Sustained-release tablets for treating nerve damage

Abstract

The present invention provides a sustained-release sheet containing a drug for treating nerve damage and a method for manufacturing the same. The sustained-release sheet containing a drug for treating nerve damage is applied to a nerve damage site, can maintain a high drug concentration for a long time, and promote nerve regeneration without irritating the nerves even if it is buried in the periphery of the nerve damage site. The sustained-release sheet for treating nerve damage is a sheet comprising a non-woven fabric formed of nanofibers containing a drug such as vitamin B12 and a biocompatible polymer such as a biodegradable aliphatic polyester, and promotes nerve regeneration by being buried in the periphery of the nerve damage site.

Description

[0001] This case is filed on March 6, 2017 、Application No. 201780015992.7(PCT / JP2017 / 008730) 、The name of the invention is Sustained-release tablets for treating nerve damage A divisional application for a patent application. Technical Field

[0002] The present invention relates to a sustained-release drug sheet for treating nerve damage (hereinafter, sometimes referred to as "the sheet of the present invention"). More specifically, it relates to a sheet containing a drug having a therapeutic effect on nerve damage and slowly releasing the drug, and a method for producing the sheet. Background Art

[0003] Peripheral nerve injury is roughly divided into discontinuous injury in which the continuity of the injured part is interrupted, and continuous nerve injury in which the nerve in the injured part has continuity, such as entrapment nerve disorder (carpal tunnel syndrome, etc.). As a treatment for discontinuous injury, direct suturing, autologous nerve transplantation, etc. can be selected. On the other hand, as a treatment for continuous nerve injury, nerve stripping and preservation treatment can be selected. So far, as a device with a regenerative effect on peripheral nerve injury, artificial nerves have been developed, but they can only be used for discontinuous nerve injury. In addition, these simply connect the defects in the injured part and do not have the effect of promoting the regeneration of nerve axons. If it takes a long time to recover after peripheral nerve injury, the muscle tissue will undergo irreversible changes, so promoting the regeneration of nerve axons becomes an important issue. In addition, as mentioned above, artificial nerves can only deal with nerve injuries where the defect exists in the injured part, and there is no equipment for continuous nerve injury, which has the largest number of patients. Therefore, a tool that can be applied to both continuous nerve injury and discontinuous nerve injury and is effective for the treatment of nerve injury is needed in the medical field.

[0004] One of the drugs contained in the sheet of the present invention is vitamin B12. Vitamin B12 is important for the nervous system to function normally, and vitamin B12 deficiency is known to cause a systemic neurological disorder called "subacute combined degeneration of the spinal cord" (Non-Patent Document 1). The inventors of the present invention reported the following: methylcobalamin at a concentration of 100 nM or more promotes neurite growth and neuronal survival; these effects are mediated by a methylation cycle as a reaction related to methylation; methylcobalamin increases the activity of Erk1 / 2 and Akt through a methylation cycle; and high-dose continuous administration of methylcobalamin improves nerve regeneration and functional recovery in a rat sciatic nerve injury model (Non-Patent Document 2).

[0005] Patent Document 1 describes a soft sheet-like biodegradable composite material for implantation in the body, which contains a biocompatible binding material such as water-soluble glass fiber and polycaprolactone. It describes that the soft sheet-like biodegradable composite material is used to wrap around a defective area of ​​a tissue in order to promote healing, and is used to prevent adhesion after surgery. In addition, it describes that the biodegradable composite material can be used as a substitute for a nerve transplant sheet and is conceived to be used for discontinuous nerve damage, but does not have the effect of promoting the regeneration of nerve axons.

[0006] As a drug that can be contained in the sheet of the present invention, there is also an extract of inflammatory skin of rabbits inoculated with vaccinia virus (hereinafter referred to as "the extract of the present invention") or its isolate. The extract of the present invention or the preparation containing the extract of the present invention is known to have a very wide range of effects and effects, but a sheet for treating nerve damage containing the extract is not known at present.

[0007] As the drug contained in the sheet of the present invention, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and the like can also be cited. These belong to the neurotrophic factor family (neurotrophin family) like neurotrophin-3 (NT-3), and are secretory proteins produced by various cells, such as nerve cells (neurons), glial cells (microglia, astrocytes, oligodendrocytes, etc.). Neurotrophic factors show effects such as maintaining the survival of nerve cells, promoting the proliferation of neurites, and promoting the synthesis of neurotransmitters. NGF is synthesized and secreted in the target cells (neurons or muscles, etc.) projected by neurons, is received by the TrkA receptors at the axon terminals of neurons, and is retrogradely transported to the cell body to exert its function. NGF specifically acts on sensory neurons (small cells of dorsal root ganglia) and sympathetic postganglionic neurons in the peripheral nervous system, and specifically acts on cholinergic neurons at the base of the forebrain that project to the cerebral cortex or hippocampus in the central nervous system. It is known that BDNF is present in the central nervous system with the hippocampus as the center, and exhibits various physiological activities in the nervous system, such as the survival and maintenance of nerve cells, the morphological regulation of neurites, the functional regulation of synapses, the control of neuroplasticity, etc. However, a sheet for treating nerve damage containing these neurotrophic factors is not known yet.

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: Japanese Patent Application No. 2005-528145

[0011] Non-patent literature

[0012] Non-patent document 1: Scalabrino et al., Lab. Invest., 62 (1990), 297-304

[0013] Non-patent document 2: Okada et al., Experimental Neurology, 222 (2010), 191-203 Summary of the invention

[0014] Technical problem to be solved by the invention

[0015] The object of the present invention is to provide a sustained-release drug sheet for treating nerve damage, which can maintain the drug concentration at an appropriate level at the damaged site for a long time, and even if embedded in the periphery of the damaged site, it will not cause stimulation that adversely affects the nerves, but promotes nerve regeneration.

[0016] Technical solutions for solving technical problems

[0017] In order to solve the above problems, the present invention includes the following inventions. In addition, more detailed contents of these inventions will be described later.

[0018] [1] A drug sustained-release sheet comprising a nonwoven fabric formed of nanofibers containing a drug for treating nerve damage and a biocompatible polymer.

[0019] [2] The drug sustained-release sheet according to [1] above, wherein the drug is vitamin B12.

[0020] [3] The drug sustained-release sheet according to [1] above, wherein the drug is an extract from inflamed skin of a rabbit inoculated with vaccinia virus or an isolate thereof.

[0021] [4] The drug sustained-release sheet according to [1] above, wherein the drug is a neurotrophic factor.

[0022] [5] The sustained-release pharmaceutical sheet according to [4] above, wherein the neurotrophic factor is NGF or BDNF.

[0023] [6] The sustained-release pharmaceutical sheet according to any one of [1] to [5] above, wherein the biocompatible polymer is a biodegradable aliphatic polyester or a polyacrylamide derivative.

[0024] [7] The drug sustained-release sheet according to [6] above, wherein the biodegradable aliphatic polyester is selected from polycaprolactone or a copolymer thereof, polylactic acid or a copolymer thereof, polyglycolic acid or a copolymer thereof, and a mixture thereof.

[0025] [8] The pharmaceutical sustained-release sheet according to [6] above, wherein the polyacrylamide derivative is selected from poly(N-isopropylacrylamide) or a copolymer thereof, poly(2-hydroxyethylmethacrylamide) or a copolymer thereof, a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide, and a mixture thereof.

[0026] [9] The pharmaceutical sustained-release sheet according to any one of [1] to [8] above, wherein the Young's modulus of the sheet is 100 kPa to 100 MPa.

[0027]

[10] The sustained-release pharmaceutical sheet according to any one of [1] to [9] above, wherein the weight of the sheet is 1 mg / cm 2 ~100mg / cm 2 .

[0028]

[11] A drug sustained-release sheet, wherein the sheet comprises a nonwoven fabric formed of nanofibers containing 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 ~100mg / cm 2 .

[0029]

[12] The drug sustained-release sheet according to

[11] above, wherein the drug is vitamin B12.

[0030]

[13] The drug sustained-release sheet according to

[11] above, wherein the drug is an extract from the inflamed skin of a rabbit inoculated with vaccinia virus or an isolate thereof.

[0031]

[14] The drug sustained-release sheet according to

[11] above, wherein the drug is a neurotrophic factor.

[0032]

[15] The sustained-release pharmaceutical sheet according to

[14] above, wherein the neurotrophic factor is NGF or BDNF.

[0033]

[16] The sustained-release pharmaceutical sheet according to any one of

[11] to

[15] above, wherein the biocompatible polymer is a biodegradable aliphatic polyester or polyacrylamide derivative.

[0034]

[17] The drug sustained-release sheet according to

[16] above, wherein the biodegradable aliphatic polyester is selected from polycaprolactone or a copolymer thereof, polylactic acid or a copolymer thereof, polyglycolic acid or a copolymer thereof, and a mixture thereof.

[0035]

[18] The pharmaceutical sustained-release sheet according to

[16] above, wherein the polyacrylamide derivative is selected from poly(N-isopropylacrylamide) or a copolymer thereof, poly(2-hydroxyethylmethacrylamide) or a copolymer thereof, a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide, and a mixture thereof.

[0036]

[19] The pharmaceutical sustained-release sheet according to any one of [1] to

[18] above, further comprising hyaluronic acid.

[0037]

[20] A method for producing a sustained-release pharmaceutical sheet, comprising the following steps (1) and (2):

[0038] (1) a step of preparing a solution comprising a pharmaceutical agent, a biocompatible polymer, and a solvent; and

[0039] (2) The solution is subjected to electrospinning to form a nonwoven fabric.

[0040]

[21] A method for producing a sustained-release pharmaceutical sheet, comprising the following steps (1) and (2):

[0041] (1) a step of preparing a solution comprising a pharmaceutical agent, a biocompatible polymer, a solvent and hyaluronic acid; and

[0042] (2) The solution is subjected to electrospinning to form a nonwoven fabric.

[0043]

[22] A method for producing a sustained-release pharmaceutical sheet, comprising the following steps (1), (2) and (3):

[0044] (1) a step of preparing a solution comprising a pharmaceutical agent, a biocompatible polymer, a solvent and hyaluronic acid;

[0045] (2) providing the solution to an electrospinning method for spinning to form a nonwoven fabric; and

[0046] (3) A step of coating nonwoven fabric with hyaluronic acid.

[0047]

[23] The method for producing a sustained-release drug sheet according to any one of

[20] to

[22] above, wherein the biocompatible polymer is a biodegradable aliphatic polyester or a polyacrylamide derivative.

[0048]

[24] The production method according to

[23] above, wherein the biodegradable aliphatic polyester is selected from polycaprolactone or a copolymer thereof, polylactic acid or a copolymer thereof, polyglycolic acid or a copolymer thereof, and a mixture thereof.

[0049]

[25] The production method according to

[23] above, wherein the polyacrylamide derivative is selected from poly(N-isopropylacrylamide) or a copolymer thereof, poly(2-hydroxyethylmethacrylamide) or a copolymer thereof, a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide, and a mixture thereof.

[0050]

[26] A method for producing a sustained-release pharmaceutical sheet, comprising the following steps (1) and (2):

[0051] (1) a step of preparing a solution comprising a drug, polycaprolactone or a copolymer thereof, and a solvent selected from TEF (2,2,2-trifluoroethanol), HFIP (1,1,1,3,3,3-hexafluoro-2-propanol), chloroform and DMF (N,N-dimethylformamide); and

[0052] (2) A step of subjecting the solution prepared in (1) to electrospinning 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 to form a nonwoven fabric on the electrode surface.

[0053]

[27] The production method according to

[26] above, wherein the polycaprolactone or its copolymer has a weight average molecular weight of 1,000 to 300,000, has 1 to 8 branches per molecule, and contains 0 to 50 mol% of DL-lactide.

[0054]

[28] The production method according to any one of

[20] to

[27] above, wherein the drug is vitamin B12.

[0055]

[29] The production method as described in any one of

[20] to

[27] above, wherein the drug is an extract of inflammatory skin of a rabbit inoculated with vaccinia virus or an isolate thereof.

[0056]

[30] The production method according to any one of

[20] to

[27] above, wherein the drug is a neurotrophic factor.

[0057]

[31] The production method according to

[30] above, wherein the neurotrophic factor is NGF or BDNF.

[0058]

[32] A method for treating nerve damage, comprising: applying a drug sustained-release sheet comprising a non-woven fabric formed of nanofibers to a patient in need of treatment, wherein the nanofibers contain a drug having a nerve damage therapeutic effect and a biocompatible polymer.

[0059]

[33] The method as described in

[32] above, wherein the drug is vitamin B12.

[0060]

[34] The method of treatment as described in

[32] above, wherein the agent is an extract of inflammatory skin of rabbits inoculated with vaccinia virus or an isolate thereof.

[0061]

[35] The treatment method described in

[32] above, wherein the drug is a neurotrophic factor.

[0062]

[36] The treatment method described in

[35] above, wherein the neurotrophic factor is NGF or BDNF.

[0063]

[37] The treatment method according to any one of

[32] to

[36] above, wherein the biocompatible polymer is a biodegradable aliphatic polyester or polyacrylamide derivative.

[0064]

[38] The method of treatment as described in

[37] above, wherein the biodegradable aliphatic polyester is selected from polycaprolactone or a copolymer thereof, polylactic acid or a copolymer thereof, polyglycolic acid or a copolymer thereof, and a mixture thereof.

[0065]

[39] The method of treatment as described in

[37] above, wherein the polyacrylamide derivative is selected from poly(N-isopropylacrylamide) or a copolymer thereof, poly(2-hydroxyethylmethacrylamide) or a copolymer thereof, a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide, and a mixture thereof.

[0066]

[40] The treatment method described in any one of

[32] to

[39] above, wherein the Young's modulus of the sheet is 100 kPa to 100 MPa.

[0067]

[41] The method according to any one of

[32] to

[40] , wherein the weight of the sheet is 1 mg / cm 2 ~100mg / cm 2 .

[0068]

[42] The treatment method according to any one of

[32] to

[41] above, wherein the method is applied by implantation in the vicinity of a nerve injury site.

[0069]

[43] Use of vitamin B12 in the manufacture of a sustained-release pharmaceutical sheet, wherein the sustained-release pharmaceutical sheet comprises a non-woven fabric formed of nanofibers containing a pharmaceutical having a therapeutic effect on nerve damage and a biocompatible polymer.

[0070]

[44] Use of an extract from inflammatory skin of a rabbit inoculated with vaccinia virus or a separate thereof in the manufacture of a sustained-release drug sheet, wherein the sustained-release drug sheet comprises a non-woven fabric formed of nanofibers containing a drug having a therapeutic effect on nerve damage and a biocompatible polymer.

[0071]

[45] Use of neurotrophic factors in the manufacture of a sustained-release drug sheet, the sustained-release drug sheet comprising a nonwoven fabric formed of nanofibers containing a drug having a therapeutic effect on nerve damage and a biocompatible polymer.

[0072]

[46] The use as described in

[45] above, wherein the neurotrophic factor is NGF or BDNF.

[0073]

[47] The use according to any one of

[43] to

[46] above, wherein the biocompatible polymer is a biodegradable aliphatic polyester or polyacrylamide derivative.

[0074]

[48] ​​The use according to

[47] above, wherein the biodegradable aliphatic polyester is selected from polycaprolactone or a copolymer thereof, polylactic acid or a copolymer thereof, polyglycolic acid or a copolymer thereof, and a mixture thereof.

[0075]

[49] The use as described in

[47] above, wherein the polyacrylamide derivative is selected from poly(N-isopropylacrylamide) or a copolymer thereof, poly(2-hydroxyethylmethacrylamide) or a copolymer thereof, a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide, and a mixture thereof.

[0076]

[50] The use according to any one of

[43] to

[49] above, wherein the Young's modulus of the sheet is 100 kPa to 100 MPa.

[0077]

[51] The use as described in any one of

[43] to

[50] above, wherein the weight of the sheet is 1 mg / cm 2 ~100mg / cm 2 .

[0078] Effects of the Invention

[0079] According to the present invention, a sustained-release sheet for treating nerve damage and a method for producing the same can be provided. The sustained-release sheet for treating nerve damage is applied by embedding around the damaged area and can maintain the drug concentration in the area at an appropriate level for a long period of time, thereby promoting nerve regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 The diagram shows the results of observing the produced polycaprolactone sheet using a scanning electron microscope (SEM).

[0081] Figure 2 The figure shows the results of SEM observation of the produced poly(N-isopropylacrylamide) sheet.

[0082] Figure 3The figure shows the result of SEM observation of the produced poly(NIPAAm-co-HMAAm) sheet.

[0083] Figure 4 The figure shows the results of SEM observation of the produced polycaprolactone sheet containing magnetic nanoparticles.

[0084] Figure 5 This is a diagram showing the results of SEM observation of the produced hyaluronic acid-coated polycaprolactone sheet.

[0085] Figure 6 This is a diagram showing the results of SEM observation of the produced polycaprolactone sheet containing the extract of the present invention.

[0086] Figure 7 The diagram shows the results of SEM observation of three types of vitamin B12-containing sheets of the present invention produced.

[0087] Figure 8 This is a graph showing the results of confirming the sustained-release properties of three types of vitamin B12-containing sheets of the present invention that were produced.

[0088] Fig. 9 This graph shows the results of measuring the blood concentration of vitamin B12 at 6 weeks after surgery in an experiment for evaluating the medicinal effect of the vitamin B12-containing sheet of the present invention using a rat sciatic nerve compression injury model.

[0089] Fig.10 The graph shows the results of evaluating motor function by measuring the sciatic function index (SFI) at 6 weeks after surgery in an experiment to evaluate the efficacy of the vitamin B12-containing sheet of the present invention using a rat sciatic nerve compression injury model.

[0090] Fig.11 This graph shows the results of evaluating sensory function by von Frey filament test at 6 weeks after surgery in an experiment to evaluate the efficacy of the vitamin B12-containing sheet of the present invention using a rat sciatic nerve compression injury model.

[0091] Fig.12 This graph shows the results of evaluating sensory function by the von Frey fiber test at 3 weeks after surgery in an experiment for evaluating the medicinal effects of the sheet of the present invention containing the extract of the present invention using a rat sciatic nerve compression injury model.

[0092] Fig.13The figures show the results of electrophysiological evaluation performed at 6 weeks after surgery in an experiment for evaluating the efficacy of the sheet of the present invention containing vitamin B12 using a rat sciatic nerve compression injury model, wherein (A) shows the result of compound muscle action potential (CMAP), (B) shows the result of terminal latency (TL), and (C) shows the result of nerve conduction velocity (NCV).

[0093] Fig.14 The graph shows the results of immunostaining using an anti-myelin basic protein (MBP) antibody as a myelin index in an experiment to evaluate the efficacy of the vitamin B12-containing sheet of the present invention using a rat sciatic nerve compression injury model. Sciatic nerves were collected 6 weeks after surgery.

[0094] Fig.15 The graphs show the results of histological evaluation performed 6 weeks after surgery in an experiment to evaluate the efficacy of the vitamin B12-containing sheet of the present invention using a rat sciatic nerve compression injury model. (A) shows the result of the total axon number, and (B) shows the result of the MBP-positive axon number / total axon number.

[0095] Fig.16 The graph shows the results of evaluating sensory function by the von Frey fiber test at 12 weeks after surgery in an experiment to evaluate the efficacy of the vitamin B12-containing sheet of the present invention on nerves connected by nerve regeneration conduits (NRC) using a rat sciatic nerve defect model.

[0096] Fig.17 This is a graph showing the results of electrophysiological evaluation (TL) performed 12 weeks after surgery in an experiment to evaluate the efficacy of the vitamin B12-containing sheet of the present invention on nerves connected by NRC using a rat sciatic nerve defect model.

[0097] Fig.18 This is a graph showing the results of electrophysiological evaluation (NCV) performed 12 weeks after surgery in an experiment to evaluate the efficacy of the vitamin B12-containing sheet of the present invention on nerves connected by NRC using a rat sciatic nerve defect model. DETAILED DESCRIPTION

[0098] The present invention provides a sustained-release drug sheet for treating nerve damage. The sustained-release drug sheet of the present invention includes a non-woven fabric formed of nanofibers containing drugs such as vitamin B12 and biocompatible polymers such as biodegradable aliphatic polyesters, and is used by embedding it in the surrounding area of ​​nerve damage. The drug contained in the sustained-release drug sheet of the present invention can be any drug that has a therapeutic effect on nerve damage, and can be selected from vitamin B12, vaccinia virus-inoculated tissue extracts or their isolates, neurotrophic factors such as NGF and BDNF, etc. By allowing these drugs to act on the nerve damage site to promote nerve regeneration, nerve damage can be effectively treated. In addition, the sustained-release drug sheet of the present invention also includes: a sustained-release drug sheet containing hyaluronic acid in addition to the above-mentioned drugs.

[0099] The object disease of the sheet material of the present invention is nerve injury. The medicament sustained-release sheet material of the present invention has a nerve regeneration promoting effect, therefore, it is very useful to consider that both continuous nerve injury and discontinuous nerve injury can be used as the treatment object. As continuous nerve injury, for example, entrapment nerve disorder (the state in which the nerve is compressed due to the surrounding tissue) can be cited. In addition, the medicament sustained-release sheet material of the present invention is also effective for promoting the regeneration of the nerve after nerve suture (the state in which direct suture is performed after nerve injury), the nerve after nerve stripping, and the nerve after nerve transplantation (the nerve injury part with defect is transplanted to make it continuous). In addition, for discontinuous nerve injury, by using the medicament sustained-release sheet material of the present invention alone or in combination with an artificial nerve, etc., it also has the effect of promoting treatment.

[0100] Vitamin B12 as a drug to be contained in the sheet of the present invention includes cobalamin and its derivatives. Specifically, methylcobalamin, cyanocobalamin, hydroxocobalamin, sulfitocobalamin, adenosylcobalamin or their salts can be listed, all of which can be suitably used as the active ingredient of the sustained-release agent of the present invention. Among them, methylcobalamin, cyanocobalamin, hydroxocobalamin or their salts are preferred, and methylcobalamin or its salts are more preferred. The content of vitamin B12 is preferably about 1% to about 30% in terms of final concentration, and more preferably about 2% to about 10%.

[0101] The sheet of the present invention includes a nonwoven fabric formed of nanofibers containing a drug and a biocompatible polymer. Nanofibers refer to fibrous substances with a diameter of about 10 nm to about 1000 nm and a length of more than 100 times the diameter. As biocompatible polymers used in the present invention, biodegradable aliphatic polyesters and polyacrylamide derivatives can be listed. As preferred biodegradable aliphatic polyesters, for example, polycaprolactone, polylactic acid, polyglycolic acid, polyglyceric acid, polyhydroxyalkanoic acid, polybutylene succinate, copolymers of these, derivatives of these, etc. can be listed. From the perspective of softness, etc., biodegradable aliphatic polyesters selected from polycaprolactone or its copolymers, polylactic acid or its copolymers, polyglycolic acid or its copolymers, and mixtures thereof are more preferably listed. As examples of the above copolymers, polylactic acid-polycaprolactone copolymers, poly(ε-caprolactone-co-DL-lactide) and the like can be listed. In addition, as polyacrylamide derivatives, for example, poly(N-isopropylacrylamide) or its copolymers, poly(2-hydroxyethylmethacrylamide) or its copolymers, or mixtures thereof can be listed. Examples of the copolymer include poly(N-isopropylacrylamide) and a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide.

[0102] In addition, as the above-mentioned nanofiber, as long as it contains at least one of the above-mentioned biocompatible polymers, it can also be a copolymer of a biocompatible polymer other than the above-mentioned biocompatible polymer and these above-mentioned biocompatible polymers. In addition, it can also be a copolymer containing multiple above-mentioned biocompatible polymers. In these cases, the form of copolymerization can be any kind in block copolymerization, random copolymerization, alternating copolymerization or graft copolymerization.

[0103] The Young's modulus of the sheet constituting the sustained-release drug sheet of the present invention is preferably about 100 kMPa to about 100 MPa, and more preferably about 1 MPa to about 50 MPa. The Young's modulus can be obtained from a stress-strain curve based on the measured value obtained using a commercially available tensile tester (e.g., EZ-S500N (manufactured by Shimadzu Corporation)). The weight of the sheet is preferably 1 mg / cm 2 ~100mg / cm 2 , more preferably 1 mg / cm 2 ~50mg / cm 2 The thickness of the sheet is preferably about 100 μm to about 1 mm, more preferably about 100 μm to about 500 μm. The area of ​​the sheet is not particularly limited, and it can be cut according to the size of the affected area when used. In addition, it can be formed into a size that is easy to use. As a molding size, for example, 0.1 cm 2 ~10cm 2 , more preferably 1 cm 2 ~4cm 2.

[0104] The sheet of the present invention is used by being embedded in the periphery of the nerve injury site. The sheet of the present invention is formed into a form with flexibility, so the mode of use can be appropriately selected. Specifically, for example, for a nerve injury site in a state where the surrounding is peeled off and the nerve is exposed, it is preferred to wind the sheet of the present invention or leave it in a covered manner. Afterwards, the surrounding tissue is restored to its original state and the skin is sutured. Since the sheet of the present invention is formed into a soft form, even if it is left in the nerve injury site, it does not bring stimulation that adversely affects the nerves or surrounding tissues. Therefore, the sheet of the present invention can be embedded in the periphery of the nerve injury site for use. Moreover, there is no need to take it out after the treatment of the injury site. In addition, the sheet of the present invention is preferably used after sterilization. The sterilization method preferably selects a method that does not decompose the agent and does not damage the shape and physical properties of the sheet. For example, radiation sterilization, plasma sterilization, etc. can be listed.

[0105] It is possible to prepare a solution containing a biocompatible polymer such as a medicament such as vitamin B12 and a biodegradable aliphatic polyester, use the solution as a raw material and utilize known spinning methods such as electrostatic spinning, self-assembly, and phase separation to make nanofibers, and utilize known methods to form a nonwoven fabric, thereby manufacturing the sheet of the present invention. In the preparation of a solution (raw solution) containing a medicament such as vitamin B12 and a biocompatible polymer such as a biodegradable aliphatic polyester, an appropriate solvent can be used. The solvent used for the raw solution is removed in the spinning process so as not to remain in the nonwoven fabric. As a solvent for the raw solution, TEF, HFIP, chloroform, DMF, etc. can be cited. TEF, etc. are preferably cited.

[0106] Biodegradable aliphatic polyesters may also contain monomer components for imparting flexibility so that the nonwoven fabric has the required flexibility. Examples of such monomer components 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 according to the situation, depending on the type of biocompatible polymer used, the type of monomer component for imparting flexibility, and the required level of flexibility. In addition, the softness of the nonwoven fabric can be improved by adjusting the molecular weight of the biocompatible polymer or by giving the polymer molecule a branched structure. The molecular weight and the number of branches are preferably appropriately set according to the type of biocompatible polymer used.

[0107] When a sheet is produced by electrospinning using polycaprolactone or a copolymer thereof as the biodegradable aliphatic polyester among biocompatible polymers, it is preferable to use a production method including the following steps (1) and (2).

[0108] (1) a step of preparing a solution comprising a pharmaceutical agent, polycaprolactone or a copolymer thereof, and a solvent selected from TEF, HEIP, chloroform and DMF; and

[0109] (2) A step of subjecting the solution prepared in (1) 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 to form a nonwoven fabric on the electrode surface.

[0110] The polycaprolactone or copolymer used in step (1) preferably has a weight average molecular weight of 1000 to 300000, 1 to 8 branches 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 10000 to 100000, 2 to 7 branches per molecule, and a DL-lactide content of 20 to 45 mol%. It is even more preferably a polycaprolactone copolymer having a weight average molecular weight of 30000 to 600000, 3 to 6 branches per molecule, and a DL-lactide content of 30 to 45 mol%. The solvent used is preferably TEF.

[0111] In step (2), the solution (raw material solution) prepared in step (1) is provided to an electrospinning method for spinning to form a nonwoven fabric 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 24 G. 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.

[0112] When a biocompatible polymer such as poly(N-isopropylacrylamide) or a copolymer thereof, or a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide is used as a polyacrylamide derivative and a sheet is produced by electrospinning, it is preferred to use a production method comprising the following steps (1) and (2).

[0113] (1) a step of preparing a solution comprising 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; and

[0114] (2) A step of subjecting the solution prepared in (1) 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 to form a nonwoven fabric on the electrode surface.

[0115] In step (2), the solution (raw material solution) prepared in step (1) is provided to an electrospinning method for spinning to form a nonwoven fabric 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 24 G. More preferably, the voltage and flow rate are a voltage of 10 to 20 kV and a flow rate of 0.3 to 1 mL / h.

[0116] The sheet of the present invention also includes a sheet containing hyaluronic acid in addition to a drug such as vitamin B12. The hyaluronic acid may be added to the solution in step (1), or the nonwoven fabric formed in step (2) may be coated by immersing it in hyaluronic acid.

[0117] By the manufacturing method as described above, a sheet of the present invention comprising a nonwoven fabric formed of nanofibers containing a drug and a biocompatible polymer can be manufactured, wherein the sheet of the present invention has a Young's modulus of 100 kPa to 100 MPa and a weight of 1 mg / cm 2 ~100mg / cm 2 The present invention also includes a method for producing such a sheet of the present invention or use of a drug, nanofiber or nonwoven fabric for producing such a sheet of the present invention.

[0118] The vitamin B12 and neurotrophic factors that can be contained in the sheet of the present invention can be manufactured by a suitable known method or purchased commercially available. In addition, hyaluronic acid can also be manufactured by a suitable known method or purchased commercially available hyaluronic acid.

[0119] The extract of the present invention that can be contained in the sheet of the present invention is an extract containing a non-proteinaceous active substance extracted and separated from the inflamed tissue of an animal inoculated with vaccinia virus and caused to develop pox.

[0120] The extract of the present invention can be obtained by inoculating vaccinia virus, crushing the inflamed tissue of the pox, adding an extraction solvent, removing the tissue pieces, performing a protein removal treatment, adsorbing the tissue to an adsorbent, and then eluting the effective ingredient. That is, for example, the following steps.

[0121] (A) Skin tissues of rabbits, mice, etc. inoculated with vaccinia virus and made to develop pox are collected, the poxed tissues are crushed, an extraction solvent such as water, phenol water, physiological saline, or phenol plus glycerol water is added, and then filtered or centrifuged to obtain an extract (filtrate or supernatant).

[0122] (B) The extract is adjusted to an acidic pH and heated to remove protein. Next, the solution after the removal of protein is adjusted to an alkaline pH and heated, and then filtered or centrifuged.

[0123] (C) The obtained filtrate or supernatant is made acidic and adsorbed onto an adsorbent such as activated carbon or kaolin.

[0124] (D) Adding an extraction solvent such as water to the adsorbent, adjusting the pH to alkaline, and eluting the adsorbed component to obtain a vaccinia virus-inoculated inflammatory tissue extract. Then, if desired, the eluate can be evaporated to dryness or freeze-dried under reduced pressure to obtain a dry solid.

[0125] As animals for inoculating vaccinia virus to obtain inflammatory tissue, various animals infected with vaccinia virus such as rabbits, cattle, horses, sheep, goats, monkeys, rats, mice, etc. can be used. As inflammatory tissue, the inflamed skin tissue of rabbits is preferred. As long as the rabbit belongs to the order Lagomorpha, any rabbit can be used. As examples, there are: Oryctolagus cuniculus, domestic rabbits (domesticated burrowing rabbits), hares (Japanese hares), pikas and snow rabbits. Among them, rabbits are suitable for use. In Japan, there are rabbits called domestic rabbits (イエウサギ) that have been frequently used as livestock or experimental animals raised in the past, which is also another name for rabbits. There are multiple breeds (breed) in rabbits, and breeds called Japanese white species and New Zealand white species (New Zealand white) can be preferably used.

[0126] The vaccinia virus may be any strain of vaccinia virus, and examples thereof include Lister strain, Dairen strain, Ikeda strain, EM-63 strain, and New York City Board of Health strain.

[0127] The extract of the present invention is liquid during manufacture, so it can also be prepared into an extract of a desired concentration by appropriate concentration and dilution, and then freeze-dried. In addition, it is also possible to obtain an isolate with a higher therapeutic effect on nerve damage by separating the extract of the present invention and use it in the sheet of the present invention.

[0128] In addition, a more specific method for producing the extract of the present invention is described in, for example, paragraphs

[0024] to

[0027] ,

[0031] , etc. of International Publication No. WO2016 / 194816.

[0129] Through the present invention, it is possible to provide a drug sustained-release sheet for treating continuous nerve damage that is not an application target of existing artificial nerves. Continuous nerve damage has the largest number of patients in peripheral nerve damage, so it can be considered that the contribution of the present invention is very high. In addition, for discontinuous nerve damage, the sheet of the present invention can also be used alone or in combination with an artificial nerve. The sheet of the present invention is used by embedding it in the periphery of the nerve damage site. Therefore, it is not necessary to increase the drug concentration in the blood by continuously administering a high dose of the drug, but it can promote nerve regeneration by continuously releasing the drug locally. In addition, the sheet of the present invention is molded using a biocompatible polymer, so it does not need to be removed after the treatment of nerve damage. Moreover, it can be molded into a very soft form, so even if it is embedded in the periphery of the nerve damage site, it will not cause stimulation that adversely affects the nerves, and it is very easy to use. In addition, a surgical microscope is required when using an artificial nerve, and the sheet of the present invention has the advantage of not requiring a surgical microscope and being very easy to operate when used alone. That is, the sheet of the present invention is very useful because it has both effectiveness and safety and is highly convenient.

[0130] Example

[0131] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

[0132] [Example 1: Preparation of a sheet material containing vitamin B12]

[0133] (1) Production of polycaprolactone sheets, etc.

[0134] 4.5 mL of HEIP was added to 900 mg of polycaprolactone, and ultrasonic treatment was performed for 3 hours to prepare a dispersion solution (polymer solution concentration: 20 wt %).

[0135] Next, the dispersed solution was completely discharged at a rate of 1.0 mL / h using a 5 mL syringe while applying a voltage of 20 kV. The spun fibers were stacked and collected on a metal substrate covered with aluminum foil to prepare a polycaprolactone sheet (syringe needle used: 22G, syringe-metal substrate distance: 13 cm).

[0136] like Figure 1 As shown in the scanning electron microscope (SEM) observation image, a polycaprolactone sheet including a nonwoven fabric formed of nanofibers was manufactured.

[0137] Similarly, a fiber sheet using poly(N-isopropylacrylamide) or a copolymer of N-isopropylacrylamide and 2-hydroxyethylmethacrylamide (hereinafter, “poly(NIPAAm-co-HMAAm)”) was prepared in the same manner (see Figure 2 and Figure 3 ).

[0138] (2) Preparation of polycaprolactone sheets containing magnetic nanoparticles

[0139] 4.5 mL of HEIP was added to 900 mg of polycaprolactone and 270 mg of magnetic nanoparticles containing γ-Fe 2 O 3 , and ultrasonic treatment was performed for 12 hours to prepare a dispersion solution (polymer solution concentration: 20 wt %).

[0140] Next, a voltage of 20 kV was applied while the dispersed solution was completely delivered at a rate of 1.0 mL / h using a 5 mL syringe. The spun fibers were stacked and collected on a metal base plate covered with aluminum foil to produce a poly(NIPAAm-co-HMAAm) sheet containing magnetic nanoparticles (the syringe needle used was 22G, and the content of magnetic nanoparticles relative to the sheet was 30%). Figure 4 As shown in the scanning electron microscope (SEM) observation image of , a polycaprolactone sheet containing magnetic nanoparticles including a nonwoven fabric formed of nanofibers was manufactured.

[0141] (3) Preparation of polycaprolactone sheet coated with hyaluronic acid

[0142] Dissolve 128 mg of hyaluronic acid in 2.5 mL of pure water to prepare a 5 wt% hyaluronic acid solution. Immerse the polycaprolactone sheet prepared in Example 1 (1) in the hyaluronic acid solution and let it stand for 24 hours. Freeze-dry for 72 hours to prepare a polycaprolactone sheet coated with hyaluronic acid. Figure 5 3 shows a scanning electron microscope (SEM) observation image of the produced hyaluronic acid-coated polycaprolactone sheet.

[0143] (4) Preparation of Sheets Containing Vitamin B12

[0144] Dissolve 600 mg of poly(ε-caprolactone-co-DL-lactide) and methylcobalamin (manufactured by Sigma) in 6 mL of TEF. A self-made poly(ε-caprolactone-co-DL-lactide) having a weight average molecular weight of 40,000, a number of branches of 4 / molecule, and a molar ratio of ε-caprolactone to DL-lactide of 60:40 was used. 6.5 mg, 13 mg, or 20 mg of methylcobalamin were dissolved to a final concentration of 1%, 2%, or 3%, respectively, to prepare three solutions. Then, nanofibers were manufactured by electrospinning, formed into a mesh shape, and a nonwoven fabric was manufactured. Specifically, the solution was extruded from a 24G needle at a flow rate of 0.5 mL / h, and a voltage of 12 kV was applied for spinning, and the nanofibers were accumulated on the electrode surface to capture them, thereby manufacturing a nonwoven fabric.

[0145] like Figure 7As shown in the scanning electron microscope (SEM) observation image of , a sheet containing vitamin B12 including a nonwoven fabric formed of nanofibers was manufactured. The thickness of the sheet was about 300 μm and the weight was about 10 mg / cm 2 .

[0146] In the same manner, sheets containing NGF and BDNF were prepared as drugs, respectively.

[0147] (5) Preparation of Sheets Containing the Extract of the Present Invention

[0148] 4.5 mL of HEIP was added to 900 mg of polycaprolactone and 270 mg of freeze-dried powder of the extract of the present invention, and ultrasonic treatment was performed for 3 hours to prepare a dispersion solution (polymer solution concentration: 20 wt %).

[0149] Next, a voltage of 20 kV was applied while the entire dispersed solution was delivered at a rate of 0.5 mL / h using a 5 mL syringe. The spun fibers were stacked and collected on a metal base plate covered with aluminum foil to prepare a sheet containing the extract of the present invention (the syringe needle used was 18G, and the amount of the extract of the present invention carried on the sheet was 30%). Figure 6 3 shows a scanning electron microscope (SEM) observation image of the prepared polycaprolactone sheet containing the extract of the present invention.

[0150] [Example 2: Confirmation of sustained-release properties of vitamin B12-containing sheets, etc.]

[0151] 3 mL of PBS was added to the tube, 10 mg of the sheet was immersed in it and kept warm at 37°C, and samples were taken over time to measure the vitamin B12 concentration. The sample volume was set to 100 μL each time, and the vitamin B12 concentration was measured by ultraviolet-visible absorbance measurement.

[0152] The results are shown in Figure 8 All three sheets showed sustained release until the 25th day. Considering the theoretical maximum value, it can be considered that the more methylcobalamin is contained, the longer the sustained release period. In addition, although not shown in the figure, it was confirmed that the three sheets also showed sustained release for up to 8 weeks (until the 56th day) thereafter.

[0153] In the same manner, the sustained release of the drug was confirmed for each of the sheets containing the extract of the present invention, NGF or BDNF.

[0154] [Example 3: Evaluation of drug efficacy using rat sciatic nerve compression injury model]

[0155] 1. Establishment of rat sciatic nerve compression injury model

[0156] All animal experiments were conducted with the approval of the Ethics Committee of the Animal Experiment Facility of Osaka University. Six-week-old male Wistar rats (weighing approximately 200 g) were used. All surgeries were performed after 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 unfolded under clean operation, and compression injury was applied with forceps at a position 5 mm away from the sciatic notch. The compression time was set to 10 seconds, the number of compressions was set to 3 times, and the interval between compression operations was set to 10 seconds. The fascia and skin were sutured with 4-0 nylon. The experimental rats were divided into the following 5 groups. That is, the following groups were used: a sham group in which the sciatic nerve was not compressed and injured but only expanded; a CTR sheet group in which the sciatic nerve was not compressed and injured but only expanded and a sheet without methylcobalamin was transplanted; an untreated group in which compression and injury were applied but no treatment was performed; a MeCbl sheet group in which compression and injury were applied and a sheet containing methylcobalamin (a sheet containing 3% methylcobalamin prepared in Example 1) was transplanted; and a MeCbl pump group in which compression and injury were applied and methylcobalamin was systemically administered (1 mg / kg / day). Systemic administration of methylcobalamin was performed by placing an osmotic minipump (Model 2ML2; Alzet, Cuperitino, CA, USA) under the skin of the back. All operations were performed by the same operator.

[0157] The same study was also conducted on the sheet containing the extract of the present invention as a drug. The experimental rats were classified into the following 4 groups. That is, it was set as: a sham control group in which the sciatic nerve was not subjected to compression injury but only expanded, a CTR sheet group in which the sciatic nerve was not subjected to compression injury but only expanded and a sheet containing no extract of the present invention was transplanted, an untreated group in which compression injury was applied but no treatment was performed, and an NTP sheet group in which compression injury was applied and a sheet containing the extract of the present invention (the sheet containing the extract of the present invention prepared in Example 1) was transplanted.

[0158] Sheets containing NGF or BDNF as a drug were similarly transplanted into model rats in which the sciatic nerve had been subjected to compression injury.

[0159] 2. Evaluation items and experimental methods

[0160] (1) Vitamin B12 blood concentration

[0161] At 6 weeks after surgery, 1 mL of blood was collected from the left ventricle of the rat under anesthesia and sedation. The collected blood was centrifuged at 800×g for 20 minutes, and the supernatant was recovered. The measurement of blood vitamin B12 concentration was commissioned to BML Co., Ltd. (Tokyo).

[0162] (2) Evaluation of motor and sensory functions

[0163] In order to evaluate motor function, the sciatic function index (SFI) was measured at 6 weeks after surgery. In order to measure the SFI, ink was applied to the hind feet of the rats, and the rats were made to walk on office paper placed on a 40 cm square horizontal platform to record their foot prints. The following items were measured to calculate the SFI. SFI = 0 indicates normal, and SFI = -100 indicates decreased function. In addition, individuals with toe necrosis and defects during the postoperative process were excluded. The SFI was calculated using the following mathematical formula. The items are shown below.

[0164] SFI=-38*((EPL-NPL) / NPL+109.5*((ETS-NTS) / NTS)+13.3*((EITS-NITS)-8

[0165] EPL: experimental print length

[0166] NPL: normal print length

[0167] ETS stands for experimental toe spread.

[0168] NTS stands for normal toe spread.

[0169] EIT: Experimental intermediary toe spread

[0170] NIT: normal intermediary toe spread

[0171] To evaluate sensory function, the mechanical hind paw withdrawal threshold was measured 6 weeks after surgery using von Frey filaments (0.008 g-26 g; TouchTest, North Coast Medical Inc, Gilroy, CA, USA). The subjects were made to walk on a metal mesh, and pressure was applied to the center of the sole until the filament began to bend, and the value of the withdrawal behavior was recorded.

[0172] (3) Electrophysiological evaluation

[0173] Rats that were 3 or 6 weeks postoperatively were sedated with anesthetics and placed in a ventral position on an operating table. The left sciatic nerve and the left tibialis anterior muscle were unfolded. The proximal sciatic nerve was stimulated with a bipolar electrode to measure the compound muscle action potential (CMAP) and the terminal latency (TL). The proximal and distal sides of the sciatic nerve compression injury were stimulated with bipolar electrodes, and the nerve conduction velocity (NCV) was calculated from each measured value. AD Instruments Power Lab 2 / 26, Stimulus isolater, Bio Amp and Chart&Scope software (all AD Instruments, Bellavista, NSW, Australis) were used in the measurement and evaluation.

[0174] (4) Histological evaluation

[0175] Six weeks after surgery, rats were sedated with anesthetics, and the left sciatic nerve was collected and fixed with 4% PFA for 5 days and 20% sucrose for 24 hours before freeze embedding. The embedded tissue was sliced ​​at a thickness of 5 μm in the short axis direction of the nerve and placed on a slide. After drying for 1 hour, it was fixed with 95% methanol for 30 minutes. After blocking, the primary antibody was reacted at 4°C overnight. The secondary antibody was reacted at room temperature for 1 hour, and the nucleus was labeled with DAPI. The primary antibody used was anti-neurofilament 200 (NF200) antibody produced in rabbit (1:1000; 102M4784, SIGMA) as an axon indicator and Anti-Myelin Basic Protein (MBP) Mouse mAb (1:1000; NE1018, CALIOCHEM) as a myelin indicator. Alexa 488_labeled goat anti-rabbit IgG antibody (1:1000; Lifetechnologies) and Alexa 568_labeled goat anti-mouse IgG antibody (1:1000; Lifetechnologies) were used as secondary antibodies. The total axon number and the number of MBP-positive axons / total axon number were evaluated.

[0176] [Example 4: Evaluation of drug efficacy using rat sciatic nerve defect model]

[0177] 1. Establishment of rat sciatic nerve defect model

[0178] Male Wistar rats (weighing about 200 g) aged 6 weeks were used. All operations were performed after 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 opened under clean operation, and the sciatic nerve was cut at a position 5 mm away from the sciatic notch and a position 10 mm further away from the position to make a 10 mm defect model.

[0179] The experimental rats were divided into the following 4 groups. (i) Nerve regeneration induction tube + MeCbl sheet group; the stump of the sciatic nerve was introduced into the two ends of the nerve regeneration induction tube with a diameter of 1.5 mm and a length of 12 mm, and sutured with 10-0 nylon, and a sheet containing methylcobalamin with a width of 10 mm and a length of 14 mm was left in place around it in a covering manner. (ii) Nerve regeneration induction tube group; the stump of the sciatic nerve was introduced into the two ends of the nerve regeneration induction tube with a length of 10-0 nylon, and sutured with 10-0 nylon. (iii) Autologous transplant sheet group; the severed sciatic nerve was reversed and sutured with 10-0 nylon. (iv) Sham control group; only the sciatic nerve was unfolded. The fascia and skin were sutured with 4-0 nylon. All operations were performed by the same surgeon.

[0180] The sheets containing the extract of the present invention, NGF or BDNF as a drug were also transplanted into sciatic nerve defect model rats in the same manner.

[0181] 2. Evaluation items and experimental methods

[0182] (1) Sensory function evaluation

[0183] In order to evaluate sensory function, the mechanical hind paw withdrawal threshold was measured at 12 weeks after surgery in the same manner as in Example 3-2(2).

[0184] (2) Electrophysiological evaluation

[0185] At 12 weeks after surgery, TL and NCV were calculated in the same manner as in Example 3-2 (3).

[0186] (3) Histological evaluation

[0187] Rats 12 weeks after surgery were sedated with anesthetics, and the left sciatic nerves were collected and fixed with 4% PFA for 7 days and 20% sucrose for 24 hours before freeze embedding. The total axon number and the number of MBP-positive axons / total axon number were evaluated in the same manner as in Example 3-2(4).

[0188] (4) Statistical processing

[0189] All numerical values ​​are expressed as mean±SEM. In Examples 3-2(1) to (4) and Example 4, statistical analysis was performed using JMP software version 11 (SAS Institute) by Tukey-Kramer HSD test.

[0190] In Test Example 3-2(5), statistical analysis was performed using SAS System Version 9.1.3 (SAS Institute), and comparison between two groups was performed using the F test, Student's t test in the case of equal variance, and Welch's test in the case of unequal variance.

[0191] 3. Results

[0192] A. Evaluation of drug efficacy using the rat sciatic nerve compression injury model

[0193] (1) Blood concentration of vitamin B12, etc.

[0194] The results are shown in Fig. 9 A significant increase in blood concentration was confirmed in the MeCbl pump group (18.35±2.27ng / mL), but no increase in blood concentration was confirmed in the MeCbl sheet group (1.73±0.05ng / mL). No increase in blood concentration was confirmed in any of the drug non-administration groups (sham control group: 1.99±0.33ng / mL, CTR sheet group: 1.48±0.05ng / mL, and untreated group: 1.50±0.07ng / mL).

[0195] Likewise, when the extract of the present invention, NGF or BDNF was used as a drug, no increase in blood concentration was observed.

[0196] (2) Evaluation of motor and sensory functions

[0197] The results of SFI are shown in Fig.10 Compared with the untreated group (-20.6±4.2), a significant improvement was observed in the MeCbl sheet group (-9.0±2.0), and a recovery trend was observed in the MeCbl pump group (-10.5±2.0).

[0198] The results of the von Frey fiber test are shown in Fig.11 Compared with the untreated group (117.8±11.7 g), significant improvements were also observed in the MeCbl sheet group (80±7.6 g) and the MeCbl pump group (77.8±7.0 g).

[0199] The results of the von Frey fiber test on the sheet containing the extract of the present invention as a drug are shown in Fig.12 A significant improvement was observed in the NTP sheet group (1.35±0.13) compared to the untreated group (2.75±0.41).

[0200] Improvements in motor function and sensory function were also observed in transplantation of sheets containing NGF or BDNF as a drug.

[0201] (3) Electrophysiological evaluation

[0202] The results are shown in Fig.13 . (A) is the result of CMAP, (B) is the result of TL, and (C) is the result of NCV. In CMAP and TL, no significant improvement was confirmed in the MeCbl sheet group (CMAP: 18.5±1.5mV, TL: 3.27±0.09ms) and the MeCbl pump group (CMAP: 19.5±1.5mV, TL: 3.24±0.07ms) compared with the untreated group (CMAP: 19.5±2.3mV, TL: 3.45±0.08ms). On the other hand, in NCV, significant improvement was also confirmed in the MeCbl sheet group (44.4±2.8m / s) and the MeCbl pump group (43.2±2.5m / s) compared with the untreated group (28.2±2.5m / s).

[0203] Similar improvement was observed in the transplantation of a sheet containing the extract of the present invention, NGF or BDNF as a drug.

[0204] (4) Histological evaluation

[0205] The results are shown in Fig.14 and Fig.15 . Figure 7 This is a microscope image of myelin sheath immunostained with anti-MBP antibody. The white part in the image is the stained myelin sheath. Fig.15 (A) is the result of total axle number, Fig.15 (B) The results are the number of MBP-positive axons / total axon numbers.

[0206] Regarding the number of regenerated axons per 1 square millimeter, in the untreated group (2843 ± 68 / mm 2 )、MeCbl sheet group (2733±142 / mm 2 )、MeCbl pump group (2735±77 / mm 2 ). On the other hand, regarding the myelination rate (MBP-positive axon number / total axon number), significant improvements were also confirmed in the MeCbl sheet group (91.0±0.8%) and the MeCbl pump group (91.5±0.6%) compared with the untreated group (85.0±0.9%).

[0207] Similar improvement was observed in the transplantation of a sheet containing the extract of the present invention, NGF or BDNF as a drug.

[0208] B. Evaluation of drug efficacy using the rat sciatic nerve defect model

[0209] (1) Sensory function evaluation

[0210] The results are shown in Fig.16 In the von Frey fiber test, significant improvement was observed in the nerve regeneration induction tube + MeCbl sheet group (84.0±9.80 g) compared with the nerve regeneration induction tube group (172.0±36.7 g).

[0211] Similar improvement was observed in the sheet containing the extract of the present invention, NGF or BDNF as a drug.

[0212] (2) Electrophysiological evaluation

[0213] The results of TL are shown in Fig.17 , and the NCV results are shown in Fig.18 In TL, improvement was observed in the Nerve Regeneration Induction Tube + MeCbl Sheet Group (3.43±0.12m / s) compared to the Nerve Regeneration Induction Tube Group (4.67±0.37m / s). In NCV, improvement was observed in the Nerve Regeneration Induction Tube + MeCbl Sheet Group (29.5±4.50m / s) compared to the Nerve Regeneration Induction Tube Group (16.3±3.13m / s).

[0214] Similar improvement was observed in the sheet containing the extract of the present invention, NGF or BDNF as a drug.

[0215] (3) Histological evaluation

[0216] Improvements were observed in the number of regenerated axons per 1 mm2 and the myelination rate (MBP-positive axon number / total axon number) in the nerve regeneration induction tube + MeCbl sheet group compared to the nerve regeneration induction tube group.

[0217] Similar improvement was observed in the sheet containing the extract of the present invention, NGF or BDNF as a drug.

[0218] By transplanting the drug-containing sheet to the local part of the nerve injury, it can be confirmed that the recovery after nerve injury is promoted without increasing the drug concentration in the blood. That is, the motor function, sensory function, and electrophysiological function after nerve injury are confirmed to be restored, and improvement is also confirmed in walking analysis. In addition, an increase in the number of myelinated axons is also confirmed in histology. In summary, the present invention is very beneficial for the functional recovery after peripheral nerve injury.

[0219] In addition, the present invention is not limited to the above-mentioned embodiments and examples, and various changes can be made within the scope indicated by the claims, and the embodiments obtained by appropriately combining the technical solutions disclosed in different embodiments are also included in the technical scope of the present invention. In addition, all academic documents and patent documents recorded in this specification are cited as references in this specification.

Claims

1. A drug sustained-release sheet for implantation in the periphery of a nerve injury site and for treating nerve injury, characterized in that: A nonwoven fabric comprising nanofibers formed by electrospinning a solution containing a biocompatible polymer selected from polycaprolactone or a copolymer thereof, The medicine is vitamin B12, The content of vitamin B12 is 2% to 10% in final concentration. The drug is released from the drug sustained-release sheet for more than 25 days. The weight of the sustained-release tablet is 1 mg / cm 2 ~50mg / cm 2 , The copolymer is poly(ε-caprolactone-co-DL-lactide).

2. The pharmaceutical sustained-release sheet according to claim 1, characterized in that: Also contains hyaluronic acid.

3. The method for producing the sustained-release pharmaceutical sheet according to claim 1 or 2, characterized in that: The method comprises the following steps (1) and (2): (1) a step of preparing a solution comprising a pharmaceutical agent, a biocompatible polymer, and a solvent; and (2) The solution is subjected to electrospinning to form a nonwoven fabric.

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