WO2019124483A1 - 神経系疾患治療剤 - Google Patents
神経系疾患治療剤 Download PDFInfo
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- WO2019124483A1 WO2019124483A1 PCT/JP2018/046945 JP2018046945W WO2019124483A1 WO 2019124483 A1 WO2019124483 A1 WO 2019124483A1 JP 2018046945 W JP2018046945 W JP 2018046945W WO 2019124483 A1 WO2019124483 A1 WO 2019124483A1
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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
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Definitions
- the present invention relates to a therapeutic agent for nervous system diseases and the like.
- Neurological diseases are diseases that occur in the brain, spinal cord, peripheral nerves and muscles. Among these, diseases that occur in the brain and spinal cord are called central nervous system diseases. Typical examples of central nervous system diseases that occur in the brain include cerebral infarction and dementia. In addition, spinal cord injuries and the like are representative of central nervous system diseases that occur in the spinal cord.
- Cerebral infarction accounts for about 60% of cerebrovascular disorder which is the fourth cause of death for one year in 2014, and it is highly likely to need nursing care after illness, and it is a disease that has a large impact on society in terms of medical expenses as well. is there.
- the area of cerebral infarction is the peripheral core (penumbra, penumbra) where blood flow can be obtained by the ischemic core (also referred to as core, simply referred to as core) and collateral circulation when the blood flow is completely blocked.
- ischemic core also referred to as core, simply referred to as core
- collateral circulation when the blood flow is completely blocked.
- nerve cells in the Core part rapidly die (primary damage), it is difficult to rescue this part, but the penumbra part may survive because it is a part that escapes cell death. It will be the liver of acute treatment of cerebral infarction whether you rescue.
- Histological changes in cerebral infarction lesions include (1) apoptosis of nerve cells, (2) induction of inflammation, and (3) disruption of blood-brain barrier (BBB).
- BBB blood-brain barrier
- Edaravone trade name: Radicut
- t-PA Thrombolytic therapy
- Peripheral nerves have regenerative ability after injury, but recovery of nerve function is not sufficient.
- Wharer's degeneration results in the removal of axons and myelin sheaths.
- the regenerating axons extend in the distal direction in the Bungner's band formed by undifferentiated Schwann cells, and target muscle renervation occurs.
- a myelin sheath is formed by Schwann cells that surround the regenerating axons.
- the extension rate of the regenerative nerve is very slow, and when the distance to the target muscle is long, muscle atrophy occurs and sufficient functional recovery can not be expected.
- macrophages play an important role. It is well known that macrophages have an inflammatory function, but they also have a phenotype with anti-inflammatory activity, and they are called M1 and M2, respectively, and they are continuous between the phenotypes. It is considered that there is a sex (a shift between M1 and M2 occurs). In general, it is said that neural regeneration is promoted by increasing the anti-inflammatory phenotype of M2 macrophages.
- vitamin B 12 is effective for treating vitamin B 12 deficiency, and that vitamin B 12 deficiency may cause neurological changes such as peripheral neuritis and spinal cord changes (Patent Document 1).
- activins show anti-inflammatory action by M2 macrophage induction (patent document 2), and that an immunosuppressant containing adipose tissue-derived mesenchymal stem cells shows M2 macrophage induction action (patent document 3) ing.
- vitamin B 12 promotes M2 macrophage / microglia induction and suppresses M1 macrophage / microglia induction, as well as ameliorating neurological diseases such as cerebral infarction.
- An object of the present invention is to provide a therapeutic agent for nervous system diseases.
- the present invention is at least selected from the group consisting of an apoptosis suppressive action, a necrosis suppressive action, an axonal extension promoting action, an M2 macrophage / microglia induction promoting action, an M1 macrophage / microglia induction inhibiting action, and a nerve regeneration promoting action.
- An object of the present invention is to provide a therapeutic agent for nervous system diseases having one type.
- vitamin B 12 has a therapeutic effect on nervous system diseases. Furthermore, it was also found that vitamin B 12 has an apoptosis suppressive action, a necrosis suppressive action, an axonal extension promoting action, an M2 macrophage / microglia induction promotion action, an M1 macrophage / microglia induction suppression action, a nerve regeneration promotion action and the like. As a result of further studies based on these findings, the present invention has been completed.
- the present invention includes the following aspects: Item 1. Containing vitamin B 12, neurological diseases therapeutics.
- Item 1A A method of treating nervous system disease comprising administering vitamin B 12 to a patient in need of nervous system disease treatment.
- Vitamin B 12 for use in the treatment of nervous system disorders.
- Vitamin B 12- containing composition for use in the treatment of nervous system disorders.
- Item 1C Use of vitamin B 12 for the manufacture of a therapeutic agent for nervous system diseases.
- Item 2 The agent for treating a nervous system disease according to item 1, which is a M2 macrophage / microglia induction promoter.
- Item 3 The agent for treating a nervous system disease according to item 1, which is a M1 macrophage / microglia induction inhibitor.
- Item 4 The agent for treating a nervous system disease according to any one of Items 1 to 3, which is a nerve regeneration promoter.
- Item 5 The agent for treating a nervous system disease according to any one of Items 1 to 4, wherein the nervous system disease is a central nervous system disease.
- Item 6 The agent for treating a nervous system disease according to item 5, wherein the central nervous system disease is a cerebrovascular disease.
- Item 7 The agent for treating a nervous system disease according to item 6, wherein the cerebrovascular disease is at least one selected from the group consisting of cerebral infarction, cerebral hemorrhage, cerebral thrombosis, cerebral arteriosclerosis and dementia.
- Item 8 The agent for treating a nervous system disease according to any one of Items 1 to 4, wherein the nervous system disease is a nerve injury.
- Item 9 The agent for treating a nervous system disease according to item 8, wherein the nerve injury is central nerve injury.
- Item 10 The agent for treating a nervous system disease according to item 9, wherein the central nerve injury is a spinal cord injury.
- Item 11 The nervous system according to any one of items 1 to 10, wherein the vitamin B 12 is at least one selected from the group consisting of methylcobalamin, cyanocobalamin, hydroxocobalamin, sulfitocobalamin, adenosyl cobalamin, and salts thereof. Disease treatment agent.
- Item 12. The agent for treating a nervous system disease according to any one of Items 1 to 11, wherein the vitamin B 12 is methylcobalamin.
- Item 13 The agent for treating a nervous system disease according to any one of Items 1 to 12, which is used by being administered continuously.
- Item 14 The agent for treating a nervous system disease according to item 13, which is a preparation for intravenous drip infusion.
- Item 15. The agent for treating a nervous system disease according to any one of Items 1 to 14, which is used to start administration after 12 to 24 hours after onset.
- Item 16 The agent for treating a nervous system disease according to any one of Items 1 to 14, which is used to start administration within 12 hours immediately after onset.
- FIG. 16 shows the results of the LDH assay of Example 2.
- the vertical axis shows the ratio of LDH activity, which is an index of necrosis, to high control.
- the horizontal axis shows the type of drug added and its presence (10 ⁇ M) and no (-). * Indicates that the p value was less than 0.05 as a result of statistical analysis by Student T test. 16 shows the results of the neurite outgrowth assay of Example 3.
- the vertical axis shows the average value of 30 or more nerve axons.
- the horizontal axis shows the concentration of the added drug (CTR is not added). * Indicates that the p value was less than 0.05 as a result of statistical analysis by Dunnet's test for CTR, and ** indicates that the p value of the same result was less than 0.01.
- the TTC staining result of Example 4 is shown.
- the vertical axis shows the infarct volume.
- the horizontal axis indicates the type of drug added and its presence (MeCbl) or no (Control). ** indicates that p value was less than 0.01 as a result of statistical analysis by Student T test. 7 shows the results of Western blot of Example 5.
- the vertical axis shows the ratio of the amount of M1 marker (left: IL-1 ⁇ protein, right: iNOS protein) to the amount of GAPDH protein.
- the horizontal axis shows the type of drug added and its presence (+ or concentration) or no (-). * Indicates that the p value was less than 0.05 as a result of statistical analysis by Dunnet's test, and ** indicates that the p value of the same result was less than 0.01. 7 shows the results of Western blot of Example 5.
- the vertical axis shows the ratio of the amount of M2 marker (left: Arginase I (Arg1) protein, right: CD206 protein) to the amount of GAPDH protein.
- the horizontal axis shows the type of drug added and its presence (+ or concentration) or no (-).
- the vertical axis shows the ratio of the amount of phosphorylated AKT protein to the amount of AKT protein.
- the horizontal axis shows the type of drug added and its presence (+ or concentration) or no (-). * Indicates that p value is less than 0.05 as a result of statistical analysis by Tukey-Kramer method, ** indicates that p value of the same result is less than 0.01, *** indicates the same result It shows that p value was less than 0.001. The result of the western blot of Example 6 is shown.
- the vertical axis shows the ratio of phosphorylated 4EBP1 protein amount to 4EBP1 protein amount.
- the horizontal axis shows the type of drug added and its presence (+ or concentration) or no (-).
- FIG. 7 Results of immunohistological evaluation of Example 7 are shown.
- the figures in each row show the results from the left, proximal 2.5 mm, injury, distal 2.5 mm, distal 5.0 mm, distal 7.5 mm.
- the vertical axis shows the number of macrophages in the upper graph
- the middle graph shows the number of M1 macrophages
- the lower graph shows the proportion of M1 macrophages.
- the horizontal axis shows the number of days elapsed after sciatic nerve injury.
- CTR indicates an untreated group
- MeCbl indicates a methylcobalamin-administered group
- Sham indicates an uninjured group subjected to sciatic nerve expansion only.
- the horizontal axis shows the number of days elapsed after sciatic nerve injury.
- CTR indicates an untreated group
- MeCbl indicates a methylcobalamin-administered group
- Sham indicates an uninjured group subjected to sciatic nerve expansion only.
- * Indicates that p value was less than 0.05 as a result of statistical analysis (CTR vs MeCbl) by Tukey-Kramer method
- # indicates a result of statistical analysis (CTR vs MeCbl) by Student T test method
- p value Indicates that it was less than 0.05.
- the vertical axis shows the number of axons in the upper figure, the number in the middle shows the number of myelinated axons, and the lower figure shows the myelination rate.
- the horizontal axis indicates the position where the nerve cross section was made (from the left side, proximal 2.5 mm, injury, distal 2.5 mm, distal 5.0 mm, distal 7.5 mm).
- CTR indicates an untreated group
- MeCbl indicates a methylcobalamin-administered group
- Sham indicates an uninjured group subjected to sciatic nerve expansion only.
- * Indicates that the p value was less than 0.05 as a result of statistical analysis (CTR v.s. MeCbl) by Tukey-Kramer method.
- the BBB score measurement result of Example 9 is shown.
- the vertical axis represents the BBB score.
- the horizontal axis indicates the number of days elapsed after surgery (0 is before surgery) for creating a spinal cord injury model.
- CTR indicates an untreated group
- MeCbl indicates a methylcobalamin-administered group
- Sham indicates an uninjured group subjected to sciatic nerve expansion only. * Indicates that the p value was less than 0.05 as a result of statistical analysis (CTR vs MeCbl) by the Steel-Dwass test method.
- 7 shows the results of the thermal alerting test of Example 9.
- the vertical axis shows the time until infrared heat stimulation is given to the right plantar and the heat draws the hind limb.
- the horizontal axis indicates the number of days elapsed after surgery (0 is before surgery) for creating a spinal cord injury model.
- CTR indicates an untreated group
- MeCbl indicates a methylcobalamin-administered group
- Sham indicates an uninjured group subjected to sciatic nerve expansion only. * Indicates that the p value was less than 0.05 as a result of statistical analysis (CTR vs MeCbl) by the Steel-Dwass test method.
- CTR vs MeCbl The result of the western blot of M1 marker (IL-1 beta protein and iNOS protein) of Example 10 is shown.
- the vertical axis shows the ratio of the amount of M1 marker (IL-1 ⁇ protein or iNOS protein) to the amount of GAPDH protein.
- the horizontal axis shows the type of drug added and its presence (+ or concentration) or no (-). * Indicates that the p value was less than 0.05 as a result of statistical analysis by Dunnet's test, and ** indicates that the p value of the same result was less than 0.01.
- the result of the western blot of M2 marker (Arg1 protein and CD206 protein) of Example 10 is shown.
- the vertical axis, the horizontal axis, and the respective symbols are the same as in FIG. 7 shows the results of immunofluorescent staining at 7 days after surgery in Example 11. Each graph represents the change by site.
- the horizontal axis of each figure shows the direction and distance from the injury.
- the vertical axis shows the number of macrophages in the upper graph
- the middle graph shows the number of M1 or M2 macrophages
- the lower graph shows the proportion of M1 or M2 macrophages.
- CTR indicates untreated group
- MeCbl indicates methylcobalamin administered group. * Indicates that the p value was less than 0.05 as a result of statistical analysis (CTR v s. MeCbl) by Mann Whitney U test, and ** indicates that the p value was less than 0.01.
- 14 shows the results of immunofluorescent staining at 14 days after surgery in Example 11. Others are similar to FIG. It shows the results of immunofluorescence staining at 28 days after surgery of Example 11. Others are similar to FIG.
- the results of immunofluorescent staining for M1 macrophages of Example 11 are shown.
- Each graph represents a change due to the number of days after surgery.
- the figures in each row show the results from the left side, 2 mm head side, 1 mm head side, 1 mm tail side, 2 mm tail side, with respect to direction and distance from the injury.
- the vertical axis shows the number of macrophages in the upper graph
- the middle graph shows the number of M1 macrophages
- the lower graph shows the proportion of M1 macrophages.
- the horizontal axis shows the number of days elapsed after surgery for creating a spinal cord injury model.
- CTR indicates untreated group
- MeCbl indicates methylcobalamin administered group.
- the vertical axis shows the M1 / M2 ratio.
- the horizontal axis shows the number of days elapsed after surgery for creating a spinal cord injury model.
- CTR indicates untreated group
- MeCbl indicates methylcobalamin administered group.
- * Indicates that the p value was less than 0.05 as a result of statistical analysis (CTR v s. MeCbl) by Mann Whitney U test, and ** indicates that the p value was less than 0.01.
- 17 shows the results of immunofluorescent staining for the M1 / M2 ratio of Example 11.
- Each graph represents the change by site. From the left side, the results after 7 days, 14 days and 28 days after surgery of spinal cord injury model creation are shown.
- the vertical axis shows the M1 / M2 ratio.
- the horizontal axis shows the number of days elapsed after surgery for creating a spinal cord injury model.
- the horizontal axis shows the direction and distance from the injury.
- CTR indicates untreated group
- MeCbl indicates methylcobalamin administered group.
- * Indicates that the p value was less than 0.05 as a result of statistical analysis (CTR v s. MeCbl) by Mann Whitney U test, and ** indicates that the p value was less than 0.01.
- 16 shows the results of the Rota-rod test of Example 12.
- the horizontal axis shows the number of days elapsed after stroke, and the vertical axis shows the relative time to fall from the rotor rod.
- CTR indicates untreated group
- MeCbl indicates methylcobalamin administered group.
- ** indicates that the p value was less than 0.01 as a result of statistical analysis (CTR vs MeCbl) by Mann Whitney U test.
- microphage / microglia means “macrophage and / or microglia” and is a term including both “macrophage and microglia” and “macrophage or microglia”.
- the present invention in one aspect thereof, comprises a vitamin B 12, neurological diseases agents, apoptosis inhibitor, necrosis inhibitor, axon extension promoter, M2 macrophages / microglia induction promoter, M1 macrophage / microglia-induced suppression
- the present invention relates to an agent, a nerve regeneration promoter and the like (also referred to herein as "the agent of the present invention”). These will be described below.
- the active ingredient vitamin B 12 is cobalamin, its derivatives, and salts thereof.
- vitamin B 12 include cobalamin, those in which cobalt ion of cobalamin is substituted, and derivatives thereof. More specific examples include methylcobalamin, cyanocobalamin, hydroxocobalamin, sulfitocobalamin, adenosyl cobalamin, salts thereof and the like. Among these, methylcobalamin, cyanocobalamin, hydroxocobalamin and salts thereof are preferable, and methylcobalamin and salts thereof are more preferable.
- the salts of cobalamin and its derivatives are not particularly limited as long as they are pharmaceutically acceptable salts, and any of acidic salts and basic salts can be adopted.
- the acid salt include mineral acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; acetate, propionate, tartrate, fumarate, maleate, apple Organic acid salts such as acid salts, citrates, methanesulfonates, para-toluenesulfonates, amino acids salts such as aspartates and glutamates, and the like.
- the basic salt include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.
- Vitamin B 12 may be in the form of a solvate.
- the solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, acetic acid and the like.
- Vitamin B 12 may be the singly or may be a combination of two or more thereof.
- Vitamin B 12 has a therapeutic effect on nervous system diseases. Therefore, vitamin B 12 can be used as an active ingredient of a therapeutic agent for nervous system diseases.
- Vitamin B 12 has an apoptosis suppressing action, a necrosis suppressing action, an axonal extension promoting action, an M2 macrophage / microglia induction promoting action, an M1 macrophage / microglia induction inhibiting action, a nerve regeneration promoting action, and the like. Therefore, vitamin B 12 is an apoptosis inhibitor, necrosis inhibitor, axonal outgrowth promoter, M2 macrophage / microglia induction promoter, M1 macrophage / microglia induction inhibitor, M1: M2 ratio (M1 to M2 macrophages / microglia It can be used as an active ingredient of a ratio of (microglia ratio) inhibitor, nerve regeneration promoter and the like.
- vitamin B 12 is a preferred embodiment of a therapeutic agent for nervous system diseases, that is, apoptosis suppressive action, necrosis suppressive action, axonal extension promoting action, M2 macrophage / microglia induction promotion action, M1 macrophage / microglia induction suppression action, and nerve It can be used as an active ingredient of a therapeutic agent for nervous system diseases based on at least one selected from the group consisting of regeneration promoting effects.
- the nervous system diseases are not particularly limited, and include central nervous system diseases and peripheral nervous system diseases.
- central nervous system diseases include cerebrovascular diseases, nerve damage and the like.
- Cerebrovascular diseases include cerebral infarction, cerebral hemorrhage, cerebral thrombosis, cerebral arteriosclerosis, dementia and the like.
- the nerve injury may be either peripheral nerve injury or central nerve injury.
- Central nerve injury also includes spinal cord injury.
- the cause of nerve damage is not particularly limited, and nerve damage due to various causes such as trauma, compression by cast, electric shock, disc herniation, radiation exposure, etc. is applicable.
- the degree of nerve damage to which the present invention is applied is not particularly limited, and in the case where axons are preserved but demyelination occurs, if Waller degeneration is involved, or nerves are anatomically ruptured, etc. Both are applicable.
- nerve damage includes various symptoms associated with it, such as motor impairment (such as motor paralysis and weakness in upper and lower limbs), sensory impairment (such as analgesia, numbness, pain, etc.) in the damaged nerve area.
- autonomic nervous disorders perspiration abnormalities, changes in skin tone, etc. and the like.
- the nervous system disease is preferably selected from the group consisting of apoptosis suppressive action, necrosis suppressive action, axonal extension promoting action, M2 macrophage / microglia induction promoting action, M1 macrophage / microglia induction inhibiting action, and nerve regeneration promotion action It is a neurological disease that can be treated based on at least one type.
- the agent of the present invention is not particularly limited as long as it contains vitamin B 12 (sometimes referred to herein simply as “active ingredient”), and may further contain other ingredients as necessary. .
- the other components are not particularly limited as long as they are pharmaceutically acceptable components.
- Other components include additives having a pharmacological action as well as additives.
- the additive for example, base, carrier, solvent, dispersant, emulsifier, buffer, stabilizer, excipient, binder, disintegrant, lubricant, thickener, humectant, coloring agent, flavor, Chelating agents and the like can be mentioned.
- Vitamin B 12 alone has a therapeutic effect on nervous system diseases, an apoptosis suppressive action, an necrosis suppressive action, an axonal extension promoting action, an M2 macrophage / microglia induction promotion action, an M1 macrophage / microglia induction suppression action (here, M1 macrophage It can not be denied that it is a shift effect from microglia to M2 macrophages / microglia. Therefore, the agent of the present invention can exert its desired effect without including other components having these effects and / or actions, but may contain other components having pharmacological actions. Good.
- the use mode of the agent of the present invention is not particularly limited, and an appropriate use mode can be adopted according to the type.
- the agent of the present invention can be used, for example, in vitro (for example, added to the culture medium for the culture) or in vivo (for example, administered to an animal), depending on its use. You can also.
- the application object of the agent of the present invention is not particularly limited, examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, deer and the like. Moreover, as a cell, an animal cell etc. are mentioned.
- the type of cells is also not particularly limited. For example, blood cells, hematopoietic stem cells / progenitor cells, gametes (sperm, egg), fibroblasts, epithelial cells, vascular endothelial cells, neurons, hepatocytes, hepatocytes, keratinocytes, myocytes And epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells and the like.
- the agent of the present invention may be in any dosage form, such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, Oral formulations such as hard capsules, soft capsules, dry syrups, solutions (including drinks, suspensions and syrups) and jellies, and preparations for injection (eg, drip injections (eg, for intravenous drip infusion) Formulations, etc.)
- Intravenous injection intramuscular injection, subcutaneous injection, intradermal injection), external preparation (eg, ointment, poultice, lotion), suppository inhalant, eye, eye ointment, point Parenteral preparations such as nasal drops, eardrops, and liposomes can be taken.
- the administration route of the agent of the present invention is not particularly limited as long as the desired effect can be obtained, and oral administration, enteral administration such as tube feeding, enteral administration, intravenous administration, transarterial administration, intramuscular administration, etc.
- enteral administration such as tube feeding, enteral administration, intravenous administration, transarterial administration, intramuscular administration, etc.
- Intracardiac administration subcutaneous administration, intradermal administration, parenteral administration such as intraperitoneal administration and the like can be mentioned.
- the content of the active ingredient in the agent of the present invention depends on the mode of use, application subject, state of application subject, etc., and is not limited, for example, 0.0001 to 100% by weight, preferably 0.001 to 50 wt. It can be%.
- the dose for administering the agent of the present invention to an animal is not particularly limited as long as it is an effective amount to exert a medicinal effect, and generally 0.1 to 1 day per day as an active ingredient weight. 1000 mg / kg body weight, preferably 0.5 to 500 mg / kg body weight per day, and for parenteral administration 0.01 to 100 mg / kg body weight per day, preferably 0.05 to 50 mg / kg body weight .
- the above dose can be appropriately increased or decreased depending on the age, condition and symptoms.
- the agent of the present invention is preferably administered continuously from the viewpoint of exerting the M2 macrophage / microglia induction promoting action, the M1 macrophage / microglia induction inhibiting action, the nerve regeneration promotion action and the like more effectively.
- the concentration of the active ingredient acting on the cells in the administration subject for example, cells of diseased part of nervous system disease, preferably macrophages / microglia
- M2 macrophage / microglia induction promoting action M1 macrophage / microglia induction inhibiting action
- nerve regeneration nerve regeneration It is possible to maintain the concentration suitable for exerting the promoting action (for example, 5 nM to 100 ⁇ M, preferably 10 nM to 50 ⁇ M, more preferably 20 nM to 10 ⁇ M, more preferably 50 nM to 5 ⁇ M, still more preferably 100 nM to 1 ⁇ M)
- the agent of this invention is based also on the application object, it is preferable that it is a drip
- the administration timing of the agent of the present invention is not particularly limited.
- the agent of the present invention is used to start administration, for example, 12 to 24 hours after onset.
- the onset is a point at which the symptom of the disease or the direct factor causing it can be confirmed, and in the case of ischemic cerebrovascular disease such as cerebral infarction, for example, the onset point of the ischemic site.
- the agent of the present invention can act on a repair mechanism that can act at a relatively late timing from ischemic site development (M2 macrophage / microglia induction promoting action, M1 macrophage / microglia induction inhibiting action, nerve regeneration promotion action), When applied to ischemic cerebrovascular disease such as cerebral infarction, it is possible to exert a therapeutic effect even when administered at the above timing (12 to 24 hours after onset).
- the agent of the present invention is used to start administration in the acute phase (eg, immediately after onset to within 12 hours of onset) of nervous system diseases such as nerve injury (preferably spinal cord injury).
- nervous system diseases such as nerve injury (preferably spinal cord injury).
- the onset is the point at which the symptom of the disease or the direct factor causing it can be confirmed, for example, in the case of nerve injury such as spinal cord injury, the point at which nerve injury occurs.
- Example 1 Apoptosis inhibitory action of cerebral cortex neurons The apoptosis inhibitory action of cerebral cortex neurons was examined by TUNEL assay using methylcobalamin. Specifically, it is as follows.
- Example 1-1 Preparation of Cerebral Cortical Nerves> Cerebral cortical nerves were collected and cultured according to a standard method.
- the cerebral cortex is dissected from the fetus of Sprague Dawley (SD) rats (day 18 of gestation) and recovered in ice cold Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin did.
- the pia and blood vessels were removed, transferred to DMEM (without FBS, containing 1% penicillin / streptomycin) and shredded to 1 mm with scissors.
- Papain final concentration 2 mg / ml was added to the cell mixture and reacted at 37 ° C.
- Example 1-2 TUNEL assay> 20 mM glutamate, 10 ⁇ M methylcobalamin (MeCbl) was added to a PLL-coated 8-well chamber slide cultured cerebral cortex nerve (Example 1-1), and after 18 hours, the percentage of apoptotic cells was evaluated with Promega deadend fluorometric TUNEL system did. It was fixed at 4 ° C. for 25 minutes with 4% paraformaldehyde (PFA). After permeabilization with 0.2% Triton X-100 for 5 minutes, incubation buffer was added and the mixture was placed for 60 minutes in the dark at 37 ° C. for labeling. Nuclei were labeled with DAPI. The total number of cells and the number of TUNEL positive cells were counted.
- PFA paraformaldehyde
- Example 1-3 Result> The results are shown in FIG. Further, the numerical values in FIG. 1 are shown in Table 1. In the TUNEL assay, the percentage of apoptotic cells was similar to that of the control when methylcobalamin alone was added. The addition of glutamate alone resulted in a significant increase in the proportion of apoptotic cells, but the combination of glutamate and methylcobalamin significantly reduced the proportion of apoptotic cells to the control level.
- Example 2 Necrosis inhibitory action of cerebral cortex neurons The necrosis inhibitory action of cerebral cortex neurons was examined by LDH assay using methylcobalamin. Specifically, it is as follows.
- Example 2-1 LDH assay> 10 ⁇ M methylcobalamin was added 30 minutes before oxygen-glucose deprivation (OGD) loading to cerebral cortex neurons (Example 1-1) cultured in PLL coated 6 well chamber slide. N-methyl-D aspartate (NMDA) was added to the standard high control. The medium was changed to earle's balanced salt solution (EBSS), and an OGD load was performed for 3 hours in an environment with an oxygen concentration of 1%. After 24 hours, the medium was returned to an oxygen concentration environment and the supernatant was collected, and LDH activity was measured using a cytotoxicity detection kit plus (SIGMA). The LDH activity of the control group and methylcobalamin-added group was calculated as a percentage (%) of the high control LDH activity.
- OGD oxygen-glucose deprivation
- Example 2-2 Result> The results are shown in FIG. The numerical values in FIG. 2 are shown in Table 2.
- the ratio of the LDH activity, which is an index of necrosis, to the high control was significantly reduced in the methylcobalamin addition group compared to the control group.
- Example 3 Axon Extension Promoting Action of Cerebral Cortical Neurons The axonal extension promoting action of cerebral cortical neurons was examined in a neurite extension assay using methylcobalamin. Specifically, it is as follows.
- Example 3-1 Neurite Outgrowth Assay> Various drugs were added 24 hours after the cortical nerve (Example 1-1) was seeded. The concentration of added drug was methylcobalamin (1 nM, 10 nM, 100 nM, 1 ⁇ M, 10 ⁇ M, 100 ⁇ M). 72 hours after cell seeding, immunofluorescent staining was carried out with anti-TuJ1 antibody, and axon length (neuron length) was measured. However, only cells not in contact with other nerves were measured, at least 30 or more nerve axons were measured in each evaluation, and the average value was calculated and used as the measurement value.
- Example 3-2 Result> The results are shown in FIG. Further, the numerical values in FIG. 3 are shown in Table 3. In the neurite outgrowth assay, axonal extension tends to be promoted in a concentration dependent manner at a peak concentration of 10 ⁇ M, and at 1 ⁇ M and 10 ⁇ M, there is a significant difference in axonal outgrowth compared with the control group without any drug I recognized the promotion.
- Example 4 Reduction of cerebral infarction volume The reduction of cerebral infarction volume was examined by TTC (2,3,5-Triphenyltetrazolium chloride) staining using methylcobalamin. Specifically, it is as follows.
- Example 4-1 Preparation of temporary middle cerebral artery occlusion (tMCAO) model, and drug administration> 8-9 week old male C57BL / 6J mice (around 24 g) were used.
- a probe for laser doppler blood flow measurement was attached on the right skull to enable monitoring of middle cerebral artery blood flow.
- the right cervix was unfolded, the external carotid artery was ligated, an incision was made in the common carotid artery, a nylon thread was inserted, and the tip was advanced while looking at the blood flow monitor.
- the tip was advanced to the middle cerebral artery bifurcation, and it was confirmed that the blood flow decreased, and after standing by at a rectum temperature of 37 ° C.
- methylcobalamin was placed in the back subcutaneous of an osmotic minipump and administered after the model was completed at a dose of 1 mg / kg / day.
- raw food was administered in the same manner. Postoperatively, the rectum temperature was maintained at 37 ° C. until anesthesia awakened.
- Example 4-3 Result> The results are shown in FIG. Further, the numerical values in FIG. 4 are shown in Table 4. Two days after tMCAO surgery on mice, cerebral infarction volume was evaluated by TTC staining. A significant reduction in infarct volume of about half was observed in the methylcobalamin-administered group compared to the control group.
- Example 5 M2 macrophage induction promoting action and M1 macrophage induction suppressing action Using methylcobalamin, M2 macrophage induction promoting action and M1 macrophage induction suppressing action were examined by Western blotting and immunohistological evaluation method. Specifically, it is as follows.
- Example 5-1 Preparation of macrophage cell line>
- the mouse-derived macrophage cell line J774A.1 (JCRB9108) was purchased from JCRB cell bank (culture resources laboratory) in Osaka Prefecture. The culture was carried out in DMEM containing 10% FBS and 1% penicillin / streptomycin.
- Example 5-2 Western blot method> J774A.1 cells (Example 5-1) were seeded in a dish of 6 cm in diameter, and 4 days later, proteins were collected using cell lysis buffer in which protease inhibitor cocktail was dissolved. After measuring the protein concentration by BCA assay, 50 ⁇ g of each sample was electrophoresed by SDS-PAGE and transferred to polyvinylidene difluoride membrane. After blocking for 1 hour with Blocking buffer, it was reacted with primary antibody at 4 ° C. over night. The secondary antibody was allowed to react for 1 hour at room temperature and detected with the ECL Western Blotting Detection System.
- Lipopolysaccharide (LPS) 100 ng / ml
- methylcobalamin were added 24 hours prior to protein collection when detecting the M1 markers iNOS and IL-1 ⁇ .
- IL-4 20 ng / ml
- methylcobalamin were added 72 hours before protein collection.
- Primary antibodies are anti-IL-1 ⁇ rabbit polyclonal antibody (Santa Cruz), anti-iNOS rabbit monoclonal antibody (Abcam), anti-Arg1 rabbit polyclonal antibody (Santa Cruz), anti-CD206 rabbit monoclonal antibody (Abcam), and secondary antibody is anti-rabbit IgG horseradish peroxidase linked whole antibody from donkey (GE Healthcare Life Sciences) was used.
- Example 5-3 Immunohistochemical evaluation method> J774A.1 cells (Example 5-1) were seeded in a dish of 6 cm in diameter, and fixed 4 days later with 4% PFA for 20 minutes. It was blocked for 30 minutes and the primary antibody was reacted at 4 ° C. over night. The secondary antibody was reacted at room temperature for 1 hour, and the nucleus was labeled with DAPI.
- iNOS which is M1 marker
- LPS 100 ng / ml
- methylcobalamin were added 24 hours before cell fixation.
- IL-4 20 ng / ml
- methylcobalamin were added 72 hours before cell fixation.
- Primary antibody is anti-iNOS rabbit monoclonal antibody (Abcam), anti-Arg1 rabbit polyclonal antibody (Santa Cruz), secondary antibody is Alexa 488 labeled goat anti rabbit IgG antibody (Lifetechnologies) or Alexa 568 labeled goat anti rabbit IgG antibody (Lifetechnologies) used.
- the numerical values in FIG. 5 are shown in Table 5 and the numerical values in FIG. 6 are shown in Table 6.
- M1 marker significant decrease in the amount of protein was observed at 100 nM and iNOS at 100 nM to 10 ⁇ M, compared to the addition of LPS alone.
- M2 marker significant increase in the amount of protein was observed at 100 nM and 1 ⁇ M compared to the addition of IL-4 alone.
- the results of the immunohistological evaluation method are shown in FIG.
- the numerical values in FIG. 7 are shown in Table 7.
- the rate of iNOS positive cells was significantly reduced in the iNOS which is the M1 marker.
- the percentage of Arg1 positive cells was significantly increased by adding 10 nM to 1 ⁇ M of methylcobalamin as compared with the addition of IL-4 alone.
- Example 6 Analysis of Mechanism of Macrophage Induction Action
- the mechanism of macrophage induction action (Example 5) by methyl cobalamin was analyzed. Specifically, activation of Akt, 4EBP1 and S6K in the Akt-mTOR pathway (one of the main signaling pathways that induce the M2 gene) 30 minutes after addition of IL-4, methylcobalamin (100 nM and 1 mM) It was evaluated by Western blot. In the pathway, upstream signals cause phosphorylation of Akt and further downstream phosphorylation of 4EBP1 and phosphorylation of S6K via mTORC1. Phosphorylation of S6K causes a negative feedback action upstream of the signal.
- FIG. 8-1 The results are shown in Fig. 8-1, Fig. 8-2, and Fig. 8-3.
- the numerical values in FIG. 8-1, FIG. 8-2, and FIG. 8-3 are shown in Table 8.
- Activation of both Akt and downstream 4EBP1 and S6K was observed by addition of IL-4.
- the combined activation of IL-4 and methylcobalamin 100 nM enhanced their activation more than the addition of IL-4 alone, but when methyl cobalamin was 1 mM, the activity of 4EBP1 and S6K was reversed, and the activity of upstream Akt was It has fallen.
- the mTOR inhibitor RAD 001 was further added to this, the activity of upstream Akt was rescued, and the suppression of downstream 4EBP1 and S6K activity was observed. From the above, it was suggested that the mechanism by which the upstream Akt activity toward M2 gene induction is suppressed by the negative feedback mechanism from the downstream when high concentration methylcobalamin is added.
- Example 7 Analysis of macrophage dynamics after sciatic nerve injury The influence of methylcobalamin on macrophage dynamics after sciatic nerve injury was analyzed by immunohistological evaluation method. Specifically, macrophages were evaluated by fluorescent immunostaining in the proximal 2.5 mm, injured area, distal 2.5 mm, 5.0 mm, and 7.5 mm nerve transversal sections at 1, 3, 7 and 14 days after sciatic nerve injury. did.
- proximal means the side from the injury on the axon to the cell body side
- distal means the side from the injury on the axon to the end of the axon
- the respective distances indicate the distances from the injury. The same applies to 8).
- Macrophages were labeled with CD68, iNOS as the M1 marker, and CD206 as the M2 marker.
- M1 macrophage ratio (%) number of M1 marker positive macrophages (number / mm 2 ) / number of macrophages (number / number 2 ) ⁇ 100. More specifically, it is as follows.
- Example 7-1 Surgical treatment (sciatic nerve crush injury rat)> Six-week-old male Wistar rats (approximately 200 g) were used. The left sciatic nerve was deployed and crush injury was made with forceps proximal to the sciatic nerve. The pressing time was 10 seconds, the number of pressings was 3 times, and the interval between pressing operations was 10 seconds. The fascia and skin were sutured with 3-0 nylon. The uninjured, untreated, and methylcobalamin-treated groups that underwent only sciatic nerve deployment were compared. For continuous administration of methylcobalamin, an osmotic minipump was placed subcutaneously at the back and administered at a dose of 1 mg / kg / day. Raw food was administered in the same way in the untreated group.
- Example 7-2 Morphological and histologic analysis> One, three, seven and fourteen days after surgery, the rat was sedated with an anesthetic and the left sciatic nerve was collected and frozen and embedded in 4% PFA for 7 days and 20% sucrose for 24 hours. .
- the embedded tissue was sliced at a thickness of 5 ⁇ m in the direction of the nerve minor axis and placed on a glass slide. As slice sites, it was performed at five points of 2.5 mm proximal to the injury, 2.5 cm distal, 5.0 mm distal, 5.0 mm distal, and 7.5 mm distal. Dry for 1 hour and fix in 95% methanol for 30 minutes. After blocking, the primary antibody was reacted at 4 ° C. over night. The secondary antibody was reacted at room temperature for 1 hour, and the nucleus was labeled with DAPI.
- Primary antibodies are anti-CD68 mouse monoclonal antibody (Abcam), anti-iNOS rabbit monoclonal antibody (Abcam), anti-CD206 rabbit monoclonal antibody (Abcam), anti-neurofilament 200 (NF200) rabbit polyclonal antibody (SIGMA) and anti-myelin Basic Protein (MBP) Mouse monoclonal antibody (CALBIOCHEM), secondary antibody labeled Alexa 488 labeled goat anti-mouse IgG antibody (Lifetechnologies), Alexa 488 labeled goat anti-rabbit IgG antibody (Lifetechnologies), Alexa 568 labeled goat anti-mouse IgG antibody (Lifetechnologies) and Alexa 568 labeled Goat anti-rabbit IgG antibody (Lifetechnologies) was used.
- Example 7-3 Result> The results are shown in FIGS.
- the numerical values in FIG. 9 are shown in Table 9-1, Table 9-2, and Table 9-3, and the numerical values in FIG. 10 are shown in Table 10-1, Table 10-2, and Table 10-3.
- the methylcobalamin-administered group showed a significant decrease in the number of accumulated macrophages at 3, 7 and 14 days after surgery in the injured area as compared with the untreated group. At the distal end, the number of macrophages increased to delay the injury, but a significant difference was observed 14 days after the operation.
- the number of M1 macrophages was significantly reduced in the methylcobalamin administration group on all evaluation days. Distal was significantly different 7 and 14 days after surgery. The same tendency was seen in the M1 macrophage ratio.
- the number of M2 macrophages significantly increased in methylcobalamin-administered groups on days 1, 7 and 14 after surgery, 7 days after surgery at 5 mm distal and 7 and 14 days after surgery at 7.5 mm distal There was a significant difference. The same tendency was seen for the M2 macrophage ratio.
- Example 8 Analysis of nerve regeneration kinetics after sciatic nerve injury The influence of methylcobalamin on nerve regeneration kinetics after sciatic nerve injury was analyzed by immunohistological evaluation method. Specifically, transverse sections of the injured sciatic nerve two weeks after sciatic nerve injury were evaluated. The evaluation sites were 2.5 mm proximal, 2.5 mm distal, 5.0 mm distal, and 7.5 mm distal similar to macrophage evaluation. Regenerating axons were labeled with NF200 and myelin was labeled with MBP.
- the myelination rate (%) myelinated axon number (pieces / mm 2 ) / axon number (pieces / mm 2 ) ⁇ 100 was calculated. More specifically, it carried out by the method similar to Example 7.
- FIG. 11 The results are shown in FIG.
- the numerical values in FIG. 11 are shown in Table 11-1, Table 11-2, and Table 11-3.
- the number of axons and myelinated axons were significantly improved in the methylcobalamin-administered group, the number of axons was 5.0 mm and 7.5 mm distally, and the number of myelinated axons was 2.5 mm distally, At 5.0 mm and 7.5 mm, significant differences were seen in the myelination rates at the distal 5.0 mm and 7.5 mm. From this result and the result of Example 7, it is suggested that methyl cobalamin promotes nerve regeneration by reducing M1 macrophages in an actual nerve regeneration process, increasing M2 macrophages and acting as an anti-inflammatory property. It was done.
- Example 9 Therapeutic Effect of Spinal Cord Injury Using methylcobalamin, the therapeutic action of spinal cord injury was examined by a BBB (Basso-Beattie-Bresnahan) score and a thermal algorithm test. Specifically, it is as follows.
- BBB Basso-Beattie-Bresnahan
- Example 9-1 Preparation of rat spinal cord injury model (Lateral Hemisection model) and drug administration> Six-week-old female Wistar rats were used. Rats were purchased from Japan Charles River (Yokohama City, Japan). Anesthesia was administered by intraperitoneal injection with dilution of the three mixed anesthetics 1:10 with saline. The anesthetic dose per administration was midazolam 0.2 mg / kg, medetomidine 0.015 mg / kg, butorphanol 0.25 mg / kg. He was placed in a prone position on the operating table and developed a back midline. The T10 lamina was resected to expose the back of the spinal cord, and the left spinal cord was cut in half with a Spitz scalpel.
- methylcobalamin administered group untreated group
- Sham group Immediately after the operation, an osmotic minipump filled with methylcobalamin (1 mg / kg / day) and saline was placed in the left back subcutaneously immediately after the operation. The Sham group only resected the Th10 lamina.
- Example 9-2 Measurement of BBB score> The rats were individually placed in cages and allowed to freely walk and observed for 5 minutes. According to the standard method, scoring was performed between 0 (no exercise) and 21 (normal exercise) in the function of the left leg. The evaluation was performed on postoperative days 1, 7, 14, 21, and 28 days after the operation.
- Example 9-3 Thermal alerting test> The rats were individually placed in their own cages, infrared thermal stimulation was given to the right plantar, and the time to heat the hindlimb was measured. The stimulation time was up to 15 seconds to avoid damage to the skin. The evaluation was performed on postoperative days 7, 14, 21 and 28 before surgery.
- Example 10 M2 microglia induction promoting action and M1 microglia induction suppressing action Using methyl cobalamin, M2 microglia induction promoting action and M1 microglia induction suppressing action were examined by Western blotting. Specifically, it is as follows.
- Example 10-1 Western blot method> Methyl cobalamin adjusted to various concentrations (1 nM to 1 mM) by adding LPS (100 ng / ml) and IL-4 (20 ng / ml) as an anti-inflammatory cytokine to a microglial cell line (6-3 cells) Were added, and the protein was recovered 1 day and 3 days after each addition. After electrophoresis, transcription to a membrane was performed, and after blocking, primary antibodies to M1 marker (iNOS, IL-1 ⁇ ) and M2 marker (Arg1, CD206) were reacted at 4 ° C. over night. The secondary antibody was reacted at room temperature for 1 hour, and band detection was performed with a detector.
- LPS 100 ng / ml
- IL-4 20 ng / ml
- M1 marker iNOS, IL-1 ⁇
- M2 marker Arg1, CD206
- the primary antibody was an anti-iNOS antibody, an anti-IL-1 ⁇ antibody, an anti-Arg1 antibody, an anti-CD206 antibody (Mannose Receptor), and the secondary antibody was Anti-Rabbit IgG, HRP-Linked Whole Ab Sheep.
- the numerical values in FIG. 14 are shown in Table 14 and the numerical values in FIG. 15 are shown in Table 15.
- M1 marker IL-1 ⁇ decreased by 1 ⁇ M and iNOS by 10 ⁇ M or more of methylcobalamin significantly decreased the amount of protein compared to addition of LPS alone.
- M2 marker Arg1 showed a significant increase in protein mass when methyl cobalamin was added at 1 nM to 10 ⁇ M, compared to the addition of IL-4 alone.
- CD206 a significant increase was observed with the addition of 10 nM to 100 nM methylcobalamin.
- Example 11 M2 macrophage induction promoting action and M1 macrophage induction suppressing action Using methyl cobalamin, M2 macrophage induction promoting action and M1 macrophage induction suppressing action were examined. Specifically, it is as follows.
- Example 11-1 Immunofluorescent staining> About the rat spinal cord injury model of Example 9-1, sedate rats after 7, 14 and 28 days after surgery with an anesthetic agent, fix by perfusion with 4% PFA, and collect the spinal cord including the injured part. After freezing for 24 hours with 20% sucrose, the cells were frozen and embedded. The embedded tissue was sliced at a thickness of 5 ⁇ m in the direction of the nerve minor axis and placed on a glass slide. Dry for 1 hour and fix in 100% methanol for 30 minutes. After blocking, the primary antibody was reacted at 4 ° C. over night. The secondary antibody was reacted at room temperature for 1 hour, and the nucleus was labeled with DAPI.
- the primary antibody used was anti-CD68 antibody, anti-iNOS antibody, anti-Arg1 antibody, and the secondary antibody used Alexa 488 labeled goat anti rabbit IgG antibody and Alexa 568 labeled goat anti mouse IgG antibody.
- FIGS. 16 to 18 show changes in the number of macrophages per unit area, the number of M1 (inflammatory type) macrophages, the number of M1 macrophages, the number of M2 (anti-inflammatory type) macrophages, and the change with the site for the ratio of M2 macrophages.
- 20 represents changes due to the number of days elapsed after these operations.
- FIG. 21 shows the change with the number of elapsed days after surgery for the M1 / M2 ratio
- FIG. 22 shows the change with this site.
- the numerical values in FIGS. 16 to 18 are sequentially shown in Tables 16 to 18, and the numerical values in FIG.
- the number of accumulated macrophages per unit area tends to be smaller than in the untreated group, and in the phenotype, the number of M1 macrophages is decreased and the number of M2 macrophages is increased. Some differences were significant.
- Example 12 About the functional recovery promotion effect of methyl cobalamin in a photocoagulation cerebral infarction model ⁇ Example 12-1.
- Target and method> A male C57BL / 6J mouse (about 24 g) of 8-10 weeks old was used, and after administration of Rose Bengal, a photocoagulation cerebral infarction model was produced by irradiating laser light.
- the skull of the mouse is drilled and drilled using a drill centered on the skull 2 mm outside the Oizumimon gate, and 5 minutes after administration of the photosensitive dye Rose Bengal, the laser light is directed to the right motor area center Irradiate and create a cerebral infarction in the right motor area.
- cerebral infarction 2 days, 4 days, 7 days, 9 days, 11 days and 14 days after operation, the Rota-rod test (accelerating velocity) was performed. The time taken for the mouse to fall from the rota rod was measured, and the ratio was calculated with Max 300 seconds as the baseline.
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Abstract
Description
項1. ビタミンB12を含有する、神経系疾患治療剤。
ビタミンB12には、コバラミン、その誘導体、及びそれらの塩が含まれる。ビタミンB12として、具体的には、コバラミン、コバラミンのコバルトイオンが置換されたもの、及びそれらの誘導体が挙げられる。より具体的な例としては、メチルコバラミン、シアノコバラミン、ヒドロキソコバラミン、スルフィトコバラミン、アデノシルコバラミン、それらの塩等が挙げられる。これらの中でも、メチルコバラミン、シアノコバラミン、ヒドロキソコバラミン、それらの塩が好ましく、メチルコバラミン、その塩がより好ましい。
ビタミンB12は、神経系疾患治療効果を有する。このため、ビタミンB12は、神経系疾患治療剤の有効成分として利用することができる。
メチルコバラミンを用いて、大脳皮質神経細胞のアポトーシス抑制作用をTUNELアッセイで調べた。具体的には以下のとおりである。
大脳皮質神経は定法に従って採取培養した。Sprague Dawley(SD)ラット(妊娠18日目)の胎仔から大脳皮質を切離し、10%ウシ胎児血清(FBS)及び1%ペニシリン/ストレプトマイシンを含有し、氷冷したダルベッコ改変イーグル培地(DMEM)に回収した。軟膜及び血管を除去し、DMEM(FBS未添加、1%ペニシリン/ストレプトマイシン含有)へ移して剪刀で1 mm大に細断した。細胞混合液にパパイン(最終濃度2 mg/ml)を添加し、37℃で30分間反応させた。Dnase I(70 U/ml)を添加し30秒間反応させたのち、10% FBS及び1%ペニシリン/ストレプトマイシンを含有したDMEMを添加し反応を停止させた。細胞混合液を800rpmで遠心分離したのち、10% FBS及び1%ペニシリン/ストレプトマイシンを含有したDMEMで再懸濁し、Poly-L lysine(PLL)でコーティングしたdishに播種した。細胞播種4時間後に、培地をNeurobasal 培地(10% B27サプリメント、1%ペニシリン/ストレプトマイシン含有)に交換した。
PLLコーティングした8 well chamber slideで培養した大脳皮質神経(実施例1-1)に20mMのグルタミン酸、10μMのメチルコバラミン(MeCbl)を添加し、18時間後にPromega deadend fluorometric TUNEL systemでアポトーシス細胞割合を評価した。4%パラホルムアルデヒド(PFA)で4℃25分間固定した。0.2% TritonX-100で5分間透過処理を行った後、incubation bufferを添加して37℃遮光下に60分間置き、標識を行った。核はDAPIで標識した。全細胞数、TUNEL陽性細胞数を計測した。
結果を図1に示す。また、図1の数値を表1に表す。TUNELアッセイにおいて、メチルコバラミン単独添加ではアポトーシス細胞割合(%)はコントロールと同様であった。グルタミン酸単独添加ではアポトーシス細胞割合の有意な増加が見られたが、グルタミン酸にメチルコバラミンを併用することにより、コントロールレベルまで有意にアポトーシス細胞割合が減少した。
メチルコバラミンを用いて、大脳皮質神経細胞のネクローシス抑制作用をLDHアッセイで調べた。具体的には以下のとおりである。
PLLコーティングした6 well chamber slideで培養した大脳皮質神経(実施例1-1)に、酸素-グルコース欠乏(OGD)負荷を行う30分前に10μMのメチルコバラミンを添加した。基準となる高コントロールにはN-メチル-Dアスパラギン酸(NMDA)を添加した。培地をearle’s balanced salt solution(EBSS)に交換し、酸素濃度1%の環境下に3時間のOGD負荷を行った。通常の培地、酸素濃度環境下に戻し24時間後に上清を採取、cytotoxicity detection kit plus(SIGMA)を用いてLDH活性を測定した。コントロール群、メチルコバラミン添加群のLDH活性度は、高コントロールのLDH活性に対する割合(%)で算出した。
結果を図2に示す。また、図2の数値を表2に表す。OGD負荷によるLDHアッセイにおいて、ネクローシスの指標となるLDH活性の高コントロールに対しての割合が、メチルコバラミン添加群でコントロール群に比し有意な低下が見られた。
メチルコバラミンを用いて、大脳皮質神経細胞の軸索伸展促進作用を神経突起伸展アッセイで調べた。具体的には以下のとおりである。
大脳皮質神経(実施例1-1)を播種して24時間後に各種薬剤を添加した。添加薬剤濃度はメチルコバラミン(1nM、10nM、100nM、1μM、10μM、100μM)とした。細胞播種72時間後に、抗TuJ1抗体で免疫蛍光染色し、軸索長(the longest neurite length per neuron)を計測した。ただし、別の神経と接していない細胞のみ計測し、各評価において少なくとも30個以上の神経軸索を測定し、その平均値を算出し計測値とした。
結果を図3に示す。また、図3の数値を表3に表す。神経突起伸展アッセイにおいて、10μM濃度をピークに濃度依存性に軸索伸展が促進される傾向を認め、1μM及び10μMにおいては薬剤未添加のコントロール群と比較し、有意差を持って軸索伸展の促進を認めた。
メチルコバラミンを用いて、脳梗塞体積の縮小作用をTTC(2,3,5-Triphenyltetrazolium chloride)染色法で調べた。具体的には以下のとおりである。
8-9週齢の雄のC57BL/6Jマウス(24g前後)を使用した。右頭蓋骨上にレーザードプラ血流計用のプローブを装着し、中大脳動脈の血流をモニタリングできるようにした。右頚部を展開し、外頚動脈を結紮後、総頸動脈に切開を加えナイロン糸を挿入、血流モニタを見ながら先端を進めた。先端が中大脳動脈分岐部まで進み血流が低下したのを確認し、その状態で1時間、直腸温37℃で待機した後、ナイロン糸を抜去し総頚動脈を結紮した。メチルコバラミンは持続投与のため、浸透圧ミニポンプを背部皮下に留置し1mg/kg/dayの用量でモデル完成後に投与した。未治療群では同様の方法で生食を投与した。術後は麻酔から覚醒するまで直腸温37℃で維持した。
術後2日のマウス(実施例4-1)をサクリファイスし、大脳を摘出した。1mm毎に冠状断でスライスし、2% TTC溶液に30分漬けた。実体顕微鏡で撮影した後、それぞれのスライスの梗塞面積を算出、全大脳スライスの梗塞面積を加える事で梗塞体積を算出した。1つのスライスでの梗塞面積は、(健側半球面積-患側健常部面積)で計算した。
結果を図4に示す。また、図4の数値を表4に表す。マウスに対するtMCAO手術後2日に、TTC染色で脳梗塞体積を評価した。メチルコバラミン投与群ではコントロール群に比し、1/2程度の有意な梗塞体積の縮小が見られた。
メチルコバラミンを用いて、M2マクロファージ誘導促進作用及びM1マクロファージ誘導抑制作用を、ウエスタンブロット法及び免疫組織学的評価法により調べた。具体的には以下のとおりである。
マウス由来マクロファージ細胞株J774A.1(JCRB9108)を、大阪府のJCRB細胞バンク(培養資源研究室)より購入した。培養は10% FBS 及び1%ペニシリン/ストレプトマイシンを含有したDMEMで行った。
J774A.1細胞(実施例5-1)を直径6cmのディッシュに播種し、4日後にprotease inhibitor cocktailを溶解したcell lysis bufferを用いてタンパク質を収集した。BCAアッセイでタンパク質濃度を測定した後、サンプル50μgずつをSDS-PAGEで電気泳動し、polyvinylidene difluoride membraneに転写した。Blocking bufferで1時間のblockingを行った後、一次抗体と4℃ over nightで反応させた。二次抗体は室温で1時間反応させ、ECLウェスタンブロッティング検出システムで検出した。M1マーカーであるiNOS及びIL-1βの検出時は、タンパク質収集の24時間前にリポ多糖(LPS)(100ng/ml)及びメチルコバラミンを添加した。M2マーカーであるArg1及びCD206の検出時は、タンパク質収集の72時間前にIL-4(20ng/ml)及びメチルコバラミンを添加した。
J774A.1細胞(実施例5-1)を直径6cmのディッシュに播種し、4日後に4% PFAで20分間固定した。30分間ブロッキングし、一次抗体は4℃ over nightで反応させた。二次抗体は室温で1時間反応させ、核をDAPIで標識した。M1マーカーであるiNOSの検出時は、細胞固定の24時間前にLPS(100ng/ml)及びメチルコバラミンを添加した。M2マーカーであるArg1の検出時は、細胞固定の72時間前にIL-4(20ng/ml)及びメチルコバラミンを添加した。
ウエスタンブロット法の結果を図5及び6に示す。また、図5の数値を表5に、図6の数値を表6に表す。M1マーカーではLPS単独添加に比し、IL-1βは100nM、iNOSは100nMから10μMで有意なタンパク質量の減少が見られた。M2マーカーではIL-4単独添加に比し、Arg1は100nMと1μMで有意なタンパク質量の増加が見られた。CD206では100nMから1μMをピークとした傾向が見られた。
メチルコバラミンによるマクロファージ誘導作用(実施例5)のメカニズムを解析した。具体的には、IL-4、メチルコバラミン(100nM及び1mM)を添加後30分のAkt-mTOR経路(M2遺伝子を誘導する主なシグナル経路の一つ)におけるAkt、4EBP1及びS6Kの活性化をウエスタンブロットで評価した。該経路においては、上流からのシグナルによって、Aktのリン酸化が起こり、さらに下流でmTORC1を介して、4EBP1のリン酸化及びS6Kのリン酸化が起こる。S6Kのリン酸化はシグナル上流にネガティブフィードバック作用をもたらす。ウエスタンブロットは、タンパク質収集の30分前にIL-4(20ng/ml)、メチルコバラミン、RAD001(200nM)を添加し、検出する一次抗体を変える以外は、実施例5-2と同様にして行った。
坐骨神経損傷後のマクロファージ動態にメチルコバラミンが与える影響を免疫組織学的評価法で解析した。具体的には、坐骨神経損傷後1、3、7、14日での近位2.5mm、損傷部、遠位2.5mm、5.0mm、7.5mmの神経横断切片で、蛍光免疫染色でマクロファージを評価した。なお、近位は軸索上の損傷部から細胞体側を意味し、遠位は軸索上の損傷部から軸索末端側を意味し、それぞれの距離は損傷部からの距離を示す(実施例8においても同様である)。マクロファージはCD68、M1マーカーとしてiNOS、M2マーカーとしてCD206で標識した。M1マクロファージ割合(%)=M1マーカー陽性マクロファージ数(個/mm2 )/マクロファージ数(個/mm2)×100で算出した。より具体的には以下のとおりである。
6週齢の雄のWistarラット(200 g前後)を使用した。左坐骨神経を展開し、坐骨神経の近位側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。筋膜及び皮膚を3-0 nylonで縫合した。坐骨神経展開のみを行った非損傷群と未治療群、メチルコバラミン投与群を比較検討した。メチルコバラミンは持続投与のため、浸透圧ミニポンプを背部皮下に留置し1mg/kg/dayの用量で投与した。未治療群では同様の方法で生食を投与した。
術後1日、3日、7日、14日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7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。スライス部位として損傷の近位2.5mm、損傷部位、遠位2.5mm、遠位5.0mm、遠位7.5mmの5箇所で行った。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℃ over nightで反応させた。二次抗体は室温で1時間反応させ、核をDAPIで標識した。
結果を図9及び10に示す。また、図9の数値を表9-1、表9-2、及び表9-3に、図10の数値を表10-1、表10-2、及び表10-3に表す。メチルコバラミン投与群は未治療群と比較して、損傷部においては術後3、7、14日で集積マクロファージ数の有意な減少を認めた。遠位では損傷部に遅れるようにマクロファージ数が増加していったが、術後14日で有意差を認めた。
坐骨神経損傷後の神経再生動態にメチルコバラミンが与える影響を免疫組織学的評価法で解析した。具体的には、坐骨神経損傷2週後の損傷坐骨神経の横断切片を評価した。評価部位はマクロファージ評価と同様に、近位2.5mm、損傷部、遠位2.5mm、5.0mm、7.5mmとした。再生軸索はNF200で標識、髄鞘をMBPで標識した。再生軸索の髄鞘化率を計算するため、髄鞘化率(%)=髄鞘化軸索数(個/mm2)/軸索数(個/mm2)×100として算出した。より具体的には、実施例7と同様の方法で行った。
メチルコバラミンを用いて、脊髄損傷の治療作用をBBB(Basso-Beattie-Bresnahan)スコア、及びThermal algesimetry testにより調べた。具体的には以下のとおりである。
6週齢、メスのWistarラットを使用した。ラットは日本チャールスリバー(横浜市、日本)から購入した。麻酔方法は3種混合麻酔薬を生理食塩水で1:10に希釈して腹腔内注射にて投与した。1回当たりの麻酔投与量はミダゾラム0.2mg/kg、メデトミジン0.015mg/kg、ブトルファノール0.25mg/kgとした。手術台に腹臥位に置き、背部正中を展開した。T10椎弓を切除し脊髄後面を露出させ、スピッツメスで左脊髄を半切した。皮膚を4-0ナイロン糸で縫合し手術を終了した。メチルコバラミン投与群、未治療群およびSham群の3群を比較した。メチルコバラミン投与群、未治療群は術直後に左背部皮下にそれぞれメチルコバラミン(1mg/kg/day)、生理食塩水を充填した浸透圧ミニポンプを留置した。Sham群はTh10椎弓の切除のみを行った。
ラットを個別にケージ内に入れ自由に歩かせ、5分間観察した。定法に従って、左下肢の機能で0点(運動なし)から21点(通常の運動)の間でスコアリングを行った。評価は術前、術後1, 7, 14, 21, 28日に行った。
ラットを個別に専用のケージ内に入れ、右足底に赤外線熱刺激を与え、熱さで後肢を引くまでの時間を測定した。皮膚へのダメージを避けるため、刺激時間は最大15秒とした。評価は術前、術後7, 14, 21, 28日に行った。
BBBスコアを図12に示す。また、図12の数値を表12に表す。メチルコバラミン投与群において未治療群に比し、術後14, 21, 28日目に左下肢運動機能の有意な改善を認めた。
メチルコバラミンを用いて、M2ミクログリア誘導促進作用及びM1ミクログリア誘導抑制作用を、ウエスタンブロット法により調べた。具体的には以下のとおりである。
ミクログリア細胞株(6-3細胞)に対し、LPS(100ng/ml)、抗炎症性サイトカインとしてIL-4(20ng/ml)を添加し、そこへ各種濃度(1nM~1mM)に調整したメチルコバラミンを加え、それぞれ添加後1日、3日でタンパク質を回収した。電気泳動、メンブレンへの転写を行い、ブロッキング後、それぞれM1マーカー(iNOS, IL-1β)、M2マーカー(Arg1, CD206)に対する1次抗体を4℃ over nightで反応させた。二次抗体は室温で1時間反応させ、検出器でバンド検出を行った。
ウエスタンブロット法の結果を図14~15に示す。また、図14の数値を表14に、図15の数値を表15に表す。ミクログリアの炎症性(M1)マーカーではLPS単独添加に比し、IL-1βは1μM、iNOSは10nM以上のメチルコバラミン添加で有意な蛋白質量の減少が見られた。抗炎症性(M2)マーカーではIL-4単独添加に比し、Arg1は1nMから10μMのメチルコバラミン添加で有意な蛋白質量の増加が見られた。CD206では10nMから100nMのメチルコバラミン添加で有意な増加が見られた。
メチルコバラミンを用いて、M2マクロファージ誘導促進作用及びM1マクロファージ誘導抑制作用を調べた。具体的には以下のとおりである。
実施例9-1のラット脊髄損傷モデルについて、術後7、14、28日経過したラットを麻酔薬で鎮静をかけ、4% PFAで灌流固定の後、損傷部を含んだ脊髄を採取して、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、100%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℃ over nightで反応させた。二次抗体は室温で1時間反応させ、核をDAPIで標識した。
免疫蛍光染色の結果を図16~22に示す。図16~18は、単位面積あたりのマクロファージ数、M1(炎症性タイプ)マクロファージ数、M1マクロファージ割合、M2(抗炎症性タイプ)マクロファージ数、M2マクロファージ割合についての部位による変化を表し、図19~20はこれらの術後の経過日数による変化を表す。図21は、M1/M2比についての術後の経過日数による変化を表し、図22はこの部位による変化を表す。また、図16~18の数値を、順に、表16~18に表し、図22の数値を表19に表す。
<実施例12-1.対象と方法>
8-10週齢の雄のC57BL/6Jマウス(24g前後)を使用し、ローズベンガルを投与後、レーザー光を照射することによる光凝固脳梗塞モデルを作製した。このモデルにおいては、マウスの頭蓋骨を大泉門より外側2mmを中心として、頭蓋骨にドリルを用いて穴を開け開頭し、光感受性色素であるローズベンガルの投与5分後に右運動野中心にレーザー光を照射し、右側運動野に脳梗塞を作製する。このように脳梗塞を作製した直後より、浸透圧ポンプ(ALZET Osmotic pumps:2週間用)を埋め込み、メチルコバラミン投与群(N=3)と未治療群(N=4)に分けて、脳梗塞術後、2日後、4日後、7日後、9日後、11日後、14日後にロタロッドテスト(accelerating velocity:加速方式)を施行した。マウスが、ロタロッドから落ちるまでの時間を計測し、Max 300秒をbaselineとして、その比率を算出した。
結果を図23に示す。また、図23の数値を表20に表す。脳梗塞術後2,4,9日後において、メチルコバラミン投与群は未治療群に比して、有意にロタロッドテストによる脳機能改善を認めた。
Claims (18)
- ビタミンB12を含有する、神経系疾患治療剤。
- M2マクロファージ/ミクログリア誘導促進剤である、請求項1に記載の神経系疾患治療剤。
- M1マクロファージ/ミクログリア誘導抑制剤である、請求項1に記載の神経系疾患治療剤。
- 神経再生促進剤である、請求項1~3のいずれかに記載の神経系疾患治療剤。
- 前記神経系疾患が、中枢神経系疾患である、請求項1~4のいずれかに記載の神経系疾患治療剤。
- 前記中枢神経系疾患が、脳血管疾患である、請求項5に記載の神経系疾患治療剤。
- 前記脳血管疾患が、脳梗塞、脳出血、脳血栓症、脳動脈硬化症及び認知症からなる群より選択される少なくとも1種である、請求項6に記載の神経系疾患治療剤。
- 前記神経系疾患が神経損傷である、請求項1~4のいずれかに記載の神経系疾患治療剤。
- 前記神経損傷が中枢神経損傷である、請求項8に記載の神経系疾患治療剤。
- 前記中枢神経損傷が脊髄損傷である、請求項9に記載の神経系疾患治療剤。
- 前記ビタミンB12がメチルコバラミン、シアノコバラミン、ヒドロキソコバラミン、スルフィトコバラミン、アデノシルコバラミン、及びそれらの塩からなる群より選択される少なくとも1種である、請求項1~10のいずれかに記載の神経系疾患治療剤。
- 前記ビタミンB12がメチルコバラミンである、請求項1~11のいずれかに記載の神経系疾患治療剤。
- 持続的に投与して用いられる、請求項1~12のいずれかに記載の神経系疾患治療剤。
- 点滴静注用製剤である、請求項13に記載の神経系疾患治療剤。
- 発症から12~24時間経過以降に投与を開始するように用いられる、請求項1~14のいずれかに記載の神経系疾患治療剤。
- 発症直後から12時間以内に投与を開始するように用いられる、請求項1~14のいずれかに記載の神経系疾患治療剤。
- 請求項1~16のいずれかに記載の神経系疾患治療剤の製造のための、ビタミンB12の使用。
- 神経系疾患の治療における使用のための、ビタミンB12含有組成物。
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CN112826827A (zh) * | 2021-01-24 | 2021-05-25 | 华中科技大学同济医学院附属协和医院 | 甲钴胺在毒物诱导的脊髓变性样疾病中的应用 |
WO2023004235A1 (en) * | 2021-07-23 | 2023-01-26 | University Of Florida Research Foundation, Incorporated | Controlling pro-inflammatory macrophage phenotype through biofunctional hydrogel design |
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JP6708869B2 (ja) | 2020-06-10 |
US11679122B2 (en) | 2023-06-20 |
CN111511373A (zh) | 2020-08-07 |
TW201929871A (zh) | 2019-08-01 |
US20220273692A1 (en) | 2022-09-01 |
KR20200101948A (ko) | 2020-08-28 |
IL275479A (en) | 2020-08-31 |
ZA202004433B (en) | 2022-01-26 |
JP6650650B2 (ja) | 2020-02-19 |
US11369626B2 (en) | 2022-06-28 |
SG11202005822PA (en) | 2020-07-29 |
TWI798320B (zh) | 2023-04-11 |
US20210008092A1 (en) | 2021-01-14 |
EP3730144A4 (en) | 2021-09-01 |
CA3086220A1 (en) | 2019-06-27 |
AU2018390261B2 (en) | 2023-03-16 |
JP2020117529A (ja) | 2020-08-06 |
EP3730144A1 (en) | 2020-10-28 |
AU2018390261A1 (en) | 2020-08-06 |
JP2020075931A (ja) | 2020-05-21 |
US20230302040A1 (en) | 2023-09-28 |
JPWO2019124483A1 (ja) | 2019-12-26 |
JP6763533B2 (ja) | 2020-09-30 |
RU2020123957A (ru) | 2022-01-26 |
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