WO2017154822A1 - 神経損傷治療用薬剤徐放シート - Google Patents
神経損傷治療用薬剤徐放シート Download PDFInfo
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- 238000007619 statistical method Methods 0.000 description 1
- 210000000331 sympathetic ganglia Anatomy 0.000 description 1
- 230000003977 synaptic function Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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- 102000015533 trkA Receptor Human genes 0.000 description 1
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Images
Classifications
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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
- A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
- A61K9/7007—Drug-containing films, membranes or sheets
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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
- A61K31/7135—Compounds containing heavy metals
- A61K31/714—Cobalamins, e.g. cyanocobalamin, i.e. vitamin B12
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
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- A—HUMAN NECESSITIES
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
Definitions
- the present invention relates to a drug sustained-release sheet for treating nerve damage (hereinafter sometimes simply referred to as “this sheet”). More specifically, the present invention relates to a sheet containing a drug having a therapeutic effect on nerve damage and gradually releasing the drug, a manufacturing method thereof, and the like.
- Peripheral nerve damage is roughly divided into discontinuous damage that breaks continuity at the damaged part and continuous nerve damage that has continuity at the damaged part such as strangulated neuropathy (carpal tunnel syndrome, etc.).
- the As a treatment method for discontinuous damage direct suture, autologous nerve transplant, or the like is selected.
- As a treatment method for continuous nerve injury nerve ablation or conservative treatment is selected.
- artificial nerves have been developed as devices having a regeneration effect on peripheral nerve damage, but this is used only for discontinuous nerve damage. Moreover, they merely bridge the defect of the damaged part and have no effect of promoting nerve axon regeneration. When a long period of time is required for recovery after peripheral nerve injury, irreversible changes occur in the muscle tissue.
- the artificial nerve is applicable only to nerve damage in which a defect exists in the damaged portion, and there is no device for continuous nerve damage having the largest number of patients. Therefore, there is a need in the medical field for tools that can be applied to both continuous nerve damage and discontinuous nerve damage and are effective in treating nerve damage.
- Vitamin B12 is important for the normal function of the nervous system, and its deficiency is known to cause systemic neuropathy called “subacute combined degeneration of the spinal cord” (Non-patent Document 1). .
- methylcobalamin at concentrations of 100 nM and higher promotes neurite outgrowth and neuronal survival, that these effects are mediated by the methylation cycle, a reaction related to methylation,
- methylation cycle a reaction related to methylation
- Patent Document 1 describes a biodegradable composite material in the form of a flexible sheet for implantation in the body, which includes a water-soluble glass fiber and a biocompatible binding material such as polycaprolactone.
- This flexible sheet-form biodegradable composite material is described to be used wrapped around a defect area of tissue to promote healing and used to prevent post-surgical adhesion formation. ing. It is also described that the biodegradable composite material can be used as a substitute for nerve grafts and is expected to be applied to discontinuous nerve injury, but promotes nerve axon regeneration Has no effect.
- the present extract As other drugs that can be contained in the sheet, there are inflammatory skin extracts of rabbits inoculated with vaccinia virus (hereinafter referred to as “the present extract”) or fractions thereof.
- the extract or the preparation containing the extract is known to have a wide variety of actions and effects, but the sheet for treating nerve damage containing the extract has not been known so far.
- Examples of the drug contained in the sheet further include nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). These belong to the neurotrophin family (neurotrophic factor family) together with neurotrophin-3 (NT-3) and the like, and various cells such as neurons (neurons), glial cells and the like (microglia, A secretory protein produced in astrocytes, oligodendrocytes, etc.). Neurotrophin exhibits actions such as maintaining survival of nerve cells, promoting neurite outgrowth, and promoting neurotransmitter synthesis.
- NNF nerve growth factor
- BDNF brain-derived neurotrophic factor
- NGF is synthesized and secreted by target cells (neurons, muscles, etc.) projected by neurons, is taken in via the TrkA receptor at the end of neurons' axons, and is retrogradely transported to the cell body to function.
- NGF specifically acts on sensory neurons (small dorsal root ganglion cells) and sympathetic ganglion neurons in the peripheral nervous system and on forebrain basal cholinergic neurons that project to the cerebral cortex and hippocampus in the central nervous system.
- BDNF is ubiquitous in the central nervous system, mainly in the hippocampus, and is known to exhibit various physiological activities in the nervous system, such as survival and maintenance of nerve cells, neurite morphology regulation, synaptic function regulation, and neuroplasticity control. ing.
- a sheet for treating nerve damage containing these neurotrophins has not been known so far.
- the present invention is capable of maintaining a drug concentration at a damaged site at an appropriate height for a long period of time, and promoting nerve regeneration without giving a stimulus that adversely affects the nerve even when embedded in the periphery of the nerve damaged site. It is an object of the present invention to provide a drug sustained-release sheet for treatment.
- a drug sustained-release sheet comprising a nonwoven fabric formed of nanofibers containing a drug for treating nerve damage and a biocompatible polymer.
- the drug sustained-release sheet according to [1], wherein the drug is vitamin B12.
- the drug sustained-release sheet according to [1], wherein the drug is a inflammatory skin extract of rabbits inoculated with vaccinia virus or a fraction thereof.
- the drug sustained-release sheet according to [1], wherein the drug is neurotrophin.
- the drug sustained-release sheet according to [4], wherein the neurotrophin is NGF or BDNF.
- the sustained-release drug sheet according to any one of [1] to [5], wherein the biocompatible polymer is a biodegradable aliphatic polyester or a polyacrylamide derivative.
- the biodegradable aliphatic polyester is selected from the group consisting of polycaprolactone or a copolymer thereof, polylactic acid or a copolymer thereof, polyglycolic acid or a copolymer thereof, and a mixture thereof [6] ]
- the polyacrylamide derivative is poly (N-isopropylacrylamide) or a copolymer thereof, poly (2-hydroxyethylmethacrylamide) or a copolymer thereof, a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide
- the drug sustained-release sheet according to the above [6] which is selected from the group consisting of a combination and a mixture thereof.
- the sustained-release drug sheet according to any one of [1] to [9], wherein the weight of the sheet is 1 mg / cm 2 to 100 mg / cm 2 .
- a drug sustained-release sheet comprising a nonwoven fabric formed of nanofibers containing a drug and a biocompatible polymer, having a Young's modulus of 100 kPa to 100 MPa, and a weight of 1 mg / cm 2 to 100 mg / cm 2 .
- the sustained-release drug sheet according to [11], wherein the drug is vitamin B12.
- the drug sustained-release sheet according to [11] wherein the drug is a inflammatory skin extract of rabbits inoculated with vaccinia virus or a fraction thereof.
- the drug sustained release sheet according to [11], wherein the drug is neurotrophin.
- the biodegradable aliphatic polyester is selected from the group consisting of polycaprolactone or a copolymer thereof, polylactic acid or a copolymer thereof, polyglycolic acid or a copolymer thereof, and a mixture thereof.
- the polyacrylamide derivative is poly (N-isopropylacrylamide) or a copolymer thereof, poly (2-hydroxyethylmethacrylamide) or a copolymer thereof, a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide
- the sustained-release drug sheet according to the above [16] which is selected from the group consisting of a combination and a mixture thereof.
- a method for producing a drug sustained-release sheet comprising the following steps (1) and (2): (1) A step of preparing a solution containing a drug, a biocompatible polymer, and a solvent (2) A step of subjecting the solution to electrospinning and spinning to form a nonwoven fabric.
- a method for producing a drug sustained-release sheet comprising the following steps (1) and (2): (1) A step of preparing a solution containing a drug, a biocompatible polymer, a solvent, and hyaluronic acid (2) A step of subjecting the solution to an electrospinning method and spinning to form a nonwoven fabric.
- a method for producing a drug sustained-release sheet comprising the following steps (1), (2) and (3): (1) A step of preparing a solution containing a drug, a biocompatible polymer, a solvent, and hyaluronic acid (2) A step of subjecting the solution to an electrospinning method to form a nonwoven fabric (3) Hyaluron on the nonwoven fabric The process of coating the acid.
- biodegradable aliphatic polyester is selected from the group consisting of polycaprolactone or a copolymer thereof, polylactic acid or a copolymer thereof, polyglycolic acid or a copolymer thereof, and a mixture thereof.
- the polyacrylamide derivative is poly (N-isopropylacrylamide) or a copolymer thereof, poly (2-hydroxyethylmethacrylamide) or a copolymer thereof, a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide
- the production method according to the above [23] which is selected from the group consisting of a coalescence and a mixture thereof.
- a method for producing a drug sustained-release sheet comprising the following steps (1) and (2): (1) a drug, polycaprolactone or a copolymer thereof, TEF (2, 2, 2-trifluoroethanol), HFIP (1, 1, 1, 3, 3, 3-hexafluoro-2-propanol), A step of preparing a solution containing a solvent selected from chloroform and DMF (N, N-dimethylformamide) (2) under the conditions of a voltage of 10 to 30 kV, a flow rate of 0.1 to 1 m / h, and a needle size of 18 to 24 G (1
- the solution prepared in (1) is subjected to an electrospinning method to spin and form a nonwoven fabric on the electrode surface.
- a method for treating nerve damage comprising applying a drug sustained-release sheet comprising a nonwoven fabric formed of nanofibers containing a drug having a nerve damage treatment effect and a biocompatible polymer to a patient in need of treatment.
- the drug is vitamin B12.
- the drug is a inflammatory skin extract of rabbit immunized with vaccinia virus or a fraction thereof.
- the treatment method according to [32] wherein the drug is neurotrophin.
- the neurotrophin is NGF or BDNF.
- the polyacrylamide derivative is poly (N-isopropylacrylamide) or a copolymer thereof, poly (2-hydroxyethylmethacrylamide) or a copolymer thereof, a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide
- the treatment method according to any of [32] to [40], wherein the weight of the sheet is 1 mg / cm 2 to 100 mg / cm 2 .
- vitamin B12 for production of a drug sustained-release sheet comprising a nonwoven fabric formed of nanofibers containing a drug having a therapeutic effect on nerve damage and a biocompatible polymer.
- neurotrophin for the manufacture of a drug sustained-release sheet comprising a nonwoven fabric formed of nanofibers containing a drug having a therapeutic effect on nerve damage and a biocompatible polymer.
- the neurotrophin is NGF or BDNF.
- the biocompatible polymer is a biodegradable aliphatic polyester or a polyacrylamide derivative.
- the biodegradable aliphatic polyester is selected from the group consisting of polycaprolactone or a copolymer thereof, polylactic acid or a copolymer thereof, polyglycolic acid or a copolymer thereof, and a mixture thereof. ] Use as described in].
- the polyacrylamide derivative is poly (N-isopropylacrylamide) or a copolymer thereof, poly (2-hydroxyethylmethacrylamide) or a copolymer thereof, a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide
- [50] The use according to any one of [43] to [49], wherein the sheet has a Young's modulus of 100 kPa to 100 MPa.
- the use according to any one of [43] to [50], wherein the weight of the sheet is 1 mg / cm 2 to 100 mg / cm 2 .
- the present invention it is possible to promote nerve regeneration by embedding and applying to the peripheral part of an injured site and maintaining the drug concentration at the site at an appropriate height for a long period of time. It is possible to provide a drug sustained-release sheet and a production method thereof.
- (A) is the figure which shows the result of having performed electrophysiological evaluation in the 6th week after an operation in the experiment which evaluates the medicinal effect of this sheet
- CMAP Compound muscle action potential
- TL Terminal latency
- NCV Nerve conduction velocity
- (A) is a figure which shows the result of having performed histological evaluation in the 6th week after an operation in the experiment which evaluates the pharmaceutical effect of this sheet
- NRC nerve regeneration induction tube
- the present invention provides a drug sustained release sheet for treating nerve damage.
- the drug sustained-release sheet of the present invention comprises a nonwoven fabric formed of nanofibers containing a drug such as vitamin B12 and a biocompatible polymer such as a biodegradable aliphatic polyester, and is used by being embedded in the periphery of a nerve injury site Is done.
- the drug to be contained in the drug sustained-release sheet of the present invention may be any drug that has a therapeutic effect on nerve damage, such as vitamin B12, vaccinia virus inoculated tissue extract or fraction thereof, neurotrophin such as NGF, BDNF, etc.
- the nerve damage can be effectively treated by acting on the nerve damage site and promoting nerve regeneration.
- the drug sustained release sheet of the present invention includes those containing hyaluronic acid in addition to the above drugs.
- the target disease of this sheet is nerve injury. Since the drug sustained release sheet of the present invention has a nerve regeneration promoting action, it is very useful in that both continuous nerve damage and discontinuous nerve damage can be treated. Examples of continuous nerve damage include strangulated neuropathy (a state in which a nerve is compressed by surrounding tissues). In addition, nerves after nerve suture (directly sutured after nerve injury), nerves after nerve detachment, nerves after nerve transplantation (neural transplantation of nerve damaged part with defect to give continuity)
- the drug sustained-release sheet of the present invention is also effective in promoting regeneration. In addition, even for discontinuous nerve damage, the drug sustained-release sheet of the present invention has an effect of promoting treatment alone or in combination with an artificial nerve.
- Vitamin B12 which is a drug contained in this sheet, contains cobalamin and its derivatives. Specific examples include methylcobalamin, cyanocobalamin, hydroxocobalamin, sulfitocobalamin, adenosylcobalamin, and salts thereof, and any of them can be suitably used as an active ingredient of the sustained-release agent of the present invention. Of these, methylcobalamin, cyanocobalamin, hydroxocobalamin, or a salt thereof is preferable, and methylcobalamin or a salt thereof is more preferable.
- the content of vitamin B12 is preferably about 1% to about 30% as a final concentration, more preferably about 2% to about 10%.
- This sheet consists of a nonwoven fabric formed of nanofibers containing a drug and a biocompatible polymer.
- Nanofiber means a fibrous material having a diameter of about 10 nm to about 1000 nm and a length of 100 times or more of the diameter.
- the biocompatible polymer used in the present invention include biodegradable aliphatic polyesters and polyacrylamide derivatives.
- Preferred biodegradable aliphatic polyesters include, for example, polycaprolactone, polylactic acid, polyglycolic acid, polyglycerol acid, polyhydroxyalkanoic acid, polybutylene succinate, copolymers thereof, derivatives thereof and the like.
- biodegradability selected from the group consisting of polycaprolactone or a copolymer thereof, polylactic acid or a copolymer thereof, polyglycolic acid or a copolymer thereof, and a mixture thereof.
- Aliphatic polyesters may be mentioned.
- the copolymer include polylactic acid-polycaprolactone copolymer, poly ( ⁇ -caprolactone-co-DL-lactide), and the like.
- the polyacrylamide derivative include poly (N-isopropylacrylamide) or a copolymer thereof, poly (2-hydroxyethylmethacrylamide) or a copolymer thereof, or a mixture thereof.
- the copolymer examples include poly (N-isopropylacrylamide), a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide, and the like.
- the nanofiber may contain at least one kind of the biocompatible polymer, and may be a biocompatible polymer other than the biocompatible polymer and a copolymer of the biocompatible polymer. Good. Further, it may be a copolymer containing a plurality of the biocompatible polymers. In these cases, the form of copolymerization may be block copolymerization, random copolymerization, alternating copolymerization, or graft copolymerization.
- the sheet constituting the drug sustained-release sheet of the present invention preferably has a Young's modulus of about 100 kPa to about 100 MPa, more preferably about 1 MPa to about 50 MPa.
- the Young's modulus can be obtained from a stress-strain curve based on a measurement value obtained using a commercially available tensile tester (for example, EZ-S500N (manufactured by Shimadzu Corporation)).
- the weight of the sheet is preferably 1 mg / cm 2 to 100 mg / cm 2 , and more preferably 1 mg / cm 2 to 50 mg / cm 2 .
- the thickness of the sheet is preferably about 100 ⁇ m to about 1 mm, more preferably about 100 ⁇ m to about 500 ⁇ m.
- the area of the sheet is not particularly limited, and it may be cut according to the size of the affected part during use. Moreover, you may shape
- the size of molding for example, preferably from 0.1 cm 2 ⁇ 10 cm 2, more preferably 1cm 2 ⁇ 4cm 2.
- This sheet is used by being embedded in the periphery of the nerve injury site. Since the present sheet is molded so as to have flexibility, the usage mode can be appropriately selected. Specifically, for example, a nerve damage site in a state where the periphery is peeled off and the nerve is exposed is preferably wound around or covered so as to be covered. Thereafter, the surrounding tissue is replaced and the skin is sutured. By forming the sheet flexibly, even if it is placed at the site of nerve injury, it does not give a stimulus that adversely affects nerves and surrounding tissues. Therefore, this sheet can be used by being embedded in the periphery of the nerve damage site. And it is not necessary to remove after treatment of a damaged part.
- the sheet is preferably used after being sterilized. As the sterilization method, it is preferable to select a method in which the drug is not decomposed and the shape and physical properties of the sheet are not impaired. Examples thereof include radiation sterilization and plasma sterilization.
- This sheet is prepared by preparing a solution containing a drug such as vitamin B12 and a biocompatible polymer such as a biodegradable aliphatic polyester, and using this solution as a raw material, such as an electrospinning method, a self-assembly method, and a phase separation method.
- the nanofiber can be produced by the spinning method described above, and the nonwoven fabric can be formed by a known method.
- An appropriate solvent can be used for the preparation of a solution (raw material solution) containing a drug such as vitamin B12 and a biocompatible polymer such as biodegradable aliphatic polyester.
- the solvent used in the raw material solution is removed in the spinning process and does not remain in the nonwoven fabric.
- the solvent used for the raw material solution include TEF, HFIP, chloroform, DMF and the like. Preferably, TEF etc. are mentioned.
- the biodegradable aliphatic polyester may contain a monomer component for imparting flexibility so that the nonwoven fabric has desired flexibility.
- a monomer component examples include DL-lactide, D-lactide, L-lactide, D-lactic acid, L-lactic acid, and the like.
- the content of the monomer component for imparting flexibility is preferably set each time depending on the type of biocompatible polymer used, the type of monomer component for imparting flexibility, and the desired flexibility level. .
- flexibility of a nonwoven fabric can be improved by adjusting the molecular weight of a biocompatible polymer, or making a branched structure in a polymer molecule.
- the molecular weight and the number of branches are preferably set as appropriate according to the type of biocompatible polymer used.
- biocompatible polymers for example, as a biodegradable aliphatic polyester, when a sheet is produced by electrospinning using polycaprolactone or a copolymer thereof, the following steps (1) and (2) are included. It is preferable to use a manufacturing method.
- Step of preparing a solution containing a drug, polycaprolactone or a copolymer thereof, and a solvent selected from TEF, HEIP, chloroform and DMF (2) Voltage 10 to 30 kV, flow rate 0.1 to 1 mL / h The step of subjecting the solution prepared in (1) to spinning using the electrospinning method under the conditions of a needle size of 18 to 24 G to form a nonwoven fabric on the electrode surface.
- the polycaprolactone or copolymer thereof used in the step (1) preferably has a weight average molecular weight of 1,000 to 300,000, a branch number of 1 to 8 per molecule, and a DL-lactide content of 0 to 50 mol%. More preferably, it is a polycaprolactone copolymer having a weight average molecular weight of 10,000 to 100,000, 2 to 7 branches per molecule, and a DL-lactide content of 20 to 45 mol%, and more preferably a weight average molecular weight of 30,000 to 600,000, A polycaprolactone copolymer having 3 to 6 branches per molecule and a DL-lactide content of 30 to 45 mol%.
- the solvent used is preferably TEF.
- step (2) the solution (raw material solution) prepared in step (1) is subjected to electrospinning to spin, and a nonwoven fabric is formed on the electrode surface.
- the conditions of the electrospinning method are preferably a voltage of 10 to 30 kV, a flow rate of 0.1 to 1 mL / h, and a needle size of 18 to 24G. More preferably, the voltage is 10 to 15 kV, the flow rate is 0.3 to 0.7 mL / h, and the needle size is 22 to 24 G.
- polyacrylamide derivatives for example, poly (N-isopropylacrylamide) or a copolymer thereof, or a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide is used.
- polyacrylamide derivatives for example, poly (N-isopropylacrylamide) or a copolymer thereof, or a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide is used.
- (1) Contains a drug, poly (N-isopropylacrylamide) or a copolymer thereof or a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide, and a solvent selected from TEF, HEIP, chloroform, and DMF Step of preparing the solution (2)
- the solution prepared in (1) was subjected to electrospinning under the conditions of a voltage of 10 to 30 kV, a flow rate of 0.1 to 1 mL / h, and a needle size of 18 to 24 G. The process of forming a nonwoven fabric.
- step (2) the solution (raw material solution) prepared in step (1) is subjected to electrospinning to spin, and a nonwoven fabric is formed on the electrode surface.
- the conditions of the electrospinning method are preferably a voltage of 10 to 30 kV, a flow rate of 0.1 to 1 mL / h, and a needle size of 18 to 24G. More preferable voltages and flow rates are a voltage of 10 to 20 kV and a flow rate of 0.3 to 1 mL / h.
- This sheet includes those containing hyaluronic acid in addition to drugs such as vitamin B12.
- Hyaluronic acid may be added to the solution in step (1), or the nonwoven fabric formed in step (2) may be dipped in hyaluronic acid for coating.
- the manufacturing method as described above is composed of a nonwoven fabric formed of nanofibers made of a drug and a biocompatible polymer, and has a Young's modulus of 100 kPa to 100 MPa and a weight of 1 mg / cm 2 to 100 mg / cm 2 .
- This sheet can be manufactured.
- Such a method for producing the present sheet, or the use of a drug or nanofiber or non-woven fabric for the production of such a present sheet is also included in the present invention.
- Drugs such as vitamin B12 and neurotrophin that can be contained in the sheet may be appropriately produced by known methods, or commercially available products may be purchased.
- hyaluronic acid may be appropriately produced by a known method, or a commercially available product may be purchased.
- the present extract that can be contained in the present sheet is an extract containing a non-protein active substance extracted and separated from the inflamed tissue of an animal that has been inoculated with vaccinia virus.
- This extract inoculates inflammatory tissue that has been inoculated with vaccinia virus, crushes the extract, removes the tissue fragment by adding an extraction solvent, adsorbs it to the adsorbent, and then elutes the active ingredient Can be obtained. That is, for example, the following steps.
- (A) Collect skin tissue such as rabbits and mice inoculated with vaccinia virus, crush the germed tissue, and add an extraction solvent such as water, phenol water, physiological saline or phenol glycerin water.
- an extract (filtrate or supernatant) is obtained by filtration or centrifugation.
- B) The extract is adjusted to an acidic pH, heated and deproteinized. Next, the deproteinized solution is adjusted to be alkaline and heated, followed by filtration or centrifugation.
- C) The obtained filtrate or supernatant is acidified and adsorbed on an adsorbent such as activated carbon or kaolin.
- D) An extract from inflamed tissue inoculated with vaccinia virus can be obtained by adding an extraction solvent such as water to the adsorbent, adjusting the pH to alkaline, and eluting the adsorbed components. Thereafter, if desired, the eluate can be made into a dried product by evaporating to dryness or freeze-drying under reduced pressure.
- mice As animals for inoculating vaccinia virus to obtain inflamed tissues, various animals infected with vaccinia virus such as rabbits, cattle, horses, sheep, goats, monkeys, rats and mice can be used. Inflamed skin tissue is preferred. Any rabbit may be used as long as it belongs to the order of rabbit eyes. Examples include rabbits, rabbits (rabbits made from rabbits), rabbits (Japanese rabbits), rabbits, snow rabbits, and the like. Of these, chira rabbits are suitable for use. In Japan, there is a rabbit called a rabbit that has been bred from the past and used as a domestic animal or experimental animal. There are many varieties (breeds) of the rabbit, but varieties such as Japanese white varieties and New Zealand white varieties (New Zealand white) can be suitably used.
- the vaccinia virus may be of any strain. Examples include Lister, Dairen, Ikeda, EM-63, and New York City Public Health Board (Health) strains.
- this extract is liquid at the time of manufacture, it can be concentrated or diluted as appropriate to obtain a desired concentration, or lyophilized. Further, by fractionating the present extract, it is possible to obtain a fraction having a higher therapeutic effect on nerve damage and to use it for the present sheet.
- a more specific method for producing the present extract is described in, for example, paragraph numbers [0024] to [0027] and [0031] of International Publication WO2016 / 194816.
- seat for treating the continuous nerve damage which was not the application object of the conventional artificial nerve can be provided. Since continuous nerve injury has the largest number of patients among peripheral nerve injury, the contribution of the present invention is considered to be very high. Also, discontinuous nerve damage can be used alone or in combination with an artificial nerve. This sheet is used by being embedded in the periphery of the nerve damage site. Therefore, it is not necessary to continuously increase the drug concentration in the blood by continuously administering a high dose of the drug, and nerve regeneration can be promoted by continuously releasing the drug locally. Furthermore, since the sheet is molded using a biocompatible polymer, it does not need to be removed after treatment of nerve damage.
- Example 1 Preparation of vitamin B12-containing sheet, etc.
- HEIP4.5mL was added to 900 mg of polycaprolactone, and ultrasonic dispersion was performed for 3 hours to prepare a dispersion solution (polymer solution concentration 20 wt%).
- a voltage of 20 kV was applied while sending out all of this dispersion solution at a rate of 1.0 mL / h using a 5 mL syringe.
- the spun fibers were laminated and collected on a metal base laid with aluminum foil to produce a polycaprolactone sheet (syringe needle used: 22G, syringe-metal base distance: 13 cm).
- a polycaprolactone sheet made of a nonwoven fabric formed of nanofibers could be produced.
- a fiber sheet using poly (N-isopropylacrylamide), a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide hereinafter referred to as “poly (NIPAAm-co-HMAAm)” is also prepared in the same manner. (See FIGS. 2 and 3).
- Fibers spun on a metal substrate laid with aluminum foil were collected and collected to produce a poly (NIPAAm-co-HMAAm) sheet containing magnetic nanoparticles (used syringe needle: 22G, magnetic for sheet) Nanoparticle content 30%).
- a polycaprolactone sheet containing magnetic nanoparticles made of a nonwoven fabric formed of nanofibers could be produced.
- FIG. 5 shows a scanning electron microscope (SEM) observation image of the prepared polycaprolactone sheet coated with hyaluronic acid.
- vitamin B12-containing sheet 600 mg of poly ( ⁇ -caprolactone-co-DL-lactide) and methylcobalamin (manufactured by Sigma) were dissolved in 6 mL of TEF.
- Poly ( ⁇ -caprolactone-co-DL-lactide) having a weight average molecular weight of 40,000, a branch number of 4 / molecule, and a molar ratio of ⁇ -caprolactone to DL-lactide of 60:40 was prepared and used.
- 6.5 mg, 13 mg, or 20 mg was dissolved to prepare 3 types of solutions so that the final concentration of methylcobalamin was 1%, 2%, or 3%.
- nanofibers were manufactured using an electrospinning method, and this was formed into a mesh shape to prepare a nonwoven fabric.
- a nonwoven fabric was produced by extruding a solution from a 24 G needle at a flow rate of 0.5 mL / h, applying a voltage of 12 kV, spinning, and collecting and capturing nanofibers on the electrode surface.
- SEM scanning electron microscope
- Example 2 Confirmation of sustained release of vitamin B12-containing sheet, etc.
- 3 mL of PBS was placed in a tube, 10 mg of a sheet was immersed in the tube, kept at 37 ° C., and sampled over time to measure vitamin B12 concentration.
- the sample amount per one time was set to 100 ⁇ L, and the vitamin B12 concentration was measured by an ultraviolet-visible absorbance measurement method.
- the results are shown in FIG. All three types of sheets showed sustained release until the 25th day. Considering the theoretical maximum value, it is considered that the sustained release period becomes longer as the amount of methylcobalamin contained increases. Although not shown in the figure, it was confirmed that the three types of sheets exhibited sustained release for 8 weeks (until the 56th day). Similarly, the sustained release of the drug was confirmed for each sheet containing the present extract, NGF or BDNF.
- Example 3 Efficacy evaluation using rat sciatic nerve crush injury model.
- rat sciatic nerve crush injury model All animal experiments were conducted with the approval of the Ethics Committee of the Osaka University Animal Experiment Facility. Six-week-old male Wistar rats (body weight about 200 g) were used. All surgeries were performed with deep sedation with a mixed anesthetic of midazolam (2 mg / kg), butorphanol (2.5 mg / kg), and medetomidine (0.15 mg / kg). The left sciatic nerve was developed under a clean operation, and crush damage was applied with a scissors at a position 5 mm distal to the sciatic notch.
- the pressing time was 10 seconds
- the pressing frequency was 3 times
- the pressing operation interval was 10 seconds. Fascia and skin were sutured with 4-0 nylon.
- the experimental rats were classified into the following 5 groups. That is, the sham group that only developed without sciatic nerve crush injury, the CTR sheet group that only developed without sciatic nerve crush injury and transplanted with a sheet not containing methylcobalamin, crush damage treated Untreated group, crush-damaged methylcobalamin-containing sheet (3% methylcobalamin-containing sheet prepared in Example 1) transplanted, crush-damaged methylcobalamin systemically administered (1 mg / kg / day) A MeCbl pump group was established.
- Methylcobalamin was administered systemically by placing osmotic minipump (Model 2ML2; Alzet, Cuperitino, CA, USA) subcutaneously on the back. All surgeries were performed by the same surgeon. A sheet containing the present extract as a drug was similarly examined.
- the experimental rats were classified into the following 4 groups. That is, the sham group in which only the sciatic nerve was expanded without crushing injury, the CTR sheet group in which the sciatic nerve was expanded without crushing injury and the sheet not containing this extract was transplanted, and crush injury was treated An untreated group that was not performed, and an NTP sheet group that was transplanted with a crush-damaged and present extract-containing sheet (the present extract-containing sheet prepared in Example 1) were provided. Similarly, a sheet containing NGF or BDNF as a drug could be transplanted into a model rat in which the sciatic nerve was crush-damaged.
- Vitamin B12 blood concentration At 6 weeks after surgery, 1 mL of blood was collected from the left ventricle of the rat under anesthesia. The collected blood was centrifuged at 800 ⁇ g for 20 minutes, and the supernatant was collected. Blood vitamin B12 concentration measurement was requested from BML (Tokyo).
- SFI sciatic function index
- the primary antibodies are anti-neurofilament 200 (NF200) antibody produced in rabbit (1: 1000; 102M4784, SIGMA), which is an axonal index, and anti-myelin basic protein (MBP) Mouse mAb (1: 1000), which is an index of myelin sheath. NE1018, CALIOCHEM) was used.
- NF200 neuroneurofilament 200
- MBP anti-myelin basic protein
- Mouse mAb (1: 1000), which is an index of myelin sheath.
- Alexa 488_labeled goat anti-rabbit IgG antibody (1: 1000; Lifetechnologies
- Alexa 568_labeled goat anti-mouse IgG antibody (1: 1000; Lifetechnologies) were used.
- Total axon number, MBP positive axon number / total axon number were evaluated.
- Example 4 Efficacy evaluation using rat sciatic nerve defect model.
- Male Wistar rats (body weight: about 200 g) 6 weeks old were used. All surgeries were performed with deep sedation with a mixed anesthetic of midazolam (2 mg / kg), butorphanol (2.5 mg / kg), and medetomidine (0.15 mg / kg).
- the left sciatic nerve was developed under a clean operation, and the sciatic nerve was cut at a position 5 mm distal from the sciatic notch and further 10 mm from there to create a 10 mm defect model.
- the experimental rats were classified into the following 4 groups.
- Nerve regeneration inducing tube + MeCbl sheet group sutured with 10-0 nylon so that the sciatic nerve stump is pulled 1 mm each at both ends of the nerve regeneration inducing tube 1.5 mm in diameter x 12 mm in length, and the width around it The methylcobalamin-containing sheet of 10 mm ⁇ length 14 mm was placed so as to cover it.
- Nerve regeneration induction tube group 10-0 nylon was sutured so that the sciatic nerve stumps were pulled into each end of the nerve regeneration induction tube by 1 mm.
- Autograft group The cut sciatic nerve was inverted and sutured with 10-0 nylon.
- sham group Only the sciatic nerve was expanded. Fascia and skin were sutured with 4-0 nylon. All surgeries were performed by the same surgeon. A sheet containing the present extract, NGF or BDNF as a drug could be similarly transplanted to a sciatic nerve deficient model rat.
- FIG. (A) is the result of CMAP
- (B) is the result of TL
- (C) is the result of NCV.
- CMAP and TL the MeCbl sheet group (CMAP: 18.5 ⁇ 1.5 mV, TL: 3) compared to the untreated group (CMAP: 19.5 ⁇ 2.3 mV, TL: 3.45 ⁇ 0.08 ms) .27 ⁇ 0.09 ms) and the MeCbl pump group (CMAP: 19.5 ⁇ 1.5 mV, TL: 3.24 ⁇ 0.07 ms) showed no significant improvement.
- FIG. 14 and FIG. FIG. 7 is a microscopic image in which the myelin sheath is immunostained using an anti-MBP antibody, and the portion that appears white in the figure is stained myelin sheath.
- FIG. 15 (A) shows the result of the total number of axons
- FIG. 15 (B) shows the result of the number of MBP positive axons / total number of axons.
- the number of regenerating axons per square millimeter is significant in the untreated group (2843 ⁇ 68 / mm 2 ), MeCbl sheet group (2733 ⁇ 142 / mm 2 ), and MeCbl pump group (2735 ⁇ 77 / mm 2 ). There was no difference.
- the myelination rate (number of MBP positive axons / total number of axons) is higher in the MeCbl sheet group (91.0 ⁇ 0.8%) than in the untreated group (85.0 ⁇ 0.9%).
- the improvement was recognized significantly similarly to the MeCbl pump group (91.5 ⁇ 0.6%). Similar improvements were observed in the transplantation of sheets containing the extract, NGF or BDNF as drugs.
- FIG. 17 shows the TL results and FIG. 18 shows the NCV results.
- TL an improvement was observed in the nerve regeneration induction tube + MeCbl sheet group (3.43 ⁇ 0.12 m / s) compared to the nerve regeneration induction tube group (4.67 ⁇ 0.37 m / s).
- NCV compared with the nerve regeneration induction tube group (16.3 ⁇ 3.13 m / s), the nerve regeneration induction tube + MeCbl sheet group (29.5 ⁇ 4.50 m / s) showed an improvement trend. Improvement was also observed in the sheet containing this extract, NGF or BDNF as a drug.
- the present invention is very useful in functional recovery after peripheral nerve injury.
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Abstract
Description
[1]神経損傷治療用の薬剤および生体適合性ポリマーを含有するナノファイバーで形成された不織布からなる薬剤徐放シート。
[2]薬剤がビタミンB12である前記[1]に記載の薬剤徐放シート。
[3]薬剤がワクシニアウイルス接種ウサギ炎症皮膚抽出物またはその分画物である前記[1]に記載の薬剤徐放シート。
[4]薬剤がニューロトロフィンである前記[1]に記載の薬剤徐放シート。
[5]ニューロトロフィンがNGFまたはBDNFである前記[4]に記載の薬剤徐放シート。
[6]生体適合性ポリマーが生分解性脂肪族ポリエステルまたはポリアクリルアミド誘導体である前記[1]~[5]のいずれかに記載の薬剤徐放シート。
[7]生分解性脂肪族ポリエステルが、ポリカプロラクトンまたはその共重合体、ポリ乳酸またはその共重合体、ポリグリコール酸またはその共重合体、およびそれらの混合物からなる群から選択される前記[6]に記載の薬剤徐放シート。
[8]ポリアクリルアミド誘導体が、ポリ(N-イソプロピルアクリルアミド)またはその共重合体、ポリ(2-ヒドロキシエチルメタクリルアミド)またはその共重合体、N-イソプロピルアクリルアミドおよび2-ヒドロキシエチルメタクリルアミドの共重合体ならびにそれらの混合物からなる群から選択される前記[6]に記載の薬剤徐放シート。
[9]シートのヤング率が100kPa~100MPaである前記[1]~[8]のいずれかに記載の薬剤徐放シート。
[10]シートの重量が1mg/cm2~100mg/cm2である前記[1]~[9]のいずれかに記載の薬剤徐放シート。
[11]シートが薬剤および生体適合性ポリマーを含有するナノファイバーで形成された不織布からなり、ヤング率が100kPa~100MPa、重量が1mg/cm2~100mg/cm2である薬剤徐放シート。
[12]薬剤がビタミンB12である前記[11]に記載の薬剤徐放シート。
[13]薬剤がワクシニアウイルス接種ウサギ炎症皮膚抽出物またはその分画物である前記[11]に記載の薬剤徐放シート。
[14]薬剤がニューロトロフィンである前記[11]に記載の薬剤徐放シート。
[15]ニューロトロフィンがNGFまたはBDNFである前記[14]に記載の薬剤徐放シート。
[16]生体適合性ポリマーが生分解性脂肪族ポリエステルまたはポリアクリルアミド誘導体である前記[11]~[15]のいずれかに記載の薬剤徐放シート。
[17]生分解性脂肪族ポリエステルが、ポリカプロラクトンまたはその共重合体、ポリ乳酸またはその共重合体、ポリグリコール酸またはその共重合体、およびそれらの混合物からなる群から選択される前記[16]に記載の薬剤徐放シート。
[18]ポリアクリルアミド誘導体が、ポリ(N-イソプロピルアクリルアミド)またはその共重合体、ポリ(2-ヒドロキシエチルメタクリルアミド)またはその共重合体、N-イソプロピルアクリルアミドおよび2-ヒドロキシエチルメタクリルアミドの共重合体ならびにそれらの混合物からなる群から選択される前記[16]に記載の薬剤徐放シート。
[19]さらにヒアルロン酸を含有させたことを特徴とする前記[1]~[18]のいずれかに記載の薬剤徐放シート。
(1)薬剤と、生体適合性ポリマーと、溶媒を含む溶液を調製する工程
(2)該溶液を電界紡糸法に供して紡糸し、不織布を形成させる工程。
[21]以下の工程(1)および(2)を含む薬剤徐放シートの製造方法:
(1)薬剤と、生体適合性ポリマーと、溶媒と、ヒアルロン酸を含む溶液を調製する工程
(2)該溶液を電界紡糸法に供して紡糸し、不織布を形成させる工程。
[22]以下の工程(1)、(2)および(3)を含む薬剤徐放シートの製造方法:
(1)薬剤と、生体適合性ポリマーと、溶媒と、ヒアルロン酸を含む溶液を調製する工程
(2)該溶液を電界紡糸法に供して紡糸し、不織布を形成させる工程
(3)不織布にヒアルロン酸をコーティングする工程。
[23]生体適合性ポリマーが生分解性脂肪族ポリエステルまたはポリアクリルアミド誘導体である前記[20]~[22]のいずれかに記載の薬剤徐放シートの製造方法。
[24]生分解性脂肪族ポリエステルが、ポリカプロラクトンまたはその共重合体、ポリ乳酸またはその共重合体、ポリグリコール酸またはその共重合体、およびそれらの混合物からなる群から選択される前記[23]に記載の製造方法。
[25]ポリアクリルアミド誘導体が、ポリ(N-イソプロピルアクリルアミド)またはその共重合体、ポリ(2-ヒドロキシエチルメタクリルアミド)またはその共重合体、N-イソプロピルアクリルアミドおよび2-ヒドロキシエチルメタクリルアミドの共重合体ならびにそれらの混合物からなる群から選択される前記[23]に記載の製造方法。
[26]以下の工程(1)および(2)を含む薬剤徐放シートの製造方法:
(1)薬剤と、ポリカプロラクトンまたはその共重合体と、TEF (2, 2, 2-トリフルオロエタノール)、HFIP (1, 1, 1, 3, 3, 3-ヘキサフルオロ-2-プロパノール)、クロロホルムおよびDMF(N,N-ジメチルホルムアミド)から選択される溶媒を含む溶液を調製する工程
(2)電圧10~30kV、流速0.1~1m/h、ニードルサイズ18~24Gの条件で(1)で調製した溶液を電界紡糸法に供して紡糸し、電極面で不織布を形成させる工程。
[27]ポリカプロラクトンまたはその共重合体が、重量平均分子量1000~300000、1分子当たりの分岐数1~8、および、DL-ラクチドを0~50モル%含む前記[26]に記載の製造方法。
[28]薬剤がビタミンB12である前記[20]~[27]のいずれかに記載の製造方法。
[29]薬剤がワクシニアウイルス接種ウサギ炎症皮膚抽出物またはその分画物である前記[20]~[27]のいずれかに記載の製造方法。
[30]薬剤がニューロトロフィンである前記[20]~[27]のいずれかに記載の製造方法。
[31]ニューロトロフィンがNGFまたはBDNFである前記[30]に記載の製造方法。
[33]薬剤がビタミンB12である前記[32]に記載の方法。
[34]薬剤がワクシニアウイルス接種ウサギ炎症皮膚抽出物またはその分画物である前記[32]に記載の治療方法。
[35]薬剤がニューロトロフィンである前記[32]に記載の治療方法。
[36]ニューロトロフィンがNGFまたはBDNFである前記[35]に記載の治療方法。
[37]生体適合性ポリマーが生分解性脂肪族ポリエステルまたはポリアクリルアミド誘導体である前記[32]~[36]のいずれかに記載の治療方法。
[38]生分解性脂肪族ポリエステルが、ポリカプロラクトンまたはその共重合体、ポリ乳酸またはその共重合体、ポリグリコール酸またはその共重合体、およびそれらの混合物からなる群から選択される前記[37]に記載の治療方法。
[39]ポリアクリルアミド誘導体がポリ(N-イソプロピルアクリルアミド)またはその共重合体、ポリ(2-ヒドロキシエチルメタクリルアミド)またはその共重合体、N-イソプロピルアクリルアミドおよび2-ヒドロキシエチルメタクリルアミドの共重合体ならびにそれらの混合物からなる群から選択される前記[37]に記載の治療方法。
[40]シートのヤング率が100kPa~100MPaである前記[32]~[39]のいずれかに記載の治療方法。
[41]シートの重量が1mg/cm2~100mg/cm2である前記[32]~[40]のいずれかに記載の治療方法。
[42]適用が神経損傷部位の周辺部に埋め込むことによるものである前記[32]~[41]のいずれかに記載の治療方法。
[44]神経損傷治療効果を有する薬剤および生体適合性ポリマーを含有するナノファイバーで形成された不織布からなる薬剤徐放シートの製造のための、ワクシニアウイルス接種ウサギ炎症皮膚抽出物またはその分画物の使用。
[45]神経損傷治療効果を有する薬剤および生体適合性ポリマーを含有するナノファイバーで形成された不織布からなる薬剤徐放シートの製造のための、ニューロトロフィンの使用。
[46]ニューロトロフィンがNGFまたはBDNFである前記[45]に記載の使用。
[47]生体適合性ポリマーが生分解性脂肪族ポリエステルまたはポリアクリルアミド誘導体である前記[43]~[46]のいずれかに記載の使用。
[48] 生分解性脂肪族ポリエステルが、ポリカプロラクトンまたはその共重合体、ポリ乳酸またはその共重合体、ポリグリコール酸またはその共重合体、およびそれらの混合物からなる群から選択される前記[47]に記載の使用。
[49]ポリアクリルアミド誘導体が、ポリ(N-イソプロピルアクリルアミド)またはその共重合体、ポリ(2-ヒドロキシエチルメタクリルアミド)またはその共重合体、N-イソプロピルアクリルアミドおよび2-ヒドロキシエチルメタクリルアミドの共重合体ならびにそれらの混合物からなる群から選択される前記[47]に記載の使用。
[50]シートのヤング率が100kPa~100MPaである前記[43]~[49]のいずれかに記載の使用。
[51]シートの重量が1mg/cm2~100mg/cm2である前記[43]~[50]のいずれかに記載の使用。
また、前記ナノファイバーとしては、前記生体適合性ポリマーの少なくとも1種を含んでいればよく、前記生体適合性ポリマー以外の生体適合性ポリマーとこれら前記生体適合性ポリマーの共重合体であってもよい。また、前記生体適合性ポリマーを複数含む共重合体であってもよい。これらの場合、共重合の形式はブロック共重合、ランダム共重合、交互共重合又はグラフト共重合のいずれであってもよい。
(1)薬剤と、ポリカプロラクトンまたはその共重合体と、TEF、HEIP、クロロホルムおよびDMFから選択される溶媒を含む溶液を調製する工程
(2)電圧10~30kV、流速0.1~1mL/h、ニードルサイズ18~24Gの条件で(1)で調製した溶液を電界紡糸法に供して紡糸し、電極面で不織布を形成させる工程。
(1)薬剤と、ポリ(N-イソプロピルアクリルアミド)もしくはその共重合体またはN-イソプロピルアクリルアミドおよび2-ヒドロキシエチルメタクリルアミドの共重合体と、TEF、HEIP、クロロホルムおよびDMFから選択される溶媒を含む溶液を調製する工程
(2)電圧10~30kV、流速0.1~1mL/h、ニードルサイズ18~24Gの条件で(1)で調製した溶液を電界紡糸法に供して紡糸し、電極面で不織布を形成させる工程。
本抽出物はワクシニアウイルスを接種して発痘した炎症組織を破砕し、抽出溶媒を加えて組織片を除去した後、除蛋白処理を行い、これを吸着剤に吸着させ、次いで有効成分を溶出することによって得ることができる。即ち、例えば、以下のような工程である。
(A)ワクシニアウイルスを接種し発痘させたウサギ、マウス等の皮膚組織等を採取し、発痘組織を破砕し、水、フェノール水、生理食塩液またはフェノール加グリセリン水等の抽出溶媒を加えた後、濾過または遠心分離することによって抽出液(濾液または上清)を得る。
(B)前記抽出液を酸性のpHに調整して加熱し、除蛋白処理する。次いで除蛋白した溶液をアルカリ性に調整して加熱した後に濾過または遠心分離する。
(C)得られた濾液または上清を酸性とし活性炭、カオリン等の吸着剤に吸着させる。
(D)前記吸着剤に水等の抽出溶媒を加え、アルカリ性のpHに調整し、吸着成分を溶出することによってワクシニアウイルス接種炎症組織抽出物を得ることができる。その後、所望に応じて、適宜溶出液を減圧下に蒸発乾固または凍結乾燥することによって乾固物とすることもできる。
なお、本抽出物のより具体的な製造方法は、例えば国際公開WO2016/194816号公報の段落番号[0024]~[0027]、[0031]等に記載されている。
〔実施例1:ビタミンB12含有シート等の作製〕
(1)ポリカプロラクトンシート等の作製
ポリカプロラクトン900 mgにHEIP4.5mLを加え、3時間超音波処理を行うことで分散溶液(ポリマー溶液濃度20wt%)を調製した。
次に、この分散溶液すべてを5 mLシリンジを用いて、1.0 mL/hの速度で送り出しながら20kVの電圧を印加した。アルミホイルを敷いた金属基盤上に紡糸された繊維を積層させて捕集し、ポリカプロラクトンシートを作製した(使用したシリンジ針: 22G、シリンジ-金属基盤距離: 13cm)。
図1の走査型電子顕微鏡(SEM)観察像に示したように、ナノファイバーで形成された不織布からなるポリカプロラクトンシートを製造することができた。
同様に、ポリ(N-イソプロピルアクリルアミド)、N-イソプロピルアクリルアミドと2-ヒドロキシエチルメタクリルアミドの共重合体(以下、「ポリ(NIPAAm-co-HMAAm)」)を用いたファイバーシートについても同様に作製した(図2および図3参照)。
ポリカプロラクトン900mgとγ―Fe2O3からなる磁性ナノ粒子270 mgに、HEIP4.5 mLを加え、12時間超音波処理を行うことで分散溶液(ポリマー溶液濃度20wt%)を調製した。
次に、この分散溶液すべてを5mLシリンジを用いて、1.0 mL/hの速度で送り出しながら20kVの電圧を印加した。アルミホイルを敷いた金属基盤上に紡糸された繊維を積層させて捕集し、磁性ナノ粒子を含有したポリ(NIPAAm-co-HMAAm)シートを作製した(使用したシリンジ針: 22G、シートに対する磁性ナノ粒子含有量30%)。図4の走査型電子顕微鏡(SEM)観察像に示したように、ナノファイバーで形成された不織布からなる磁性ナノ粒子を含有したポリカプロラクトンシートを製造することができた。
ヒアルロン酸128mgを純水2.5mLに溶解し、5wt%のヒアルロン酸溶液を調製した。実施例1(1)で作製したポリカプロラクトンシートをヒアルロン酸溶液に浸漬させ、24時間静置した。凍結乾燥を72時間行い、ヒアルロン酸コーティングポリカプロラクトンシートを作製した。図5に作製されたヒアルロン酸でコーティングされたポリカプロラクトンシートの走査型電子顕微鏡(SEM)観察像を示した。
6mLのTEFに、600mgのポリ(ε-カプロラクトン-co-DL-ラクチド)およびメチルコバラミン(Sigma社製)を溶解した。ポリ(ε-カプロラクトン-co-DL-ラクチド)は重量平均分子量が40000、分岐数が4/分子、ε-カプロラクトンとDL-ラクチドのモル比が60:40のものを自製して使用した。メチルコバラミンの終濃度が1%、2%または3%になるように、それぞれ6.5mg、13mgまたは20mgを溶解し、3種類の溶液を調製した。次に、電界紡糸法を用いてナノファイバーを製造し、これを網目状に形成して不織布を作製した。具体的には、24Gのニードルから0.5mL/hの流速で溶液を押し出し、12kVの電圧を印加して紡糸し、電極面にナノファイバーを集積させて捕捉することにより、不織布を作製した。
図7の走査型電子顕微鏡(SEM)観察像に示したように、ナノファイバーで形成された不織布からなるビタミンB12含有シートを製造することができた。このシートの厚さは約300μm、重量は約10mg/cm2あった。
同様にして、薬剤としてNGFおよびBDNFについても、それぞれを含有するシートを作製することができた。
ポリカプロラクトン900mgと本抽出物凍結乾燥粉末270mgに、HEIP4.5 mLを加え、3時間超音波処理を行うことで分散溶液(ポリマー溶液濃度20wt%)を調製した。
次に、この分散溶液すべてを5mLシリンジを用いて、0.5mL/hの速度で送り出しながら20kVの電圧を印加した。アルミホイルを敷いた金属基盤上に紡糸された繊維を積層させて捕集し、本抽出物含有シートを作製した(使用したシリンジ針: 18G、シートに対する本抽出物担持量30%)。図6に作製した本抽出物を含有したポリカプロラクトンシートの走査型電子顕微鏡(SEM)観察像を示した。
チューブに3mLのPBSを入れ、その中に10mgのシートを浸漬して37℃に保温し、経時的にサンプリングしてビタミンB12濃度を測定した。1回あたりのサンプル量は100μLとし、ビタミンB12濃度は紫外可視吸光度測定法により行った。
結果を図8に示した。3種類のシートは、いずれも25日目まで徐放性を示した。理論最大値を考慮すれば、含有するメチルコバラミン量が多いほど、徐放期聞が長くなると考えられる。なお、図には示さないが、3種類のシートはその後も8週間にわたり(56日目まで)徐放性を示すことを確認した。
同様にして、本抽出物、NGFまたはBDNFを含有させたシートについても、それぞれ薬剤の徐放性を確認した。
1.ラット坐骨神経圧挫損傷モデルの作製
すべての動物実験は大阪大学動物実験施設の倫理委員会の承認を得て進めた。6週齢の雄のWistar系ラット(体重約200g)を使用した。すべての手術は、midazolam(2mg/kg)、butorphanol(2.5mg/kg)、medetomidine(0.15mg/kg)の混合麻酔薬で深鎮静をかけて行った。清潔操作下に左坐骨神経を展開し、坐骨切痕から遠位5mmの位置に鑷子で圧挫損傷を加えた。圧挫時間は10秒間、圧挫回数は3回とし、圧挫操作の間隔は10秒間とした。筋膜および皮膚を4-0ナイロンで縫合した。実験ラットは以下の5群に分類した。すなわち、坐骨神経を圧挫損傷せず展開のみ行ったsham群、坐骨神経を圧挫損傷せず展開のみ行いメチルコバラミンを含有していないシートを移植したCTR sheet群、圧挫損傷し治療を行わなかったuntreated群、圧挫損傷しメチルコバラミン含有シート(実施例1で作製した3%メチルコバラミン含有シート)を移植したMeCbl sheet群、圧挫損傷しメチルコバラミンを全身投与(1mg/kg/day)したMeCbl pump群を設けた。メチルコバラミンの全身投与は、osmotic minipump(Model 2ML2; Alzet, Cuperitino, CA, USA)を背部皮下に留置することにより行った。すべての手術は同一の術者が行った。
薬剤として本抽出物を含有したシートについても同様に検討した。実験ラットは以下の4群に分類した。すなわち、坐骨神経を圧挫損傷せず展開のみ行ったsham群、坐骨神経を圧挫損傷せず展開のみ行い本抽出物を含有していないシートを移植したCTR sheet群、圧挫損傷し治療を行わなかったuntreated群、圧挫損傷し本抽出物含有シート(実施例1で作製した本抽出物含有シート)を移植したNTP sheet群を設けた。
薬剤として、NGFまたはBDNFを含有したシートについても、同様に坐骨神経を圧挫損傷したモデルラットに移植することができた。
(1)ビタミンB12血中濃度
術後6週時に、麻酔鎮静下にてラット左心室から血液を1mL採取した。採取した血液を800×gで20分間遠心分離し上清を回収した。血中ビタミンB12濃度測定はBML社(東京)に依頼した。
運動機能の評価のため、術後6週時点でsciatic function index(SFI)を測定した。SFI測定のため、ラットの後足にインクをつけ40cm四方の水平台の上に置いたoffice paper上を歩行させfoot printを記録した。以下の項目を計測しSFIを計算した。SFI=0が正常、SFI=-100が機能低下を示す。また、術後経過中に足趾の壊死・欠損を生じた個体は除外した。SFIは以下の数式で計算した。各項目は以下に示すとおりである。
SFI=-38*((EPL-NPL)/NPL+109.5*((ETS-NTS)/NTS)+13.3* ((EITS-NITS)-8
EPL: experimental print length
NPL: normal print length
ETS: experimental toe spread
NTS: normal toe spread
EIT: experimental intermediary toe spread
NIT: normal intermediary toe spread
術後3週または6週経過したラットを麻酔薬で鎮静をかけ、手術台に腹臥位とした。左坐骨神経および左前脛骨筋を展開した。Compound muscle action potential(CMAP)とTerminal latency(TL)は、坐骨神経近位を双極電極で刺激して測定した。Nerve conduction velocity(NCV)は坐骨神経圧挫損傷部の近位側および遠位側をそれぞれ双極電極で刺激して、各測定値から算出した。測定および評価には、AD Instruments Power Lab 2/26、Stimulus isolater、Bio AmpおよびChart&Scope software(いずれも、AD Instruments,Bel la Vista,NSW,Australis)を用いた。
術後6週経過したラットを麻酔薬で鎮静をかけ、左坐骨神経を採取して4%PFAで5日間、20%スクロースで24時間固定後に凍結包埋した。包埋した組織を神経短軸方向に5μm厚でスライスしglass slideに置いた。1時間乾燥させて、95%メタノールで30分間固定した。ブロッキング後に1次抗体を4℃で一夜反応させた。二次抗体は室温で1時間反応させ、核をDAPIで標識した。一次抗体は軸索の指標であるanti-neurofilament 200(NF200)antibody produced in rabbit(1:1000;102M4784,SIGMA)および髄鞘の指標であるAnti-Myelin Basic Protein(MBP)Mouse mAb(1:1000;NE1018,CALIOCHEM)を使用した。二次抗体はAlexa 488 _ labeled goat anti-rabbit IgG antibody(1:1000;Lifetechnologies)およびAlexa 568 _ labeled goat anti-mouse IgG antibody(1:1000;Lif etechnologies)を使用した。全軸索数、MBP陽性軸索数/全軸索数を評価した。
1.ラット坐骨神経欠損モデルの作製
6週齢の雄のWistar系ラット(体重約200g)を使用した。すべての手術は、midazolam(2mg/kg)、butorphanol(2.5mg/kg)、medetomidine(0.15mg/kg)の混合麻酔薬で深鎮静をかけて行った。清潔操作下に左坐骨神経を展開し、坐骨切痕から遠位5mmの位置と、そこからさらに遠位10mmの位置で坐骨神経を切断し10mmの欠損モデルを作成した。
実験ラットは以下の4群に分類した。(i) 神経再生誘導チューブ+MeCblシート群;径1.5mm×長さ12mmの神経再生誘導チューブの両端に坐骨神経断端をそれぞれ1mmずつ引き込むように10-0 ナイロンで縫合し、その周りに幅10mm×長さ14mmのメチルコバラミン含有シートを覆うように留置した。(ii) 神経再生誘導チューブ群;神経再生誘導チューブの両端に坐骨神経断端をそれぞれ1 mmずつ引き込むように10-0 ナイロンで縫合した。(iii) 自家移植片群;切断した坐骨神経を反転して10-0 ナイロンで縫合した。(iv) sham群;坐骨神経の展開のみを行った。筋膜および皮膚を4-0 ナイロンで縫合した。すべての手術は同一の術者が行った。
薬剤として本抽出物、NGFまたはBDNFを含有したシートについても、同様に坐骨神経欠損モデルラットに移植することができた。
(1)感覚機能評価
感覚機能の評価のため、術後12週時点で実施例3-2(2)と同様にmechanical hind paw withdrawal thresholdを測定した。
術後12週時点で実施例3-2(3)と同様にTLおよびNCVを算出した。
術後12週経過したラットを麻酔薬で鎮静させ、左坐骨神経を採取して4%PFAで7日間、20%スクロースで24時間固定後に凍結包埋した。実施例3-2(4)と同様に全軸索数、MBP陽性軸索数/全軸索数を評価した。
すべての数値はmean±SEMで表記した。実施例3-2(1)乃至(4)、実施例4においては、統計処理はJMP software version 11(SAS Institute社)を用いてTukey-Kramer HSD検定にて施行した。
試験例3-2(5)においては、統計解析は、SAS System Version 9.1.3(SAS Institute社)を用いて2群間比較はF検定にて施行し、等分散の場合はStudentのt検定、不等分散の場合はWelchの検定にて施行した。
A. ラット坐骨神経圧挫損傷モデルを用いた薬効評価
(1)ビタミンB12等の血中濃度
結果を図9に示した。MeCbl pump群(18.35±2.27ng/mL)では血中濃度の有意な上昇を認めたが、MeCbl sheet群(1.73±0.05ng/mL)では血中濃度の上昇を認めなかった。薬剤非投与群(sham群:1.99±0.33ng/mL、CTR sheet群:1.48±0.05ng/mLおよびuntreated群:1.50±0.07ng/mL)はいずれも血中濃度上昇を認めなかった。
薬剤として本抽出物、NGFまたはBDNFを用いた場合も、同様に血中濃度の上昇を認めなかった。
SFIの結果を図10に示した。untreated群(-20.6±4.2)と比較してMeCbl sheet群(-9.0±2.0)は有意に改善を認め、MeCbl pump群(-10.5±2.0)で回復傾向を認めた。
von Frey filament testの結果を図11に示した。untreated群(117.8±11.7g)と比較してMeCbl sheet群(80±7.6g)はMeCbl pump群(77.8±7.0g)と同様に有意な改善を認めた。
薬剤として本抽出物を含有したシートのvon Frey filament testの結果を図12に示した。untreated群(2.75±0.41)と比較してNTP sheet群(1.35±0.13)は有意な改善を認めた。
薬剤としてNGFまたはBDNFを含有したシートの移植でも、運動機能および感覚機能において改善が認められた。
結果を図13に示した。(A)がCMAPの結果、(B)がTLの結果、(C)がNCVの結果である。CMAPおよびTLにおいては、untreated群(CMAP:19.5±2.3mV、TL:3.45±0.08ms)と比較してMeCbl sheet群(CMAP:18.5±1.5mV、TL:3.27±0.09ms)、MeCbl pump群(CMAP:19.5±1.5mV、TL:3.24±0.07ms)ともに有意な改善は認めなかった。一方、NCVにおいては、untreated群(28.2±2.5m/s)と比較して、MeCbl sheet群(44.4±2.8m/s)はMeCbl pump群(43.2±2.5m/s)群と同様に有意に改善を認めた。
薬剤として本抽出物、NGFまたはBDNFを含有したシートの移植でも、同様に改善が認められた。
結果を図14および図15に示した。図7は抗MBP抗体を用いて髄鞘を免疫染色した顕微鏡画像であり、図中白みを帯びて見える部分が染色された髄鞘である。図15(A)は全軸索数の結果、図15(B)はMBP陽性軸索数/全軸索数の結果である。
1平方ミリメートル当たりの再生軸索の本数は、untreated群(2843±68本/mm2)、MeCbl sheet群(2733±142/mm2)、MeCbl pump群(2735±77/mm2)で有意な差を認めなかった。一方、髄鞘化率(MBP陽性軸索数/全軸索数)はuntreated群(85.0±0.9%)と比較して、MeCbl sheet群(91.0±0.8%)はMeCbl pump群(91.5±0.6%)と同様に有意に改善を認めた。
薬剤として本抽出物、NGFまたはBDNFを含有したシートの移植でも、同様に改善が認められた。
(1)感覚機能評価
結果を図16に示した。von Frey filament testにおいて、神経再生誘導チューブ群(172.0±36.7g)と比較して神経再生誘導チューブ+MeCblシート群(84.0±9.80g)は有意に改善を認めた。
薬剤として、本抽出物、NGFまたはBDNFを含有したシートでも、同様に改善を認めた。
TLの結果を図17に、NCVの結果を図18に示した。TLにおいて、神経再生誘導チューブ群(4.67±0.37m/s)と比較して神経再生誘導チューブ+MeCblシート群(3.43±0.12m/s)は改善を認めた。NCVにおいては、神経再生誘導チューブ群(16.3±3.13m/s)と比較して神経再生誘導チューブ+MeCblシート群(29.5±4.50m/s)は改善傾向を認めた。
薬剤として、本抽出物、NGFまたはBDNFを含有したシートでも、同様に改善を認めた。
1平方ミリメートル当たりの再生軸索の本数および髄鞘化率(MBP陽性軸索数/全軸索数)において、神経再生誘導チューブ群と比較して神経再生誘導チューブ+MeCblシート群は改善を認めた。
薬剤として、本抽出物、NGFまたはBDNFを含有したシートでも、同様に改善を認めた。
Claims (26)
- 神経損傷治療用の薬剤および生体適合性ポリマーを含有するナノファイバーで形成された不織布からなる薬剤徐放シート。
- 薬剤がビタミンB12である請求項1に記載の薬剤徐放シート。
- 薬剤がワクシニアウイルス接種炎症ウサギ皮膚抽出物またはその分画物である請求項1に記載の薬剤徐放シート。
- 薬剤がニューロトロフィンである請求項1に記載の薬剤徐放シート。
- ニューロトロフィンがNGFまたはBDNFである請求項4に記載の薬剤徐放シート。
- 生体適合性ポリマーが生分解性脂肪族ポリエステルまたはポリアクリルアミド誘導体である請求項1~5のいずれかに記載の薬剤徐放シート。
- 生分解性脂肪族ポリエステルが、ポリカプロラクトンまたはその共重合体、ポリ乳酸またはその共重合体、ポリグリコール酸またはその共重合体、およびそれらの混合物からなる群から選択される請求項6に記載の薬剤徐放シート。
- ポリアクリルアミド誘導体が、ポリ(N-イソプロピルアクリルアミド)またはその共重合体、ポリ(2-ヒドロキシエチルメタクリルアミド)またはその共重合体、N-イソプロピルアクリルアミドおよび2-ヒドロキシエチルメタクリルアミドの共重合体ならびにそれらの混合物からなる群から選択される請求項6に記載の薬剤徐放シート。
- さらにヒアルロン酸を含有することを特徴とする請求項1~8のいずれかに記載の薬剤徐放シート。
- シートのヤング率が100kPa~100MPaである請求項1~9のいずれかに記載の薬剤徐放シート。
- シートの重量が1mg/cm2~100mg/cm2である請求項1~10のいずれかに記載の薬剤徐放シート。
- シートが薬剤および生体適合性ポリマーを含有するナノファイバーで形成された不織布からなり、ヤング率が100kPa~100MPa、重量が1mg/cm2~100mg/cm2である薬剤徐放シート。
- 薬剤がビタミンB12である請求項12に記載のシート。
- 薬剤がワクシニアウイルス接種ウサギ炎症皮膚組織抽出物またはその分画物である請求項12に記載の薬剤徐放シート。
- 薬剤がニューロトロフィンである請求項12に記載の薬剤徐放シート。
- ニューロトロフィンがNGFまたはBDNFである請求項15に記載のシート。
- 生体適合性ポリマーが生分解性脂肪族ポリエステルまたはポリアクリルアミド誘導体である請求項12~16のいずれかに記載の薬剤徐放シート。
- 生分解性脂肪族ポリエステルが、ポリカプロラクトンまたはその共重合体、ポリ乳酸またはその共重合体、ポリグリコール酸またはその共重合体、およびそれらの混合物からなる群から選択される請求項17に記載の薬剤徐放シート。
- ポリアクリルアミド誘導体がポリ(N-イソプロピルアクリルアミド)またはその共重合体、ポリ(2-ヒドロキシエチルメタクリルアミド)またはその共重合体、N-イソプロピルアクリルアミドおよび2-ヒドロキシエチルメタクリルアミドの共重合体ならびにそれらの混合物からなる群から選択される請求項17に記載の薬剤徐放シート。
- さらにヒアルロン酸を含有することを特徴とする請求項12~19のいずれかに記載の薬剤徐放シート。
- 以下の工程(1)および(2)を含む薬剤徐放シートの製造方法:
(1)薬剤と、生体適合性ポリマーと、溶媒を含む溶液を調製する工程
(2)該溶液を電界紡糸法に供して紡糸し、不織布を形成させる工程。 - 以下の工程(1)および(2)を含む薬剤徐放シートの製造方法:
(1)薬剤と、生体適合性ポリマーと、溶媒と、ヒアルロン酸を含む溶液を調製する工程
(2)該溶液を電界紡糸法に供して紡糸し、不織布を形成させる工程。 - 以下の工程(1)、(2)および(3)を含む薬剤徐放シートの製造方法:
(1)薬剤と、生体適合性ポリマーと、溶媒と、ヒアルロン酸を含む溶液を調製する工程
(2)該溶液を電界紡糸法に供して紡糸し、不織布を形成させる工程
(3)不織布にヒアルロン酸をコーティングする工程。 - 生体適合性ポリマーが生分解性脂肪族ポリエステルまたはポリアクリルアミド誘導体である請求項21~23のいずれかに記載の薬剤徐放シートの製造方法。
- 生分解性脂肪族ポリエステルが、ポリカプロラクトンまたはその共重合体、ポリ乳酸またはその共重合体、ポリグリコール酸またはその共重合体、およびそれらの混合物からなる群から選択される請求項24に記載の製造方法。
- ポリアクリルアミド誘導体が、ポリ(N-イソプロピルアクリルアミド)またはその共重合体、ポリ(2-ヒドロキシエチルメタクリルアミド)またはその共重合体、N-イソプロピルアクリルアミドおよび2-ヒドロキシエチルメタクリルアミドの共重合体ならびにそれらの混合物からなる群から選択される請求項24に記載の製造方法。
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ZA201805801B (en) | 2019-05-29 |
EP3427726B1 (en) | 2023-05-03 |
JP2020169201A (ja) | 2020-10-15 |
IL261533B (en) | 2021-12-01 |
CN116473948A (zh) | 2023-07-25 |
PL3427726T3 (pl) | 2023-07-10 |
CN108883077A (zh) | 2018-11-23 |
EP3427726A1 (en) | 2019-01-16 |
KR20180120219A (ko) | 2018-11-05 |
IL261533A (en) | 2018-10-31 |
CA3016391A1 (en) | 2017-09-14 |
ES2946262T3 (es) | 2023-07-14 |
AU2017230387B2 (en) | 2022-07-21 |
JP7057573B2 (ja) | 2022-04-20 |
TW201737901A (zh) | 2017-11-01 |
CA3016391C (en) | 2024-06-04 |
US20190083415A1 (en) | 2019-03-21 |
US11324704B2 (en) | 2022-05-10 |
JP6750820B2 (ja) | 2020-09-02 |
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