 and 13 and Reference Examples 1 and 5 into the joint space of 0.1 mL / kg with reference to the methods described in References 1 to 8 above. Set.
- ⁇ Method for measuring pain suppression effect For the measurement of the weight distribution of both hind paws, an analgesic evaluation device for small animals, Incapacitance Tester (manufactured by Linton Instrument, UK) was used. This device accurately detects the weight distribution between the right and left legs of the animal placed in the main body container in grams using a dual-channel sensor pad installed on the bottom of the container. Averaged over the set time. The main body container used was for rabbits. The measurement setting time was 5 seconds when the animal was stationary.
- the left rear foot weight distribution ratio (%) was calculated from the left and right weights (loads) according to the following formula (6).
- Left rear foot weight distribution ratio (%) ⁇ left load (g) / (right load (g) + left load (g)) ⁇ 100 ⁇ (6)
- the average value of the left hind paw weight distribution ratio (%) calculated three times was defined as the left hind paw weight distribution ratio (%) per measurement.
- FIG. 11 it was found that the self-crosslinking hyaluronic acid of Examples 12 and 13 had an improved pain suppressing effect as compared with Reference Example 1.
- * and ** indicate that there is a significant difference as compared with the negative control group (physiological saline administration) which is Reference Example 5. (*: P ⁇ 0.05, **: p ⁇ 0.01 (t test))
- the above five test samples were placed in a constant temperature environment tester (manufactured by ESPEC) set at 60 ° C. and heated for a predetermined time.
- the heating time was different for the five test samples. That is, one test sample was taken out at each time point 0, 2, 4, 6, and 10 hours after the start of heating.
- the cross-linked hyaluronic acid composition (0.4 ml) was centrifuged at 5 ° C., 2,000 rpm for 30 minutes using a centrifugal filter unit (0.45 micrometer pore size, manufactured by Millipore) to remove the solvent.
- the filter unit was dried for 20 hours to obtain the weight of the self-crosslinked hyaluronic acid from which the solvent had been removed and the weight of the dried self-crosslinked hyaluronic acid, and the equilibrium swelling ratio was calculated.
- the solvent was 10 mM phosphate buffered saline (pH 6.0), the NaCl concentration was 0.9 wt%, the sample was swollen at 5 ° C. for 1 day, and measured after reaching an equilibrium swollen state.
- the crosslinked hyaluronic acid composition having a reduced degree of crosslinking by increasing the heating time also has a 60 ° C. solubility half-life and a quadratic function correlation.
- the equilibrium swelling ratio increased as the cross-linked hyaluronic acid composition with a lower degree of cross-linking, indicating a correlation similar to the Flory-Lehner equation.
- Comparative Example 12 has a low average sedimentation concentration of 5.2% by weight, indicating that the concentration cannot be increased as compared with Examples 14-17.
- a crosslinked hyaluronic acid composition capable of obtaining a sufficient therapeutic effect for osteoarthritis of the knee even when the number of administrations is less than before, and self-crosslinked hyaluronic acid particles used therefor can do.
Abstract
Description
ゲル分率(%)=(1-(溶解ヒアルロン酸量/全ヒアルロン酸量))×100・・・(1)
平衡膨潤倍率=1+(ρ/ρ0)×(Qw-1)・・・(2)
(ρ:自己架橋ヒアルロン酸粒子の密度、ρ0:水系溶媒(緩衝液)の密度)
平衡沈降濃度=C×(V0/V)・・・(3)
<自己架橋ヒアルロン酸の合成>
2Nの硝酸75gを自公転型混練り装置(プライミクス製)に入れ、-10℃に冷却し、シャーベット状の硝酸凍結物を得た。硝酸凍結物に粘度平均分子量220万のヒアルロン酸ナトリウムの粉末22.5g(水分含量10%)を投入し、-10℃、100rpmで均一なゴム状になるまで1時間練り混ぜた(ヒアルロン酸ナトリウム20.8質量%)。
上記のとおり得られた自己架橋ヒアルロン酸を中和後30分間静置し、上清をデカンテーションで除き、沈降した自己架橋ヒアルロン酸に対して、9倍の重量の50mMリン酸緩衝液を加えた。次に、この架橋ヒアルロン酸懸濁液を高速回転装置(製品名:クレアミクスダブルモーション、エムテクニック(株)製)に投入し、装置のローターを順方向20,000rpm、スクリーンを逆方向に18,000rpmで回転させ、50℃未満になるよう冷却しながら15分間微粒化した。ローターは後退角度0度ローターを使用し、スクリーン上に存在するスリットの巾が1.0mmのものを使用した。
実施例1と同様に自己架橋ヒアルロン酸を合成し、得られた自己架橋ヒアルロン酸について、処理時間を30分とした以外は、実施例1と同様に粒子化させた。その結果、得られた自己架橋ヒアルロン酸粒子の平均体積粒径は52μmだった。
実施例1と同様に自己架橋ヒアルロン酸を合成し、得られた自己架橋ヒアルロン酸について、処理時間を120分とした以外は、実施例1と同様に粒子化させた。その結果、得られた自己架橋ヒアルロン酸粒子の平均体積粒径は41μmだった。
実施例1で得た自己架橋ヒアルロン酸粒子を、高圧型破砕装置(製品名:ナノマイザー、ナノマイザー(株)製)を用いて破砕した。装置破砕部にはφ100μmの衝突型ジェネレーターを取り付け、自己架橋ヒアルロン酸粒子が直ちに室温以下となるように冷却しながら、200MPaで3回処理した。この自己架橋ヒアルロン酸粒子の粒子径を、レーザー回折・散乱式粒度分布計(製品名:SALD-7000、島津製作所製)を用いて測定した。測定条件として、試料の屈折率を1.300とし、分散媒には10mMリン酸緩衝生理食塩水を使用した。その結果、得られた自己架橋ヒアルロン酸粒子の平均体積粒径は5μmだったが、収率が非常に低く実用的でなかった。
また、上述の自己架橋ヒアルロン酸の合成方法に従って調製した自己架橋ヒアルロン酸を用い、中和後30分間静置し、上清をデカンテーションで除き、沈降した自己架橋ヒアルロン酸に対して、9倍の重量の50mMリン酸緩衝液を加えた。次に、架橋ヒアルロン酸懸濁液を高速破砕装置(製品名:T.K.ホモミックス、プライミクス(株)製)に投入し、ローターを16,000rpmとし、50℃未満になるよう冷却しながら60分間破砕した。この自己架橋ヒアルロン酸粒子の粒子径について、粒度・形状分布測定器PITA-1(セイシン企業製)を用いて定量したが、大粒子混在のため測定できなかった。そのため、開き目0.2mmのふるいを用いて分級したところ、重量ベースで90%以上ふるい上に残留したため、平均体積粒径は200μm以上とした。
上記で得られた実施例1~3及び比較例1~2の自己架橋ヒアルロン酸粒子について、溶解性半減期を測定した。pH7.4のリン酸緩衝液を用いて、60℃環境で加熱を行い、5時間間隔でサンプル採取した。採取したサンプルを希釈し、遠心分離により上清と沈殿部に分け、それぞれの部分のヒアルロン酸濃度を測定し、ゲル分率を算出した。加熱時間に対するゲル分率の挙動を読み取り、ゲル分率50%に到達する加熱時間を求めた。
生理的食塩水に10mM濃度でリン酸緩衝成分を加え、pH7.4のリン酸緩衝化食塩水を調整した。このリン酸緩衝化生理的食塩水100mlに対して実施例1~3及び比較例1~2の自己架橋ヒアルロン酸粒子を添加し、自己架橋ヒアルロン酸が完全に溶解するまで30日間、37.0℃で浸漬した。
(実施例4)
実施例1で得られた自己架橋ヒアルロン酸粒子を、5℃、pH7.0の10mMリン酸緩衝生理食塩水に投入し、一時間おきに10mMリン酸緩衝生理食塩水を交換することを2回繰り返した。この架橋ヒアルロン酸組成物の全体積に対する、自己架橋ヒアルロン酸粒子の乾燥重量(濃度)が6w/v%になるように以下のように調整した。
架橋ヒアルロン酸組成物の全体積に対する自己架橋ヒアルロン酸粒子の乾燥重量が3w/v%になるように、pH7.0の10mMリン酸緩衝生理食塩水に投入して調整した以外は、実施例4と同様にして架橋ヒアルロン酸組成物を調製した。
自己架橋ヒアルロン酸の粒子化に高速回転装置(製品名:クレアミクスシングルモーション、エムテクニック(株)製)を使用し、装置のローターを順方向に10,000rpm回転させ、架橋ヒアルロン酸が30℃未満になるよう冷却しながら6分間微粒化した以外は、実施例1と同様にして平均体積粒径が300μmの自己架橋ヒアルロン酸粒子を調製した。さらに、実施例4と同様にして自己架橋ヒアルロン酸粒子の濃度が、6w/v%の架橋ヒアルロン酸組成物を調製した。
自己架橋ヒアルロン酸の粒子化に高速回転装置(製品名:クレアミクスシングルモーション、エムテクニック(株)製)を使用し、装置のローターを順方向に20,000rpmで回転させ、架橋ヒアルロン酸が30℃未満になるよう冷却しながら4分間微粒化した以外は、実施例1と同様にして平均体積粒径が153μmの自己架橋ヒアルロン酸粒子を調製した。さらに、実施例4と同様にして自己架橋ヒアルロン酸粒子の濃度が、6w/v%の架橋ヒアルロン酸組成物を調製した。
自己架橋ヒアルロン酸の粒子化に高速回転装置(製品名:クレアミクスシングルモーション、エムテクニック(株)製)を使用し、装置のローターを順方向に20,000rpmで回転させ、自己架橋ヒアルロン酸の冷却を行わずに20分間微粒化し、平均体積粒径100μmの自己架橋ヒアルロン酸粒子を調製した。この時、自己架橋ヒアルロン酸の温度は85℃まで上昇した。さらに、実施例4と同様にして自己架橋ヒアルロン酸粒子の濃度が、6w/v%の架橋ヒアルロン酸組成物を調製した。
ヒアルロン酸関節製剤「スベニール」(商品名、中外製薬(株)製)
(粘度平均分子量200万、ヒアルロン酸濃度1w/v%)
ヒアルロン酸関節製剤「アルツ」(商品名、生化学工業(株)製)
(粘度平均分子量80万、ヒアルロン酸濃度1w/v%)
ヒアルロン酸関節製剤「Synvisc」(商品名、ジェンザイムコーポレーション社製)
(ヒアルロン酸濃度0.8w/v%)
ヒアルロン酸関節製剤「Durolane」(商品名、Q-MED社製)
(ヒアルロン酸濃度2.0w/v%)
生理食塩水「大塚生食注」(商品名、大塚製薬(株)製)
粘度測定装置であるレオメーターとして、MCR300(商品名、Physica製)を使用した。コーンプレートはコーン角1.009°(D=49.938mm)を用い、せん断速度50S-1、25℃にて測定した。実施例4及び5並びに比較例3の架橋ヒアルロン酸組成物、参考例1~5の粘度比較を行った。表3に粘度の測定結果を示す。
架橋ヒアルロン酸組成物1mlを内径0.45cmの注射器テルモシリンジSS-01T(商品名、テルモ社製)に詰め、内径0.40mm、針の長さ25mmの注射針23G(テルモ社製)を付けた。押し出し圧測定機EZ-TEST(商品名、島津製作所(株)製)を用い、温度25℃で速度50mm/分の吐出条件で、実施例4、比較例3及び比較例4の架橋ヒアルロン酸組成物、参考例1~5のこのシリンジにかかる圧力を測定した。表4に測定結果を示す。
吐出圧測定(1)で使用した「注射針23G(内径0.40mm)よりも細い24G、25G及び27Gの注射針を使用し、実施例4及び参考例1~5のサンプル1mlを注射針のついたシリンジ(テルモ社製)に詰め、吐出圧測定(1)と同様にしてシリンジにかかる圧力を測定した。その結果を以下の表5及び図2に示す。
実施例4及び5並びに比較例5のサンプルについて、自己架橋ヒアルロン酸に換算して10mg分を、0.1N水酸化ナトリウム溶液1mlに投入し、0℃で30分間静置し、自己架橋ヒアルロン酸を溶解した。この溶解液に、0.1N塩酸1mlを添加して中和し、GPC溶媒で濃度を0.01質量%になるように希釈調製し、0.2μmのメンブランフィルターでろ過した後、GPC装置に0.1ml注入して、一次分子量として粘度平均分子量の測定を行った。
生理的食塩水に10mM濃度でリン酸緩衝成分を加え、pH7.4のリン酸緩衝化食塩水を調整した。このリン酸緩衝化生理的食塩水100mlに対して実施例4及び5並びに比較例5のサンプル0.5mlを添加し、自己架橋ヒアルロン酸が完全に溶解するまで30日間、37.0℃で浸漬した。リン酸緩衝化生理的食塩水中に溶出したヒアルロン酸の粘度平均分子量は、上記架橋ヒアルロン酸の粘度平均分子量測定と同様に行った。
ウサギ(日本白色種 オス)、重量約3kgを麻酔(麻酔組成:ケタミン(4ml)+キシラジン(3ml)+生食(5ml))し、後足両膝に実施例4及び5、比較例3及び5、並びに、参考例1及び3のサンプルを、内径0.45cmのシリンジに23G注射針を用い、投与量0.1ml/kgで注入した。
実施例4及び5並びに参考例1及び5の関節腔内注射が疼痛に及ぼす作用を、ウサギの膝関節半月板部分切除による実験的変形性関節症を用いて測定した。
動物としては、13週齢のKbl:JW(SPF)系ウサギ(雄)を実施例・参考例毎に8羽ずつ用いるために計32羽を準備した。動物を入荷後3~8日の毎日、評価装置に対する訓化として、小動物用鎮痛評価装置Incapacitance Tester(Linton Instrument製)の本体容器(ホルダー)に入れ、5秒間静止させる操作を行った。
半月板部分切除手術日の前日に、群分けを行った。群分け日に、全例の体重と両後足重量配分を測定した。測定した両後足重量配分より左後足重量配分比((左荷重/両側合計荷重)×100(%))を算出した。左後足重量配分比を基準とし、個体値が平均値に近い順に選択した。選択した動物は、左後足重量配分比による層別連続無作為化法を用いて各群に割り付けた。左後足重量配分比の平均値が各群で同様の値を示し、群間に差がないことを確認後、体重についても平均値が各群で同様の値を示し、群間に差がないことを確認した。
半月板部分切除手術は群分け日の翌日に行い、半月板部分切除手術日を術後0日と定義した。14~15週齢の動物を用い、参考文献1~3に記載の方法を参考にして、半月板部分切除変形性関節症モデルを作製した。
参考文献2:T. Kikuchi, H. Yamada and M. Shimmei:Effect of high molecular weight hyaluronan on cartilage degeneration in a rabbit model of osteoarthritis,Osteoarthritis and Cartilage, Vol.4,No.2, pp.99-110(1996)
参考文献3:菊池 寿幸,山田 治基,堀田 拓,館田 智昭,小松 修一, 新名 正由:家兎変形性関節症モデルにおける高分子hyaluronanの軟骨変性抑制作用,関節外科,Vol.15,No.11,92-98(1996)
参考文献4:舘田 智昭,永峯 春代,岩館 克治,中村 亨:ウサギの実験的変形性関節症(OA)モデルおよび固定関節拘縮(PS)モデルにおけるヒアルロン酸ナトリウム製剤(ME3710)の薬効薬理試験,薬理と治療,23,833-841(1995)
参考文献5:野地 裕美,八木 直美,小田 康弘,岩館 克治,田元 浩一,関川 彬:
室温保存可能な新規高分子ヒアルロン酸ナトリウム製剤の生物学的同等性試験,薬理と治療,33,303-312(2005)
参考文献6:渡辺 耕志,並木 脩,豊島 弘道,楠本 剛夫:固定関節に対する高分子ヒアルロン酸の影響,整形外科基礎科学,Vol.9,77-79(1982)
参考文献7:宮崎 匡輔,長野 聖,鈴木 啓太郎,後藤 幸子,山口 敏二郎,並木 脩:ウサギ固定関節に対するヒアルロン酸ナトリウムの影響,整形外科基礎科学,Vol.11,125-127(1984)
参考文献8:T. kawano, H. Miura, T. Mawatari, T. Moro-Oka, Y. Nakanishi, H. Higashi and Y. Iwamoto:Mechanical Effects of the Intraarticular Administration of High Molecular Weight Hyaluronic Acid Plus Phospholipid on Synovial Joint Lubrication and Prevention of Articular Cartilage Degeneration in Experimental Osteoarthritis, Arthritis & Rheumatism, Vol.48,No.7, pp.1923 - 1929 (2003)
参考文献1~8に記載の方法を参考にして、実施例4及び5並びに参考例1及び5の注射剤を0.1mL/kg関節腔内に注入する4群を、表8のとおり設定した。
両後足重量配分の測定には小動物用鎮痛評価装置Incapacitance Tester(英国Linton Instrument社製)を用いた。本装置は、本体容器に入れた動物の左右の脚への重量配分を、容器底面に設置したデュアルチャンネルのセンサーパッドにより、左右それぞれの重量をグラム単位で正確に検出し、その値を試験者が設定した時間にて平均化した。本体容器はウサギ用のものを使用した。測定設定時間は動物の静止状態で5秒とした。
左後足重量配分比(%)={左荷重(g)/(右荷重(g)+左荷重(g))×100}・・・(4)
実施例1で得られた自己架橋ヒアルロンを50mMリン酸緩衝生理食塩水(pH7.4)の溶媒に交換し、6wt%の懸濁状の架橋ヒアルロン酸組成物を調製した。得られた架橋ヒアルロン酸組成物7mlを採取し、容器に移し密閉した。同様の架橋ヒアルロン酸組成物採取を繰り返し、5本の試験サンプルを得た。
架橋ヒアルロン酸組成物0.4mlを、遠心式フィルターユニット(0.45マイクロメートル孔径,ミリポア社製)を用いて5℃、2,000rpm、30分間遠心分離して溶媒を除去し、さらに遠心式フィルターユニットごと20時間乾燥することで、溶媒除去した自己架橋ヒアルロン酸重量と乾燥した自己架橋ヒアルロン酸重量を求め、平衡膨潤倍率を算出した。溶媒は、10mMリン酸緩衝生理食塩水(pH6.0)で、NaCl濃度は0.9wt%であり、5℃で1日間膨潤させ、平衡膨潤状態に到達した後に、測定した。
実施例6~9及び比較例6の架橋ヒアルロン酸組成物について、実施例1~3及び比較例1~2における測定方法と同様に、60℃溶解性半減期を測定した。
実施例6~9及び比較例6の架橋ヒアルロン酸組成物の一次分子量として、実施例1~3及び比較例1~2における測定方法と同様に、粘度平均分子量を測定した。
自己架橋ヒアルロン酸中の架橋度(自己架橋エステル化度)は、プロトン核磁気共鳴法(NMR)による化学シフト強度(ピーク面積値)によって求めた。測定のためには、あらかじめヒアルロン酸構造を加水分解し、低分子量化する必要があったため、自己架橋ヒアルロン酸の主鎖構造のみを選択的に加水分解するヒアルロン酸加水分解酵素による加水分解処理を行った。ヒアルロン酸加水分解酵素は、Hyaluronidaze from sheep testes TypeV,lyophilized powder、activity:>1,500 units/mg solid(シグマアルドリッチ社製)を用い、不純物を除くため陽イオン交換カラムMono S 5/50GL(GEヘルスケア社製)を用い精製した。精製は、上記の酵素を0.1g/mL濃度になるよう10mM、pH5.0酢酸緩衝液に溶解し、この溶液0.1mLを同緩衝液で平衡化した上記のカラムに通液し、NaCl濃度0.05~0.15mol/Lで溶出してくる精製画分の酵素溶液0.8mLを得た。加水分解処理は、架橋ヒアルロン酸を10mM、pH5.0酢酸緩衝生理食塩水1.0mLに濃度3wt%になるように調整し、上記の精製酵素溶液0.2mLを添加し、37℃160rpm振とう条件下で24時間反応させ、その後、酵素溶液0.2mLを加えて同条件下でさらに24時間反応させた。反応後の溶液は、-30℃で凍結し、その後18時間凍結乾燥させた後、NMR測定試料とした。
測定で得られたスペクトル図より、架橋エステル基に相当する化学シフト(Ha:4.18ppm)及びアセチルメチル基に相当する化学シフト(Hb:2.05ppm)の積分値を求め、以下の式(5)より架橋度を算出した。
架橋度=100×([Ha]×2)/([Hb]/3)・・・(5)
実施例6~9及び比較例6の架橋ヒアルロン酸組成物の溶媒を、10mMリン酸緩衝化生理食塩水(pH6.0)に交換し、自己架橋ヒアルロン酸の濃度が6w/v%となるように調整した。60℃環境での加熱中、適当な間隔でサンプル採取を行い、遊離したヒアルロン酸量を測定し、ゲル分率を測定した。加熱時間に対するゲル分率の挙動を読み取り、ゲル分率97%に到達する加熱時間を求めた。また、ゲル分率95%に到達する加熱時間も上記と同様に求めた。その測定結果を以下の表10及び図8~9に示す。
また、実施例6~9及び比較例6の架橋ヒアルロン酸懸濁液について、平均沈降濃度の測定を行った。平衡沈降濃度は、懸濁液のヒアルロン酸濃度[C]、懸濁液の体積[V0]及び沈澱の体積[V]を測定し、下記式(6)のとおり求めた。
平衡沈降濃度=C×(V0/V)・・・(6)
<ヒアルロン酸原料中のエタノール除去前処理>
粘度平均分子量220万のヒアルロン酸ナトリウムの粉末を通気フィルター付き容器に入れ、室温下でポンプにより吸引し、3日間通気してエタノールを除去した。
2Nの硝酸75gを自公転型混練り装置(プライミクス製)に入れ、-10℃に冷却し、シャーベット状の硝酸凍結物を得た。硝酸凍結物に上記処理を施したヒアルロン酸ナトリウムの粉末22.5g(水分含量10%)を投入し、-10℃、100rpmで均一なゴム状になるまで1時間練り混ぜた(ヒアルロン酸ナトリウム20.8質量%)。
上記のとおり得られた自己架橋ヒアルロン酸を中和後30分間静置し、上清をデカンテーションで除き、沈降した自己架橋ヒアルロン酸に対して、9倍の重量の50mMリン酸緩衝液を加えた。次に、この架橋ヒアルロン酸懸濁液を高速回転装置(製品名:クレアミクスダブルモーション、エムテクニック(株)製)に投入し、装置のローターを順方向20,000rpm、スクリーンを逆方向に18,000rpmで回転させ、50℃未満になるよう冷却しながら15分間微粒化した。ローターは後退角度0度ローターを使用し、スクリーン上に存在するスリットの巾が1.0mmのものを使用した。
1000ppmのエタノールを含有するように調整したヒアルロン酸原料を用い、実施例10と同様にして、自己架橋ヒアルロン酸粒子を得た。
31000ppmのエタノールを含有するように調整したヒアルロン酸原料を用い、実施例10と同様にして、自己架橋ヒアルロン酸粒子を得た。
116000ppmのエタノールを含有するように調整したヒアルロン酸原料を用い、実施例10と同様にして、自己架橋ヒアルロン酸粒子を得た。
自己架橋ヒアルロン酸中の自己架橋エステル化度(架橋度)は、プロトン核磁気共鳴法(NMR)による化学シフト強度(ピーク面積値)によって求めた。測定のためには、あらかじめヒアルロン酸構造を加水分解し、低分子量化する必要があったため、自己架橋ヒアルロン酸の主鎖構造のみを選択的に加水分解するヒアルロン酸加水分解酵素による加水分解処理を行った。ヒアルロン酸加水分解酵素は、Hyaluronidaze from sheep testes TypeV,lyophilized powder、activity:>1,500 units/mg solid(シグマアルドリッチ社製)を用い、不純物を除くため陽イオン交換カラムMono S 5/50GL(GEヘルスケア社製)を用い精製した。精製は、上記の酵素を0.1g/mL濃度になるよう10mM、pH5.0酢酸緩衝液に溶解し、この溶液0.1mLを同緩衝液で平衡化した上記のカラムに通液し、NaCl濃度0.05~0.15mol/Lで溶出してくる精製画分の酵素溶液0.8mLを得た。加水分解処理は、架橋ヒアルロン酸を10mM、pH5.0酢酸緩衝生理食塩水1.0mLに濃度3wt%になるように調整し、上記の精製酵素溶液0.2mLを添加し、37℃160rpm振とう条件下で24時間反応させ、その後、酵素溶液0.2mLを加えて同条件下でさらに24時間反応させた。反応後の溶液は、-30℃で凍結し、その後18時間凍結乾燥させた後、NMR測定試料とした。
架橋度=100×([Ha]×2)/([Hb]/3)・・・(4)
上記で得られた実施例10~11及び比較例7~8の自己架橋ヒアルロン酸粒子について、エチルエステル量をNMRにより測定した。
ヒアルロン酸のエチルエステル量(モル%)=100×([Ha]/2)/([Hb]/3)・・・(5)
上記で得られた実施例10~11及び比較例7~8の自己架橋ヒアルロン酸粒子について、溶解性半減期を測定した。pH7.4のリン酸緩衝液を用いて、60℃環境で加熱を行い、5時間間隔でサンプル採取した。採取したサンプルを希釈し、遠心分離により上清と沈殿部に分け、それぞれの部分のヒアルロン酸濃度を測定し、ゲル分率を算出した。加熱時間に対するゲル分率の挙動を読み取り、ゲル分率50%に到達する加熱時間を求めた。
生理的食塩水に10mM濃度でリン酸緩衝成分を加え、pH7.4のリン酸緩衝化食塩水を調整した。このリン酸緩衝化生理的食塩水100mlに対して実施例10~11及び比較例7~8の自己架橋ヒアルロン酸粒子を添加し、自己架橋ヒアルロン酸が完全に溶解するまで30日間、37.0℃で浸漬した。
実施例10で得られた自己架橋ヒアルロン酸粒子の粒子径について、粒度・形状分布測定器PITA-1(セイシン企業製)を用い定量した。前処理として自己架橋ヒアルロン酸をメチレンブルーにより染色(染色液濃度:1w/v%、染色時間:1分以上)した。PITA-1の測定条件としては、キャリア液は蒸留水を用い、測定粒子数は10,000個、対物レンズ4倍で測定した。その結果、得られた自己架橋ヒアルロン酸粒子の平均体積粒径は65μmだった。
(実施例12)
実施例10で得られた自己架橋ヒアルロン酸粒子を、5℃、pH7.0の10mMリン酸緩衝生理食塩水に投入し、一時間おきに10mMリン酸緩衝生理食塩水を交換することを2回繰り返した。この架橋ヒアルロン酸組成物の全体積に対する、自己架橋ヒアルロン酸粒子の乾燥重量(濃度)が6w/v%になるように以下のように調整した。
架橋ヒアルロン酸組成物の全体積に対する自己架橋ヒアルロン酸粒子の乾燥重量が3w/v%になるように、pH7.0の10mMリン酸緩衝生理食塩水に投入して調整した以外は、実施例12と同様にして架橋ヒアルロン酸組成物を調製した。
自己架橋ヒアルロン酸の粒子化に高速回転装置(製品名:クレアミクスシングルモーション、エムテクニック(株)製)を使用し、装置のローターを順方向に10,000rpmで回転させ、架橋ヒアルロン酸が30℃未満になるよう冷却しながら6分間微粒化した以外は、実施例10と同様にして平均体積粒径が300μmの自己架橋ヒアルロン酸粒子を調製した。さらに、実施例12と同様にして自己架橋ヒアルロン酸粒子の濃度が、6w/v%の架橋ヒアルロン酸組成物を調製した。
自己架橋ヒアルロン酸の粒子化に高速回転装置(製品名:クレアミクスシングルモーション、エムテクニック(株)製)を使用し、装置のローターを順方向に20,000rpmで回転させ、架橋ヒアルロン酸が30℃未満になるよう冷却しながら4分間微粒化した以外は、実施例10と同様にして平均体積粒径が153μmの自己架橋ヒアルロン酸粒子を調製した。さらに、実施例12と同様にして自己架橋ヒアルロン酸粒子の濃度が、6w/v%の架橋ヒアルロン酸組成物を調製した。
自己架橋ヒアルロン酸の粒子化に高速回転装置(製品名:クレアミクスシングルモーション、エムテクニック(株)製)を使用し、装置のローターを順方向に20,000rpmで回転させ、自己架橋ヒアルロン酸の冷却を行わずに20分間微粒化し、平均体積粒径100μmの自己架橋ヒアルロン酸粒子を調製した。この時、自己架橋ヒアルロン酸の温度は85℃まで上昇した。さらに、実施例12と同様にして自己架橋ヒアルロン酸粒子の濃度が、6w/v%の架橋ヒアルロン酸組成物を調製した。
ヒアルロン酸関節製剤「スベニール」(商品名、中外製薬(株)製)
(粘度平均分子量200万、ヒアルロン酸濃度1w/v%)
ヒアルロン酸関節製剤「アルツ」(商品名、生化学工業(株)製)
(粘度平均分子量80万、ヒアルロン酸濃度1w/v%)
ヒアルロン酸関節製剤「Synvisc」(商品名、ジェンザイムコーポレーション社製)
(ヒアルロン酸濃度0.8w/v%)
ヒアルロン酸関節製剤「Durolane」(商品名、Q-MED社製)
(ヒアルロン酸濃度2.0w/v%)
生理食塩水「大塚生食注」(商品名、大塚製薬(株)製)
粘度測定装置であるレオメーターとして、MCR300(商品名、Physica製)を使用した。コーンプレートはコーン角1.009°(D=49.938mm)を用い、せん断速度50S-1、25℃にて測定した。実施例12~13並びに比較例9の架橋ヒアルロン酸組成物、参考例1~5の粘度比較を行った。表13に粘度の測定結果を示す。
架橋ヒアルロン酸組成物1mlを内径0.45cmの注射器テルモシリンジSS-01T(商品名、テルモ社製)に詰め、内径0.40mm、針の長さ25mmの注射針23G(テルモ社製)を付けた。押し出し圧測定機EZ-TEST(商品名、島津製作所(株)製)を用い、温度25℃で速度50mm/分の吐出条件で、実施例12及び比較例9~10の架橋ヒアルロン酸組成物、参考例1~5のこのシリンジにかかる圧力を測定した。表14に測定結果を示す。
吐出圧測定(1)で使用した「注射針23G(内径0.40mm)よりも細い24G、25G及び27Gの注射針を使用し、実施例12及び参考例1~5のサンプル1mlを注射針のついたシリンジ(テルモ社製)に詰め、吐出圧測定(1)と同様にしてシリンジにかかる圧力を測定した。その結果を以下の表15及び図10に示す。
実施例12及び13並びに比較例11のサンプルについて、自己架橋ヒアルロン酸に換算して10mg分を、0.1N水酸化ナトリウム溶液1mlに投入し、0℃で30分間静置し、自己架橋ヒアルロン酸を溶解した。この溶解液に、0.1N塩酸1mlを添加して中和し、GPC溶媒で濃度を0.01質量%になるように希釈調製し、0.2μmのメンブランフィルターでろ過した後、GPC装置に0.1ml注入して、実施例10~11及び比較例7~8と同様に、一次分子量として粘度平均分子量の測定を行った。測定結果を表16に示す。
生理的食塩水に10mM濃度でリン酸緩衝成分を加え、pH7.4のリン酸緩衝化食塩水を調整した。このリン酸緩衝化生理的食塩水100mlに対して実施例12及び13並びに比較例11のサンプル0.5mlを添加し、自己架橋ヒアルロン酸が完全に溶解するまで30日間、37.0℃で浸漬し、実施例10~11及び比較例7~8と同様に、粘度平均分子量の測定を行った。測定結果を表16に示す。
ウサギ(日本白色種 オス)、重量約3kgを麻酔(麻酔組成:ケタミン(4ml)+キシラジン(3ml)+生食(5ml))し、後足両膝に実施例12及び13、比較例9及び11、並びに、参考例1及び3のサンプルを、内径0.45cmのシリンジに23G注射針を用い、投与量0.1ml/kgで注入した。
実施例12及び13並びに参考例1及び5の関節腔内注射が疼痛に及ぼす作用を、ウサギの膝関節半月板部分切除による実験的変形性関節症を用いて測定した。
動物としては、13週齢のKbl:JW(SPF)系ウサギを実施例・参考例毎に8羽ずつ用いるために計32羽を準備した。動物を入荷後3~8日の毎日、評価装置に対する訓化として、動物を小動物用鎮痛評価装置Incapacitance Tester(Linton Instrument製)の本体容器(ホルダー)に入れ、5秒間静止させる操作を行った。
半月板部分切除手術日の前日に、群分けを行った。群分け日に、全例の体重と両後足重量配分を測定した。測定した両後足重量配分より左後足重量配分比((左荷重/両側合計荷重)×100(%))を算出した。左後足重量配分比を基準とし、個体値が平均値に近い順に選択した。選択した動物は、左後足重量配分比による層別連続無作為化法を用いて各群に割り付けた。左後足重量配分比の平均値が各群で同様の値を示し、群間に差がないことを確認後、体重についても平均値が各群で同様の値を示し、群間に差がないことを確認した。
半月板部分切除手術は群分け日の翌日に行い、半月板部分切除手術日を術後0日と定義した。14~15週齢の動物を用い、上述の参考文献1~3に記載の方法を参考にして、半月板部分切除変形性関節症モデルを作製した。
上述の参考文献1~8に記載の方法を参考にして、実施例12及び13並びに参考例1及び5の注射剤を0.1mL/kg関節腔内に注入する4群を、表18のとおり設定した。
両後足重量配分の測定には小動物用鎮痛評価装置Incapacitance Tester(英国Linton Instrument社製)を用いた。本装置は、本体容器に入れた動物の左右の脚への重量配分を、容器底面に設置したデュアルチャンネルのセンサーパッドにより、左右それぞれの重量をグラム単位で正確に検出し、その値を試験者が設定した時間にて平均化した。本体容器はウサギ用のものを使用した。測定設定時間は動物の静止状態で5秒とした。
左後足重量配分比(%)={左荷重(g)/(右荷重(g)+左荷重(g))×100}・・・(6)
実施例10で得られた自己架橋ヒアルロンを50mMリン酸緩衝生理食塩水(pH7.4)の溶媒に交換し、6wt%の懸濁状の架橋ヒアルロン酸組成物を調製した。得られた架橋ヒアルロン酸組成物7mlを採取し、容器に移し密閉した。同様の架橋ヒアルロン酸組成物採取を繰り返し、5本の試験サンプルを得た。
架橋ヒアルロン酸組成物0.4mlを、遠心式フィルターユニット(0.45マイクロメートル孔径,ミリポア社製)を用いて5℃、2,000rpm、30分間遠心分離して溶媒を除去し、さらに遠心式フィルターユニットごと20時間乾燥することで、溶媒除去した自己架橋ヒアルロン酸重量と乾燥した自己架橋ヒアルロン酸重量を求め、平衡膨潤倍率を算出した。溶媒は、10mMリン酸緩衝生理食塩水(pH6.0)で、NaCl濃度は0.9wt%であり、5℃で1日間膨潤させ、平衡膨潤状態に到達した後に、測定した。
実施例14~17及び比較例12の架橋ヒアルロン酸組成物について、実施例10~11及び比較例7~8における測定方法と同様に、60℃溶解性半減期を測定した。
実施例14~17及び比較例12の架橋ヒアルロン酸組成物の一次分子量として、実施例10~11及び比較例7~8における測定方法と同様に、粘度平均分子量を測定した。
実施例14~17及び比較例12の架橋ヒアルロン酸組成物について、実施例10~11及び比較例7~8における測定方法と同様に、架橋度(自己架橋エステル化度)を測定した。なお、測定条件を以下のとおりとした。機器:AVANCEIII 500、観測幅:500.232MHz、パルス幅:10.5μs(90°)、測定モード:13Cデカップル-1Hノンデカップル法、積算回数:7600回、測定温度:30℃。
実施例14~17及び比較例12の架橋ヒアルロン酸組成物の溶媒を、10mMリン酸緩衝化生理食塩水(pH6.0)に交換し、自己架橋ヒアルロン酸の濃度が6w/v%となるように調整した。60℃環境での加熱中、適当な間隔でサンプル採取を行い、遊離したヒアルロン酸量を測定し、ゲル分率を測定した。加熱時間に対するゲル分率の挙動を読み取り、ゲル分率97%に到達する加熱時間を求めた。また、ゲル分率95%に到達する加熱時間も上記と同様に求めた。その測定結果を以下の表20及び図16~17に示す。
また、実施例14~17及び比較例12の架橋ヒアルロン酸懸濁液について、平均沈降濃度の測定を行った。平衡沈降濃度は、懸濁液のヒアルロン酸濃度[C]、懸濁液の体積[V0]及び沈澱の体積[V]を測定し、下記式(7)のとおり求めた。
平衡沈降濃度=C×(V0/V)・・・(7)
Claims (13)
- 平衡膨潤倍率が3~10倍の自己架橋ヒアルロン酸の粒子と、水系溶媒とを含有する架橋ヒアルロン酸組成物であって、
前記架橋ヒアルロン酸組成物の全体積に対する、前記自己架橋ヒアルロン酸の粒子の乾燥重量は3~8w/v%である、架橋ヒアルロン酸組成物。 - 前記自己架橋ヒアルロン酸の粒子は、平均体積粒径が10~100μmである、請求項1記載の架橋ヒアルロン酸組成物。
- 前記自己架橋ヒアルロン酸の粒子は、一次分子量が80万以上であり、自己架橋エステル化度が0.05~0.50mol%である請求項2記載の架橋ヒアルロン酸組成物。
- 前記自己架橋ヒアルロン酸の粒子は、エチルエステル量が0.05mol%以下であり、自己架橋エステル化度が0.05~0.50mol%である、請求項1記載の架橋ヒアルロン酸組成物。
- 前記自己架橋ヒアルロン酸の粒子は、一次分子量が80万以上である請求項4記載の架橋ヒアルロン酸組成物。
- 請求項1~5のいずれか一項記載の架橋ヒアルロン酸組成物を含有する注射剤。
- 1回あたり1.25mg/kg体重以上の前記自己架橋ヒアルロン酸が投与されるように用いられる、請求項6記載の注射剤。
- 1回あたり75mg以上の前記自己架橋ヒアルロン酸が投与されるように用いられる、請求項6記載の注射剤。
- 請求項6~8のいずれか一項に記載の注射剤を収容した、プレフィルドシリンジ製剤。
- 平均体積粒径が10~100μmであり、平衡膨潤倍率が3~10倍である自己架橋ヒアルロン酸粒子。
- 一次分子量が80万以上であり、自己架橋エステル化度が0.05~0.50mol%である請求項10記載の自己架橋ヒアルロン酸粒子。
- エチルエステル量が0.05mol%以下、自己架橋エステル化度が0.05~0.50mol%であり、平衡膨潤倍率が3~10倍である自己架橋ヒアルロン酸粒子。
- 一次分子量が80万以上である、請求項12記載の自己架橋ヒアルロン酸粒子。
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WO2016167229A1 (ja) * | 2015-04-15 | 2016-10-20 | キユーピー 株式会社 | カルボキシメチル基含有修飾ヒアルロン酸および/またはその塩の架橋物およびその製造方法 |
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CN103124558B (zh) | 2015-06-24 |
CA2809228C (en) | 2018-01-23 |
CA2809228A1 (en) | 2012-03-01 |
EP2609924A4 (en) | 2014-01-22 |
AU2011294295A1 (en) | 2013-03-21 |
US9216193B2 (en) | 2015-12-22 |
KR20140000206A (ko) | 2014-01-02 |
EP2609924A1 (en) | 2013-07-03 |
JPWO2012026468A1 (ja) | 2013-10-28 |
CN103124558A (zh) | 2013-05-29 |
JP5824455B2 (ja) | 2015-11-25 |
AU2011294295B2 (en) | 2014-06-26 |
US20130203697A1 (en) | 2013-08-08 |
KR101834588B1 (ko) | 2018-03-05 |
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