RU2825666C2 - Therapeutic agent for treating nervous system disease - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to use of methylcobalamin in promoting nerve regeneration and can be used in medicine for treating diseases of the nervous system. Presented is a method involving administering an agent consisting of methylcobalamin as an active ingredient by continuous intravenous drop infusion.

EFFECT: invention provides effective treatment of such nervous system diseases as cerebral infarction or spinal cord injury.

5 cl, 23 dwg, 20 tbl, 12 ex

Description

[Field of technology]

[0001]

The present invention relates to a therapeutic agent for treating diseases of the nervous system or the like.

[State of the art]

[0002]

Diseases of the nervous system occur in the brain, spinal cord, peripheral nerves, and muscles. Diseases that affect the brain and spinal cord are called central nervous system diseases. Common examples of central nervous system diseases that occur in the brain include cerebral infarction and dementia. Common examples of central nervous system diseases that occur in the spinal cord include spinal cord injury.

[0003]

Cerebral infarction accounted for about 60 percent of cerebrovascular accident cases, which was the fourth leading cause of death in 2014. Given that patients are likely to require care after suffering a cerebral infarction, the disease greatly impacts society in terms of health care costs. The areas of cerebral infarction are divided into the ischemic core (also called simply the core), in which blood flow is completely blocked, and the peripheral ischemic penumbra (penumbral zone), in which blood flow is maintained by collateral circulation. Although it is difficult to save the central part, where nerve cells quickly die (primary injury), the penumbral region can survive because the cells remain alive in this part. Thus, the method of saving the penumbral region is a vital point in the treatment of the acute phase of cerebral infarction. Histological changes in the cerebral infarction site include (1) apoptosis of nerve cells, (2) induction of inflammation, and (3) destruction of the blood-brain barrier (BBB). Examples of drugs for the treatment of cerebral infarction currently used in Japan include urokinase, anticoagulants, and antiplatelet agents, as well as blood thinners and edema reducers. Edaravone (trade name Radicut) was approved as a free radical scavenger in Japan in 2001, but has not been approved in Europe, the United States, etc. Thrombolytic therapy (tissue plasminogen activator [tPA] therapy) was approved in 2005, but its use is limited to treatment within 4.5 hours of the onset of the disease. Then, no additional therapeutic agents for cerebral infarction have been available since 2005.

[0004]

It is reported that there are approximately 5,000 new patients with spinal cord injury in Japan every year. Pain, numbness, impaired coordination, etc. are associated with extremely reduced quality of life for patients. The pathogenesis of spinal cord injury involves damage to nerve cells and vascular tissue due to direct external impact during injury (primary injury), followed by a series of reactions involving the destruction of the barrier between the blood and the spinal cord (secondary injury), resulting in the expansion of the lesion area. Since primary injury is inevitable, how to reduce secondary injury is very important in the treatment of acute or subacute phase of spinal cord injury. However, at present, there is no safe and effective therapeutic drug for the treatment of acute phase of spinal cord injury in clinical settings. The treatment guidelines for acute spinal cord injury issued in the United States clearly state that conventional treatments using high doses of methylprednisolone should not be used routinely due to its severe side effects. Thus, there is an unmet medical need for a new therapeutic agent effective in spinal cord injury.

[0005]

Peripheral nerves have the ability to regenerate after injury, but this is not sufficient to restore the functions of the nervous system. When a nerve is injured, Wallerian degeneration occurs, i.e., phagocytic elimination of axons and myelin sheaths. In the subsequent regeneration process, the regenerated axon extends distally along the Büngner band formed by undifferentiated Schwann cells, leading to reinnervation of the target muscle. Eventually, the myelin sheath is formed by Schwann cells that surround the regenerated axon. However, the regenerated nerve extends at a very slow rate, and muscle atrophy occurs if the distance to the target muscle is large; thus, sufficient functional recovery cannot be expected. In recent years, it has been discovered that macrophages play an important role in each step of the regeneration process, and this finding has attracted attention. Although the proinflammatory function of macrophages is well known, there is also another phenotype that has an anti-inflammatory function opposite to it, and these two phenotypes, designated as M1 and M2, respectively, are considered as a continuum (a switch can occur between M1 and M2). In general, it is said that nerve regeneration can be stimulated by an increase in M2 macrophage, which is an anti-inflammatory phenotype.

[0006]

On the other hand, it is known that the central nervous system has a lower regenerative capacity than the peripheral nervous system. It is known that after central nerve injury, axon growth inhibitor is expressed in oligodendrocytes, which are cells that form the myelin sheath around the axon, and that macrophage/microglia, astrocyte, etc. form a glial scar that has an inhibitory effect on axon growth. Therefore, it is said that suppression of the proinflammatory effect of macrophage/microglia is important after central nerve injury, and that nerve regeneration can be promoted by increasing M2 macrophage/microglia, which is an anti-inflammatory phenotype, as in the case of the peripheral nervous system.

[0007]

It is known that vitamin B ₁₂ is effective for the treatment of vitamin B ₁₂ deficiency and that neurological changes such as peripheral neuritis or spinal cord changes can occur in vitamin B ₁₂ deficiency (Patent Document 1).

[0008]

In addition, patients with cerebral ischemia have elevated blood homocysteine levels compared with healthy individuals, indicating a relationship between blood homocysteine levels and cerebral ischemia. Furthermore, blood homocysteine levels were found to be reduced by the administration of folic acid, vitamin B ₆ and vitamin B ₁₂ , suggesting that lowering homocysteine levels may potentially reduce the risk of cerebral ischemia (Non-patent document 1).

[0009]

In addition, it is known that activins have an anti-inflammatory effect by inducing M2 macrophage (Patent Document 2), and that an immunosuppressive agent containing adipose tissue-derived mesenchymal stem cells induces M2 macrophage (Patent Document 3).

[0010]

However, it has not been disclosed that vitamin B ₁₂ promotes the induction of M2 macrophage/microglia, inhibits the induction of M1 macrophage/microglia, and alleviates neurological diseases such as cerebral infarction.

[Prior Art Documents]

[Patent documents]

[0011]

Patent Document 1: Japanese Patent Laid-Open No. 2016-513694

Patent Document 2: International Publication No. WO 2011/149036

Patent Document 3: International Publication No. WO 2011/043136

[Non-patent document]

[0012]

Non-Patent Document 1: Current Medicinal Chemistry, 2007, Vol. 14, No. 3, p. 249-263

[Essence of the invention]

[Problem solved by the invention]

[0013]

The aim of the present invention is to provide a therapeutic agent for treating a disease of the nervous system. Preferably, the aim of the present invention is to provide a therapeutic agent for treating a disease of the nervous system, which has at least one selected from the group consisting of an apoptosis-inhibiting action, a necrosis-inhibiting action, an axonal growth-promoting action, an M2 macrophage/microglia induction-promoting action, an M1 macrophage/microglia induction-inhibiting action, and a nerve regeneration-promoting action.

[Problem Solving Tools]

[0014]

The present inventors conducted extensive studies in view of the above-mentioned problems and found that vitamin B ₁₂ has a therapeutic effect on diseases of the nervous system. In addition, the present inventors also found that vitamin B ₁₂ has an apoptosis-inhibiting action, a necrosis-inhibiting action, an axonal growth-promoting action, an M2 macrophage/microglia-inducing action, an M1 macrophage/microglia-inducing action, a nerve regeneration-promoting action, and the like. The present inventors conducted further studies based on these results and completed the present invention.

[0015]

In particular, the present invention covers the following aspects:

Item 1. A therapeutic agent for the treatment of a disease of the nervous system, including vitamin B ₁₂ .

[0016]

Item 1A. A method of treating a disease of the nervous system comprising administering vitamin B ₁₂ to a patient in need of treatment for the disease of the nervous system.

[0017]

Item 1B1. Vitamin B ₁₂ for use in the treatment of diseases of the nervous system.

[0018]

Item 1B2. A composition comprising vitamin B ₁₂ for use in the treatment of a disease of the nervous system.

[0019]

Item 1C. Use of vitamin B ₁₂ for the production of a therapeutic agent for the treatment of a disease of the nervous system.

[0020]

Item 2. A therapeutic agent for treating a disease of the nervous system according to item 1, wherein the therapeutic agent for treating a disease of the nervous system is an agent that promotes the induction of M2 macrophage/microglia.

[0021]

Item 3. The therapeutic agent for treating a disease of the nervous system according to item 1, wherein the therapeutic agent for treating a disease of the nervous system is an agent that inhibits the induction of M1 macrophage/microglia.

[0022]

Item 4. A therapeutic agent for treating a disease of the nervous system according to any one of items 1-3, wherein the therapeutic agent for treating a disease of the nervous system is an agent that promotes nerve regeneration.

[0023]

Item 5. A therapeutic agent for treating a disease of the nervous system according to any of items 1-4, wherein the disease of the nervous system is a disease of the central nervous system.

[0024]

Item 6. A therapeutic agent for the treatment of a disease of the nervous system according to item 5, wherein the disease of the central nervous system is a cerebrovascular disease.

[0025]

Item 7. A therapeutic agent for treating a disease of the nervous system according to item 6, wherein the cerebrovascular disease is at least one disease selected from the group consisting of cerebral infarction, intracerebral hemorrhage, cerebral thrombosis, cerebral atherosclerosis and dementia.

[0026]

Item 8. A therapeutic agent for treating a disease of the nervous system according to any of items 1-4, wherein the disease of the nervous system is a nerve injury.

[0027]

Item 9. A therapeutic agent for treating a disease of the nervous system according to item 8, wherein the nerve damage is damage to the central nerve.

[0028]

Item 10. A therapeutic agent for the treatment of a disease of the nervous system according to item 9, wherein the damage to the central nerve is a spinal cord injury.

[0029]

Item 11. A therapeutic agent for treating a nervous system disease according to any one of items 1-10, wherein vitamin B ₁₂ is at least one selected from the group consisting of methylcobalamin, cyanocobalamin, hydroxocobalamin, sulfitocobalamin, adenosylcobalamin and salts thereof.

[0030]

Item 12. A therapeutic agent for treating a disease of the nervous system according to any of items 1-11, wherein the vitamin B ₁₂ is methylcobalamin.

[0031]

Item 13. A therapeutic agent for treating a disease of the nervous system according to any one of items 1-12, wherein the therapeutic agent for treating a disease of the nervous system is used for continuous administration.

[0032]

Item 14. A therapeutic agent for treating a disease of the nervous system according to item 13, wherein the therapeutic agent for treating a disease of the nervous system is a dosage form for intravenous drip infusion.

[0033]

Item 15. A therapeutic agent for treating a disease of the nervous system according to any of items 1-14, wherein the therapeutic agent for treating a disease of the nervous system is used in such a way that administration begins 12-24 hours after the onset of the disease.

[0034]

Item 16. A therapeutic agent for treating a disease of the nervous system according to any of items 1-14, wherein the therapeutic agent for treating a disease of the nervous system is used in such a way that administration begins immediately after the onset of the disease or no later than 12 hours after the onset of the disease.

[Brief description of figures]

[0035]

Figure 1 shows the results of the TUNEL assay in Example 1. The vertical axis indicates the proportion of apoptotic cells. The horizontal axis indicates the types of drugs added and whether drugs were added (+) or not (-). Two asterisks “**” indicate that the result of the statistical analysis using the Tukey-Kramer method had a p value less than 0.01.

Fig. 2 shows the results of the lactate dehydrogenase (LDH) assay in Example 2. The vertical axis indicates the LDH activity, which is an indicator of necrosis, as a percentage compared with that of the high control. The horizontal axis indicates the type of drug added and whether the drug (10 μM) was added or not (-). An asterisk "*" indicates that the result of the statistical analysis using Student's t-test had a p value of less than 0.05.

Fig. 3 shows the results of the neurite outgrowth assay in Example 3. The vertical axis indicates the average length of 30 or more neuronal axons. The horizontal axis indicates the concentrations of the added drug. The control (CTR) does not contain the drug. An asterisk "*" indicates that the result of the statistical analysis using Dunnett's test against CTR had a p value of less than 0.05, and "**" indicates that the result had a p value of less than 0.01.

Fig. 4 shows the results of 2,3,5-triphenyltetrazolium chloride (TTC) staining in Example 4. The vertical axis indicates the volume of ischemic lesion. The horizontal axis indicates the type of drug added and whether the drug was added (MeCbl) or not (control). Two asterisks “**” indicate that the result of statistical analysis using Student’s t-test had a p value less than 0.01.

Figure 5 shows the results of Western blot analysis in Example 5. The vertical axes indicate the ratio of the amount of M1 marker (left graph, IL-1β protein; right graph, iNOS protein) to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. The horizontal axes indicate the types of drugs added and whether drugs were added (+ or concentration) or not (-). An asterisk "*" indicates that the statistical analysis result using Dunnett's test had a p value less than 0.05, and "**" indicates that the result had a p value less than 0.01.

Figure 6 shows the results of Western blot analysis in Example 5. The vertical axes indicate the ratio of the amount of M2 marker (left graph, arginase I (Arg1) protein; right graph, CD206 protein) to the amount of GAPDH protein. The horizontal axes indicate the types of drugs added and whether drugs were added (+ or concentration) or not (-). An asterisk "*" indicates that the statistical analysis result using Dunnett's test had a p value less than 0.05, and "**" indicates that the result had a p value less than 0.01.

Fig. 7 shows the results of immunohistological analysis in Example 5. The vertical axes indicate the percentage of M1 macrophages (percentage of iNOS-positive cells) on the left graph and the percentage of M2 macrophages (percentage of Arg1-positive cells) on the right graph. The horizontal axis indicates the types of drugs added and whether drugs were added (+ or concentration) or not (-). Two asterisks “**” indicate that the result of statistical analysis using Dunnett’s test had a p value less than 0.01.

Fig. 8-1 shows the results of Western blot analysis in Example 6. The vertical axis indicates the ratio of the amount of phosphorylated Akt protein to the amount of Akt protein. The horizontal axis indicates the types of drugs added and whether drugs were added (+ or concentration) or not (-). An asterisk "*" indicates that the result of statistical analysis using the Tukey-Kramer method had a p value of less than 0.05, "**" indicates that the result had a p value of less than 0.01, and "***" indicates that the result had a p value of less than 0.001.

Fig. 8-2 shows the results of Western blot analysis in Example 6. The vertical axis indicates the ratio of the amount of phosphorylated 4EBP1 protein to the amount of 4EBP1 protein. The horizontal axis indicates the types of drugs added and whether drugs were added (+ or concentration) or not (-). An asterisk "*" indicates that the result of statistical analysis using the Tukey-Kramer method had a p value of less than 0.05, "**" indicates that the result had a p value of less than 0.01, and "***" indicates that the result had a p value of less than 0.001.

Fig. 8-3 shows the results of Western blot analysis in Example 6. The vertical axis indicates the ratio of the amount of phosphorylated S6K protein to the amount of S6K protein. The horizontal axis indicates the types of drugs added and whether drugs were added (+ or concentration) or not (-). An asterisk "*" indicates that the result of statistical analysis using the Tukey-Kramer method had a p value of less than 0.05, "**" indicates that the result had a p value of less than 0.01, and "***" indicates that the result had a p value of less than 0.001.

Figure 9 shows the results of the immunohistological analysis in Example 7. The graphs in the left row show the results at 2.5 mm proximal to the injury site, the injury site, and 2.5, 5.0, and 7.5 mm distally. The vertical axes indicate the number of macrophages in the upper graphs, the number of M1 macrophages in the middle graphs, and the proportion of M1 macrophages in the lower graphs. The horizontal axes indicate the number of days since the sciatic nerve injury. CTR refers to the untreated group, MeCbl refers to the methylcobalamin treatment group, and Sham refers to the uninjured group that underwent sciatic nerve exteriorization only. An asterisk "*" indicates that the result of statistical analysis using the Tukey-Kramer method (CTR vs. MeCbl) had a p- value less than 0.05, and "#" indicates that the result of statistical analysis using the Student's t-test (CTR vs. MeCbl) had a p- value less than 0.05.

Fig. 10 shows the results of the immunohistological analysis in Example 7. The graphs in the left row show the results at 2.5 mm proximal to the injury site, the injury site, and 2.5, 5.0, and 7.5 mm distally. The vertical axes indicate the number of macrophages in the upper graphs, the number of M2 macrophages in the middle graphs, and the proportion of M2 macrophages in the lower graphs. The horizontal axes indicate the number of days since the sciatic nerve injury. CTR refers to the untreated group, MeCbl refers to the methylcobalamin treatment group, and Sham refers to the uninjured group that underwent sciatic nerve exteriorization only. An asterisk "*" indicates that the result of statistical analysis using the Tukey-Kramer method (CTR vs. MeCbl) had a p- value less than 0.05, and "#" indicates that the result of statistical analysis using the Student's t-test (CTR vs. MeCbl) had a p- value less than 0.05.

Fig. 11 shows the results of immunohistological analysis in Example 8. The vertical axes indicate the number of axons in the upper graph, the number of myelinated axons in the middle graph, and myelination in the lower graph. The horizontal axes indicate the positions at which the nerve cross sections were obtained (left, 2.5 mm proximal to the injury site, injury site, and 2.5, 5.0, and 7.5 mm distal). CTR refers to the untreated group, MeCbl refers to the methylcobalamin treated group, and Sham refers to the uninjured group that underwent sciatic nerve exteriorization only. An asterisk "*" indicates that the result of the statistical analysis using the Tukey-Kramer method (CTR vs. MeCbl) had a p value less than 0.05.

Fig. 12 shows the measurement results of the BBB indices in Example 9. The vertical axis indicates the BBB index. The horizontal axis indicates the number of days passed after the spinal cord injury model operation, where 0 indicates before the operation. CTR refers to the untreated group, MeCbl refers to the methylcobalamin treatment group, and Sham refers to the no-injury group that only underwent sciatic nerve exteriorization. An asterisk "*" indicates that the statistical analysis result using the Steele-Dwass test (CTR vs. MeCbl) had a p value of less than 0.05.

Fig. 13 shows the results of thermal algesimetry in Example 9. The vertical axis indicates the time elapsed after the infrared irradiation of the right sole of the hind limb of the rat model until the rat withdrew the limb due to the applied heat. The horizontal axis indicates the number of days elapsed after the operation to create the spinal cord injury model, where 0 means before the operation. CTR refers to the untreated group, MeCbl refers to the methylcobalamin treatment group, and Sham refers to the no-injury group that only underwent sciatic nerve exteriorization. The asterisk "*" indicates that the result of the statistical analysis using the Steele-Dwass test (CTR vs. MeCbl) had a p value of less than 0.05.

Fig. 14 shows the results of Western blot analysis of M1 markers (IL-1β protein and iNOS protein) in Example 10. The vertical axis indicates the ratio of the amount of M1 marker (IL-1β protein or iNOS protein) to the amount of GAPDH protein. The horizontal axis indicates the types of drugs added and whether the drugs were added (+ or concentration) or not (-). An asterisk "*" indicates that the result of statistical analysis using Dunnett's test had a p value of less than 0.05, and "**" indicates that the result had a p value of less than 0.01.

Fig. 15 shows the results of Western blotting of M2 markers (Arg1 protein and CD206 protein) in Example 10. The vertical axes, horizontal axes and each symbol have the same meaning as described for Fig. 14.

Fig. 16 shows the results of immunofluorescence staining 7 days after the operation in Example 11. Each graph shows the difference according to the position. The horizontal axis in each graph indicates the direction and distance from the lesion site. The vertical axes indicate the number of macrophages in the upper graphs, the number of M1 or M2 macrophages in the middle graphs, and the proportion of M1 or M2 macrophages in the lower graph. CTR refers to the untreated group, and MeCbl refers to the methylcobalamin treatment group. The asterisk "*" means that the statistical analysis result using the Mann-Whitney U test (CTR vs. MeCbl) had a p value of less than 0.05, and "**" means that the result had a p value of less than 0.01.

Fig. 17 shows the results of immunofluorescence staining 14 days after the operation in Example 11 and is otherwise the same as in Fig. 16.

Fig. 18 shows the results of immunofluorescence staining 28 days after the operation in Example 11 and is otherwise the same as in Fig. 16.

Fig. 19 shows the results of immunofluorescence staining of M1 macrophages in Example 11. Each graph shows the difference according to the number of days passed after the operation. The graphs in each row show the results according to the direction and distance from the injury site: 2 and 1 mm from the head side and 1 and 2 mm from the tail side. The vertical axes indicate the number of macrophages in the upper graphs, the number of M1 macrophages in the middle graphs, and the proportion of M1 macrophages in the lower graph. The horizontal axes indicate the number of days passed after the operation to create the spinal cord injury model. CTR refers to the untreated group, and MeCbl refers to the methylcobalamin treatment group. The asterisk "*" means that the result of the statistical analysis using the Mann-Whitney U test (CTR vs. MeCbl) had a p value less than 0.05, and "**" means that the result had a p value less than 0.01.

Fig. 20 shows the results of immunofluorescence staining of M2 macrophages in Example 11 and is otherwise the same as in Fig. 19.

Fig. 21 shows the results of immunofluorescence staining for the M1/M2 ratio in Example 11. Each graph shows the difference according to the number of days passed after the surgery. The graphs show the results according to the direction and distance from the injury site: 2 and 1 mm from the head side and 1 and 2 mm from the tail side. The vertical axes indicate the M1/M2 ratio. The horizontal axes indicate the number of days passed after the surgery to create the spinal cord injury model. CTR refers to the untreated group, and MeCbl refers to the methylcobalamin treatment group. The asterisk "*" means that the statistical analysis result using the Mann-Whitney U test (CTR vs. MeCbl) had a p value of less than 0.05, and "**" means that the result had a p value of less than 0.01.

Fig. 22 shows the results of immunofluorescence staining for the M1/M2 ratio in Example 11. Each graph shows the difference depending on the position. The graphs on the left show the results at 7, 14, and 28 days after the spinal cord injury model surgery. The vertical axes indicate the M1/M2 ratio. The horizontal axes indicate the number of days after the spinal cord injury model surgery. The horizontal axes indicate the direction and distance from the injury site. CTR refers to the untreated group, and MeCbl refers to the methylcobalamin treatment group. The asterisk "*" means that the statistical analysis result using the Mann-Whitney U test (CTR vs. MeCbl) had a p value of less than 0.05, and "**" means that the result had a p value of less than 0.01.

Fig. 23 shows the results of the rotarod test in Example 12. The horizontal axis indicates the number of days elapsed after the operation that caused the cerebral infarction, and the vertical axis indicates the relative time until the mouse model fell off the rotarod. CTR refers to the untreated group, and MeCbl refers to the methylcobalamin treatment group. Two asterisks “**” indicate that the result of the statistical analysis using the Mann-Whitney U test (CTR vs. MeCbl) had a p value of less than 0.01.

[Method of carrying out the invention]

[0036]

As used herein, the terms "comprising" and "including" include the terms "comprising," "including," "substantially comprising," and "consisting of."

[0037]

As used herein, the term "macrophage/microglia" refers to "macrophages and/or microglia" and encompasses both the meaning of "macrophages and microglia" and the meaning of "macrophages or microglia".

[0038]

In one aspect, the present invention relates to a therapeutic agent for treating diseases of the nervous system, an apoptosis inhibiting agent, a necrosis inhibiting agent, an axon growth promoting agent, an M2 macrophage/microglia induction promoting agent, an M1 macrophage/microglia induction inhibiting agent, a nerve regeneration promoting agent, or the like, which includes vitamin B ₁₂ (hereinafter, may also be referred to as the "agent of the present invention"). These agents will be described below.

[0039]

1. Active ingredient

Vitamin B ₁₂ includes cobalamin, its derivatives, and salts thereof. Specific examples of vitamin B ₁₂ include cobalamin, the cobalt substitution product of cobalamin, and its derivatives. More specific examples include methylcobalamin, cyanocobalamin, hydroxocobalamin, sulfitocobalamin, adenosylcobalamin, and their salts. Among them, methylcobalamin, cyanocobalamin, hydroxocobalamin, and their salts are preferred, and methylcobalamin and its salts are more preferred.

[0040]

Cobalamin salts and derivatives thereof are not particularly limited, provided that the salts are pharmacologically acceptable, and both acidic salts and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochlorides, hydrobromides, sulfates, nitrates and phosphates; organic acid salts such as acetates, propionates, tartrates, fumarates, maleates, malates, citrates, methanesulfonates and p-toluenesulfonates; and amino acid salts such as aspartates and glutamates. Examples of basic salts include alkali metal salts such as sodium salts and potassium salts; and alkaline earth metal salts such as calcium salts and magnesium salts.

[0041]

Vitamin B ₁₂ may be in the form of solvates. Solvents are not particularly limited as long as they are pharmacologically acceptable, and examples include water, ethanol, glycerol, and acetic acid.

[0042]

As vitamin B _12, one type can be used alone or two or more types can be used in combination.

[0043]

2. Application

Vitamin B ₁₂ has a therapeutic effect on diseases of the nervous system. Therefore, vitamin B ₁₂ can be used as an active ingredient in a therapeutic agent for diseases of the nervous system.

[0044]

Vitamin B ₁₂ has an apoptosis inhibiting effect, a necrosis inhibiting effect, an axonal growth promoting effect, an M2 macrophage/microglia induction promoting effect, an M1 macrophage/microglia induction inhibiting effect, a nerve regeneration promoting effect, and the like. Therefore, vitamin B ₁₂ can be used as an active ingredient of an agent such as an apoptosis inhibiting agent, a necrosis inhibiting agent, an axonal growth promoting agent, an M2 macrophage/microglia induction promoting agent, an M1 macrophage/microglia induction inhibiting agent, a nerve regeneration promoting agent, an M1:M2 ratio restoring agent (the ratio of M1 macrophage/microglia to M2 macrophage/microglia), or a nerve regeneration promoting agent.

[0045]

In addition, vitamin B ₁₂ can be used as an active ingredient in a preferred form of a therapeutic agent for treating diseases of the nervous system, which is an active ingredient in a therapeutic agent for treating diseases of the nervous system based on at least one selected from the group consisting of an apoptosis inhibiting action, a necrosis inhibiting action, an axon growth promoting action, an M2 macrophage/microglia induction promoting action, an M1 macrophage/microglia induction inhibiting action, and a nerve regeneration promoting action.

[0046]

The nervous system disease is not specifically limited and includes central nervous system disease and peripheral nervous system disease. Examples of central nervous system disease include cerebrovascular disease and central nervous system injury.

[0047]

Examples of cerebrovascular disease include cerebral infarction, intracerebral hemorrhage, cerebral thrombosis, cerebral atherosclerosis, and dementia.

[0048]

Nerve injury may be peripheral nerve injury or central nerve injury. Central nerve injury includes spinal cord injury. The causes of nerve injury are not particularly limited, and nerve injury due to various causes such as traumatic injury, pressure caused by a plaster cast, electrical injury, disc herniation, or radiation may be involved. The severity of the corresponding nerve injury is not particularly limited, and the corresponding cases include all cases, including the case where the axons are preserved but demyelination has occurred, the case where Wallerian degeneration is present, and the case where the nerves are separated anatomically. Nerve injury covers various symptoms associated with nerve injury, including, for example, dyskinesia (eg, motor paralysis and muscle weakness in the upper and lower limbs), sensory disorder (eg, hypoesthesia, numbness and pain), autonomic nervous system disorder (eg, dyshidrosis, skin color change) or the like in the damaged innervation area.

[0049]

The disease of the nervous system is preferably a disease of the nervous system that can be treated by at least one selected from the group consisting of an apoptosis-inhibiting action, a necrosis-inhibiting action, an axonal growth-promoting action, an M2 macrophage/microglia induction-promoting action, an M1 macrophage/microglia induction-inhibiting action, and a nerve regeneration-promoting action.

[0050]

The agent of the present invention is not particularly limited, as long as the agent contains vitamin B ₁₂ (used herein, may be referred to simply as "active ingredient") and may contain other ingredients as needed. Other ingredients are not limited as long as they are pharmacologically acceptable. Other ingredients include additives in addition to the ingredient having a pharmacological effect. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, flavors, and chelating agents.

[0051]

Vitamin _B12 used alone can exert a therapeutic effect on diseases of the nervous system, an apoptosis inhibiting effect, a necrosis inhibiting effect, an axon growth promoting effect, an M2 macrophage/microglia induction promoting effect, an M1 macrophage/microglia induction inhibiting effect (herein, the effect of switching the M1 macrophage/microglia to the M2 macrophage/microglia is not excluded), a nerve regeneration promoting effect, and the like. Therefore, the agent of the present invention can exert the desired effect without containing other ingredients having these effects and/or actions. However, the agent of the present invention may contain other ingredients having a pharmacological effect.

[0052]

The application methods for the agent of the present invention are not particularly limited, and a suitable application method can be selected according to the type of the agent. The agent of the present invention can be used in vitro (for example, added to the medium of cultured cells) or in vivo (for example, administered to animals), for example, depending on the purpose.

[0053]

The targets for using the agent of the present invention are not particularly limited. Examples of target mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats and deer. Examples of target cells include animal cells. Cell types are also not particularly limited, and examples include blood cells, hematopoietic stem cells, progenitor cells, gametes (sperm and egg), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, liver cells, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells and cancer cells.

[0054]

The agent of the present invention may be in any dosage form. Examples of the dosage form include oral dosage forms such as tablets (including orodispersible tablets, chewable tablets, foam tablets, lozenges and gelatin drop formulations), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrup preparations, liquids (including drinkable dosage forms, suspensions and syrups) and jelly dosage forms; and parenteral dosage forms such as injectable formulations (e.g., drip infusion [e.g., intravenous drip infusion formulations], intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical formulations (e.g., ointments, patches, and lotions), suppositories, inhalation formulations, ophthalmic formulations, ophthalmic ointments, nasal drops, ear drops, and liposomal dosage forms.

[0055]

The administration methods of the agent of the present invention are not particularly limited as long as the desired effect can be achieved, and examples include oral administration and parenteral administration including enteral administration such as administration by feeding tube and enema, intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration and intraperitoneal administration.

[0056]

The content of the active ingredient in the agent of the present invention is not limited and varies depending on the method of use, the purpose of use, the conditions of the purpose of use, etc. For example, the content may be from 0.0001 to 100% by mass, and preferably from 0.001 to 50% by mass.

[0057]

The dose of the agent of the present invention for administration to animals is not particularly limited as long as the dose is an effective dose that gives a therapeutic effect. The usual dose (weight of the active ingredient) is 0.1 to 1000 mg/kg body weight per day, and preferably 0.5 to 500 mg/kg body weight per day for oral administration; and 0.01 to 100 mg/kg body weight per day, and preferably 0.05 to 50 mg/kg body weight per day for parenteral administration. The above-described doses can be appropriately adjusted depending on age, pathological conditions and symptoms.

[0058]

The agent of the present invention is preferably used by continuous administration from the viewpoint of further effectively achieving an action promoting the induction of M2 macrophage/microglia, an action inhibiting the induction of M1 macrophage/microglia, a nerve regeneration promoting action or the like. With continuous administration, the concentration of the active ingredient that acts on the target cells for administration (for example, cells in the affected site of a nervous system disease, preferably macrophage/microglia) can be maintained within the concentration range suitable for achieving an action promoting the induction of M2 macrophage/microglia, an action inhibiting the induction of M1 macrophage/microglia, and a nerve regeneration promoting action (for example, 5-100 μM, preferably 10-50 μM, more preferably 20-10 μM, still more preferably 50-5 μM, and most preferably 100-1 μM), and the effects can be achieved more effectively. In addition, the agent of the present invention is preferably a drip infusion and more preferably a dosage form for intravenous drip infusion, although depending on the purpose of use.

[0059]

The time of administration of the agent according to the present invention is not particularly limited.

[0060]

As an example, the agent of the present invention is used in such a way that the administration of the agent is started 12 to 24 hours after the onset of the disease. The onset is the time when the symptom of the disease or the factor directly causing the disease can be recognized. For example, the onset is the time when ischemic injury develops in an ischemic cerebrovascular disease such as cerebral infarction. Since the agent of the present invention can act on the repair mechanism that can act relatively long after the development of ischemic injury (an action promoting the induction of macrophage/microglia M2, an action inhibiting the induction of macrophage/microglia M1, and an action promoting nerve regeneration), therapeutic effects can be achieved even if the agent is administered at the above-described time (12 to 24 hours after the onset) when administering the agent for an ischemic cerebrovascular disease such as cerebral infarction.

[0061]

As another example, the agent of the present invention is used such that the administration of the agent is started during the acute phase (for example, immediately after the onset or within 12 hours after the onset) of a nervous system disease, such as a nerve injury (preferably a spinal cord injury). The onset is the time when a symptom of the disease or a factor directly causing the disease can be recognized. For example, in the case of nerve injury, such as a spinal cord injury, the onset is the time when the nerve is damaged.

[Examples]

[0062]

The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0063]

Example 1. Apoptosis inhibitory effect on cerebral cortical neurons

The apoptosis inhibitory effect of methylcobalamin on cerebral cortical neurons was investigated using TUNEL analysis. Specifically, the study was carried out as follows.

[0064]

<Example 1-1. Obtaining cerebral cortical neurons>

Cerebral cortical neurons were collected and cultured routinely. The cerebral cortex was dissected from fetal Sprague-Dawley (SD) rats (gestation day 18) and placed in ice-cold Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The pia mater and blood vessels were removed, and the remaining cerebral cortex was transferred to DMEM (containing 1% penicillin/streptomycin and without FBS) and cut into small 1 mm pieces with surgical scissors. Papain (final concentration, 2 mg/ml) was added to the cell mixture, and the mixture was allowed to react at 37°C for 30 min. DNase I (70 U/ml) was added to the reaction mixture and the reaction was allowed to proceed for 30 seconds. Then, DMEM containing 10% FBS and 1% penicillin/streptomycin was added to stop the reaction. The cell mixture was centrifuged at 800 rpm, the pellet was resuspended in DMEM containing 10% FBS and 1% penicillin/streptomycin, and the suspension was plated on a poly-L-lysine (PLL)-coated dish. Four hours after cell seeding, the medium was replaced with Neurobasal medium (containing 10% B27 supplement and 1% penicillin/streptomycin).

[0065]

<Example 1-2. TUNEL analysis>

Cerebral cortical neurons (Sample 1-1) cultured on an 8-well PLL-coated slide were supplemented with 20 mM glutamic acid and 10 μM methylcobalamin (MeCbl). After 18 h, the proportion of apoptotic cells was assessed using Promega's DeadEnd Fluorometric TUNEL System. Cells were fixed with 4% paraformaldehyde (PFA) at 4°C for 25 min. After cells were permeabilized with 0.2% Triton X-100 for 5 min, incubation buffer was added and the mixture was left to stand at 37°C protected from light for 60 min to label permeabilized cells. Nuclei were labeled with 4'6-diamidino-2-phenylindole (DAPI). The total number of cells and the number of TUNEL-positive cells were determined.

[0066]

<Example 1-3. Results>

The results are shown in Fig. 1. The values corresponding to the data in the graph of Fig. 1 are shown in Table 1. In the TUNEL assay, when methylcobalamin alone was added, the proportion of apoptotic cells (%) was similar to that of the control. When glutamic acid alone was added, the proportion of apoptotic cells increased significantly. However, when methylcobalamin was added together with glutamic acid, the proportion of apoptotic cells decreased significantly to the level of the control.

[0067]

[Table 1]

Glutamate - - + + MeCbl - + - + Average 6.0 5.9 15.0 5.6 Standard error 1,2 1.3 1.3 0.6 Standard deviation 2.0 2,2 2,2 1,1

[0068]

Example 2. Necrosis inhibitory effect on cerebral cortical neurons

The necrosis inhibitory effect of methylcobalamin on cerebral cortical neurons was assessed using the LDH assay. Specifically, the study was performed as follows.

[0069]

<Example 2-1. LDH analysis>

Cerebral cortical neurons (Sample 1-1) cultured on a 6-well PLL-coated slide were supplemented with 10 μM methylcobalamin 30 min before exposure to oxygen-glucose deprivation (OGD) stress. N-methyl-D-aspartic acid (NMDA) was added to the high control as a reference. The medium was replaced with Earle's balanced salt solution (EBSS), and the neurons were exposed to OGD stress in an atmosphere of 1% oxygen for 3 h. The medium was replaced with Earle's balanced salt solution (EBSS), and the neurons were exposed to OGD stress in an atmosphere of 1% oxygen for 3 h. The neurons were returned to normal medium and an atmosphere of normal oxygen. After 24 hours, the supernatant was collected and the LDH activity was measured using the Cytotoxicity Detection Kit ^PLUS (SIGMA). The LDH activity in the control group and the methylcobalamin supplementation group was calculated as the proportion (%) relative to the LDH activity in the high control group.

[0070]

<Example 2-2. Results>

The results are shown in Fig. 2. The values corresponding to the data in the graph of Fig. 2 are shown in Table 2. In the LDH assay under OGD stress, the proportion of LDH activity in the methylcobalamin supplemented group relative to the high control was significantly reduced compared with that in the control group.

[0071]

[Table 2]

MeCbl(-) MeCbl(+) Average 3.1 1.3 Standard error 0.6 0.5 Standard deviation 1.0 0.8

[0072]

Example 3. Axon growth promoting effect on cerebral cortical neurons

The axon growth-promoting effect of methylcobalamin on cerebral cortical neurons was investigated using a neurite outgrowth assay. Specifically, the study was performed as follows.

[0073]

<Example 3-1. Neurite extension analysis>

Cerebral cortical neurons (Example 1-1) were seeded and the drug was added at different concentrations after 24 hours. The concentrations of added methylcobalamin were 1, 10, and 100 nM and 1, 10, and 100 μM. Immunofluorescence staining was performed using anti-TuJ1 antibody 72 hours after cell seeding and the axon length (the longest neurite length per neuron) was measured. The measurement was performed on cells that were not in contact with other cells. At least 30 neuronal axons were measured for each assessment and the average of the obtained values was calculated and designated as the measured value.

[0074]

<Example 3-2. Results>

The results are shown in Fig. 3. The values corresponding to the data in the graph of Fig. 3 are given in Table 3. The results of the neurite outgrowth assay showed concentration-dependent trends in promoting axonal outgrowth with a peak at 10 μM methylcobalamin. At concentrations of 1 and 10 μM, axonal outgrowth was significantly increased compared to the drug-free control group.

[0075]

[Table 3]

[0076]

Example 4. The effect of reducing the volume of ischemic brain damage

The effect of methylcobalamin on reducing the volume of ischemic brain damage was investigated using the 2,3,5-triphenyltetrazolium chloride (TTC) staining method. Specifically, the study was conducted as follows.

[0077]

<Example 4-1. Construction of transient middle cerebral artery occlusion (tMCAO) model and drug administration>

Male C57BL/6J mice aged 8 to 9 weeks (approximately 24 g) were used. The probe of the laser Doppler volumetric blood flow meter was placed on the right side of the skull so that the blood flow in the middle cerebral artery could be monitored. The right neck region was cut and exposed, and the external carotid artery was ligated. Then, the common carotid artery was cut and a nylon thread was inserted into the common carotid artery and the tip was advanced while the blood flow was monitored with a blood flow monitor. When the tip reached the bifurcation of the middle cerebral artery and a decrease in blood flow was observed, this condition was maintained for 1 hour at a rectal temperature of 37°C. Then, the nylon thread was removed and the common carotid artery was ligated. For continuous administration of methylcobalamin, an osmotic minipump was placed and left in the dorsal subcutaneous space. After the model was established, methylcobalamin was administered at a dose of 1 mg/kg/day. In the untreated group, saline was administered instead of methylcobalamin according to the same procedure. After the operation, the temperature was maintained at a rectal temperature of 37°C until the mouse recovered from anesthesia.

[0078]

<Example 4-2. TTS staining method>

Two days after the operation, the mouse (Example 4-1) was sacrificed and the brain was removed. The brain was cut at 1 mm intervals to obtain coronal sections, and the cut sections were immersed in 2% TTS solution for 30 minutes. Images were obtained using a stereoscopic microscope, the area of ischemic damage in each section was calculated, and all the areas of ischemic damage in the brain sections were summed to calculate the volume of ischemic damage. The area of ischemic damage in each section was calculated using the following formula: area of the unaffected hemisphere - area of the unaffected part of the affected side.

[0079]

<Example 4-3. Results>

The results are shown in Fig. 4. The values corresponding to the data in the graph of Fig. 4 are shown in Table 4. Two days after the tMCAO operation, the volume of ischemic brain lesion was evaluated using TTC staining. In the methylcobalamin-treated group, the volume of ischemic lesion was significantly reduced by about 1/2 compared with the control group.

[0080]

[Table 4]

Control MeCbl Average 59.1 28.8 Standard error 8.0 3.2 Standard deviation 22.6 9.0

[0081]

Example 5. Action promoting the induction of M2 macrophages and action inhibiting the induction of M1 macrophages

The M2 macrophage induction promoting effect and M1 macrophage induction inhibiting effect of methylcobalamin were examined by Western blotting and immunohistological analysis. Specifically, the study was conducted as follows.

[0082]

<Example 5-1. Preparation of macrophage cell line>

Murine macrophage cell line J774A.1 (JCRB9108) was purchased from JCRB Cell Bank (Laboratory of Cell Cultures), Osaka, Japan. Cells were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin.

[0083]

<Example 5-2. Western blot>

J774A.1 cells (example 5-1) were seeded in a 6 cm dish. After 4 days, proteins were harvested using cell lysis buffer containing protease inhibitor cocktail. Protein concentration was measured by BCA assay. Then, 50 μg of each sample was subjected to SDS-PAGE and electrophoretic proteins were transferred to polyvinylidene difluoride membrane. The membrane was blocked with blocking buffer for 1 h and then the proteins were reacted with primary antibodies at 4°C overnight. The proteins were then reacted with secondary antibody at room temperature for 1 h and detected using the ECL Western Blotting Detection System. For detection of M1 markers iNOS and IL-1β, lipopolysaccharide (LPS) (100 ng/mL) and methylcobalamin were added 24 h before protein harvesting. For detection of M2 markers Arg1 and CD206, IL-4 (20 ng/ml) and methylcobalamin were added 72 h before protein collection.

[0084]

The primary antibodies used were rabbit polyclonal anti-IL-1 (Santa Cruz), rabbit monoclonal anti-iNOS (Abcam), rabbit polyclonal anti-Arg1 (Santa Cruz), and rabbit monoclonal anti-CD206 (Abcam). The secondary antibody was donkey anti-rabbit IgG, horseradish peroxidase-coupled whole antibody (GE Healthcare Life Sciences).

[0085]

<Example 5-3. Immunohistological analysis method>

J774A.1 cells (example 5-1) were seeded in a 6 cm dish. After 4 days, the cells were fixed with 4% PFA for 20 min. Blocking was performed for 30 min and the cells were allowed to react with primary antibodies at 4°C overnight. The cells were then allowed to react with secondary antibody at room temperature for 1 h so that the nuclei were labeled with DAPI. For detection of the M1 marker iNOS, LPS (100 ng/ml) and methylcobalamin were added 24 h before cell fixation. For detection of the M2 marker Arg1, IL-4 (20 ng/ml) and methylcobalamin were added 72 h before cell fixation.

[0086]

The primary antibodies used were a rabbit monoclonal antibody against iNOS (Abcam) and a rabbit polyclonal antibody against Arg1 (Santa Cruz). The secondary antibodies were a goat anti-rabbit IgG antibody labeled with Alexa 488 (Life Technologies) or a goat anti-rabbit IgG antibody labeled with Alexa 568 (Life Technologies).

[0087]

<Example 5-4. Results>

The results of Western blotting are shown in Fig. 5 and 6. The values corresponding to the data in the graphs in Fig. 5 are shown in Table 5, and the values corresponding to the data in the graphs in Fig. 6 are shown in Table 6. The levels of M1 marker were as follows: compared with LPS alone, IL-1β level was significantly decreased by the addition of 100 nM methylcobalamin in combination, and iNOS level was significantly decreased by the addition of 100 nM to 10 μM methylcobalamin in combination. Compared with IL-4 alone, M2 marker Arg1 level was significantly increased by the addition of 100 nM and 1 μM methylcobalamin in combination. CD206 levels peaked with the addition of methylcobalamin at 100 nM to 1 μM.

[0088]

[Table 5]

[0089]

[Table 6]

[0090]

The results of the immunohistological analysis are shown in Fig. 7. The values corresponding to the data in the graphs in Fig. 7 are shown in Table 7. Compared with LPS alone, the proportion of cells positive for the M1 marker iNOS was significantly reduced by the addition of 10 nM to 100 μM methylcobalamin in combination. Compared with the addition of IL-4 alone, the proportion of cells positive for the M2 marker Arg1 was significantly increased by the addition of 10 nM to 1 μM methylcobalamin in combination.

[0091]

Immunofluorescence staining showed a transition from M1 to M2 with a peak at approximately 100 nM methylcobalamin, as in the Western blot described above.

[0092]

[Table 7]

[0093]

Example 6. Analysis of the mechanism of action of macrophage induction

The mechanism of macrophage induction (example 5) by methylcobalamin was analyzed. Specifically, the analysis was performed as follows. Thirty minutes after the addition of IL-4 and methylcobalamin (100 nM and 1 mM), the activation of Akt, 4EBP1, and S6K in the Akt-mTOR pathway (one of the main signaling pathways that induces M2 gene expression) was assessed using Western blotting. In this pathway, Akt phosphorylation occurs in response to an upstream signal, and 4EBP1 phosphorylation and S6K phosphorylation then occur via mTORC1 in a downstream direction. S6K phosphorylation provides negative feedback to the upstream signaling pathway. Western blotting was performed as in Example 5-2, except that IL-4 (20 ng/ml), methylcobalamin, and RAD001 (200 nM) were added 30 min before protein collection and the primary antibodies for detection were changed.

[0094]

The results are shown in Figs. 8-1, 8-2, and 8-3. The corresponding values to the data in the graphs in Figs. 8-1, 8-2, and 8-3 are shown in Table 8. When IL-4 was added, activation of Akt and both 4EBP1 and S6K in the downstream direction were observed. Compared with IL-4 alone, activation of 4EBP1 and activation of S6K were enhanced when IL-4 and 100 nM methylcobalamin were used in combination. However, when the concentration of methylcobalamin was 1 mM, the upstream activity of Akt was decreased although 4EBP1 and S6K were activated. When RAD001, which is an mTOR inhibitor, was further added in addition to IL-4 and methylcobalamin, the upstream activity of Akt was restored and the downstream activity of 4EBP1 and S6K was weakened. Thus, a mechanism was proposed in which the upstream activity of Akt in inducing the M2 gene was suppressed by a downstream negative feedback mechanism when high concentrations of methylcobalamin were added.

[0095]

[Table 8]

[0096]

Example 7. Analysis of macrophage phenotypes after sciatic nerve injury

The effects of methylcobalamin on the phenotype of macrophages after sciatic nerve injury were analyzed by immunohistological analysis. Specifically, the analysis was performed as follows. Using transverse sections of the nerve at a distance of 2.5 mm proximally, at the injury site, and at a distance of 2.5, 5.0, and 7.5 mm distally, macrophages were assessed by immunofluorescence staining at 1, 3, 7, and 14 days after sciatic nerve injury. The proximal direction refers to the side of the axon cell body relative to the injury site, and the distal direction refers to the terminal side of the axon relative to the injury site. Each of the distances indicates the distance from the injury site (the same applies to example 8). Macrophages were labeled with CD68, the M1 marker iNOS, and the M2 marker CD206. The proportion of M1 macrophages was calculated using the following formula: proportion of M1 macrophages (%) = number of M1 marker-positive macrophages per ^mm2 / number of macrophages per ^mm2 × 100. A more detailed description will be provided below.

[0097]

<Example 7-1. Surgical treatment (sciatic nerve injury model in rats)>

Six-week-old male Wistar rats (about 200 g) were used. The left sciatic nerve was exposed, and crush injury of the sciatic nerve was performed on the proximal side using forceps. The nerve was crushed for 10 seconds three times at 10-second intervals. The fascia and skin were sutured using 3-0 nylon. A no-injury group, which underwent only sciatic nerve exposure, an untreated group, and a methylcobalamin-treated group were compared. For continuous administration of methylcobalamin, an osmotic minipump was placed and left in the dorsal subcutaneous space, and methylcobalamin was administered at a dose of 1 mg/kg/day. In the untreated group, saline was administered instead of methylcobalamin by the same method.

[0098]

<Example 7-2. Morphological and histological analysis>

At 1, 3, 7, and 14 days after surgery, rats were anesthetized with anesthetic. The left sciatic nerve was collected, fixed with 4% PFA for 7 days and 20% sucrose for 24 hours, and the fixed nerve was frozen. The embedded tissue was cut into a 5 μm thick section in the short axis direction of the nerve and placed on a glass slide. Sections were prepared from 5 positions 2.5 mm proximal to the lesioned area, the injured area, and 2.5, 5.0, and 7.5 mm distal. Sections were dried for 1 hour and fixed with 95% methanol for 30 minutes. Fixed sections were blocked and allowed to react with primary antibodies at 4°C overnight. Then, cells were allowed to react with secondary antibody at room temperature for 1 hour to label the nuclei with DAPI.

[0099]

Mouse monoclonal antibody against CD68 (Abcam), rabbit monoclonal antibody against iNOS (Abcam), rabbit monoclonal antibody against CD206 (Abcam), rabbit polyclonal antibody against neurofilament 200 (NF 200) (SIGMA), and mouse monoclonal antibody against myelin basic protein (MBP) (Calbiochem) were used as primary antibodies. Alexa 488-labeled goat anti-mouse IgG (Life Technologies), Alexa 488-labeled goat anti-rabbit IgG (Life Technologies), Alexa 568-labeled goat anti-mouse IgG (Life Technologies), and Alexa 568-labeled goat anti-rabbit IgG (Life Technologies) were used as secondary antibodies.

[0100]

<Example 7-3. Results>

The results are shown in Figs. 9 and 10. The values corresponding to the data in the graphs in Fig. 9 are shown in Tables 9-1, 9-2, and 9-3, and the values corresponding to the data in the graphs in Fig. 10 are shown in Tables 10-1, 10-2, and 10-3. In the methylcobalamin-treated group, the number of macrophages accumulated at the injury site was significantly reduced at 3, 7, and 14 days after the operation compared with the untreated group. At the distal positions, the number of macrophages was increased behind the injury site, and there was a significant difference at 14 days after the operation.

[0101]

In the methylcobalamin-treated group, the number of M1 macrophages was significantly reduced on all evaluation days. In the distal regions, there were significant differences at 7 and 14 days after surgery. Similar trends were observed for the proportions of M1 macrophages.

[0102]

In the methylcobalamin-treated group, the number of M2 macrophages increased significantly at 1, 7, and 14 days after surgery at the injury site. There was a significant difference at 5 mm distal at 7 days after surgery and at 7.5 mm distal at 7 and 14 days after surgery. Similar trends were observed for the proportions of M2 macrophages.

[0103]

[Table 9-1]

[0104] [Table 9-2]

[0105] [Table 9-3]

[0106] [Table 10-1]

[0107] [Table 10-2]

[0108] [Table 10-3]

[0109]

Example 8. Analysis of nerve regeneration after sciatic nerve injury

The effect of methylcobalamin on nerve regeneration after sciatic nerve injury was analyzed by immunohistological analysis. Specifically, the analysis was performed as follows. Cross sections of the injured sciatic nerve obtained 2 weeks after sciatic nerve injury were used for the analysis. Similar to the macrophage analysis, the analysis was performed for sections at a distance of 2.5 mm in the proximal direction from the injury site, the injury site, and 2.5, 5.0, and 7.5 mm in the distal direction. Regenerated axons were labeled with NF 200, and myelin sheaths were labeled with MBP. The percentage of myelination of regenerated axons was calculated according to the following formula: Percentage of myelination (%) = number of myelinated axons per mm ² / number of axons per mm ² × 100. More specifically, the analysis was performed in the same manner as in Example 7.

[0110]

The results are shown in Fig. 11. The values corresponding to the data in the graphs in Fig. 11 are shown in Tables 11-1, 11-2, and 11-3. In the methylcobalamin treatment group, the number of axons and the number of myelinated axons were significantly increased at the injury site. There were significant differences in the number of axons at 5.0 and 7.5 mm in the distal direction, the number of myelinated axons at 2.5, 5.0, and 7.5 mm in the distal direction, and the percentage of myelination at 5.0 and 7.5 mm in the distal direction. These results and the results in Example 7 indicated that methylcobalamin promoted nerve regeneration in an anti-inflammatory manner by decreasing M1 macrophages and increasing M2 macrophages in the actual nerve regeneration process.

[0111] [Table 11-1]

[0112] [Table 11-2]

[0113][Table 11-3]

[0114]

Example 9. Therapeutic effect on spinal cord injury

The therapeutic effects of methylcobalamin on spinal cord injury were assessed using the Basso-Beattie-Bresnahan (BBB) scale and thermal algesimetry. Specifically, the study was conducted as follows.

[0115]

<Example 9-1. Construction of a rat spinal cord injury model (lateral hemisection model) and administration of a drug>

Six-week-old female Wistar rats were used. The rats were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan). Anesthesia was performed as follows. A 1:10 dilution of a mixture of three anesthetics in saline was administered by intraperitoneal injection. One dose included 0.2 mg/kg midazolam, 0.015 mg/kg medetomidine, and 0.25 mg/kg butorphanol. The rat was fixed in the prone position on the operating table, and a dorsal midline incision was made. The T10 vertebral arch was removed to expose the posterior surface of the spinal cord, and the left spinal nerve was trimmed using a sharp surgical scalpel (spitz mess). The skin was sutured using 4-0 nylon suture, and the surgery was completed. Three groups were compared: methylcobalamin-treated group, untreated group, and sham group. Immediately after surgery, an osmotic minipump filled with methylcobalamin (1 mg/kg/day) or saline was inserted in the methylcobalamin-treated group and untreated group, respectively, and left in the left dorsal subcutaneous space. The sham group underwent resection of the T10 vertebral arch only.

[0116]

<Example 9-2. Determining the BBB Index>

Each rat was individually placed in a cage, allowed to move freely, and observed for 5 minutes. According to the standard method, left lower limb function was assessed using a score from 0 (no locomotor activity) to 21 (normal locomotor activity). Assessments were made before surgery and at 1, 7, 14, 21, and 28 days after surgery.

[0117]

<Example 9-3. Thermal algesimetry>

Each rat was placed in a dedicated cage, individually, infrared stimulation was applied to the right sole and the time until the rat retracted the hind limb was measured. To avoid skin damage, stimulation was continued for a maximum of 15 seconds. Assessments were made preoperatively and at 7, 14, 21 and 28 days postoperatively.

[0118]

<Example 9-4. Results>

The BBB scores are shown in Fig. 12. The values corresponding to the data in the graph of Fig. 12 are shown in Table 12. Compared with the untreated group, the motor function of the left lower limb was significantly improved in the methylcobalamin administration group at 14, 21, and 28 days after the operation.

[0119]

[Table 12]

[0120]

The results of thermal algesimetry are shown in Fig. 13. The values corresponding to the data in the graph of Fig. 13 are shown in Table 13. In the methylcobalamin treatment group, hyperesthesia of the right lower limb was significantly improved at 21 and 28 days after the operation.

[0121]

[Table 13]

[0122]

Example 10. M2 microglia induction promoting action and M1 microglia induction inhibiting action

The M2 microglia induction promoting effect and M1 microglia induction inhibiting effect of methylcobalamin were investigated by Western blotting. Specifically, the study was conducted as follows.

[0123]

<Example 10-1. Western Blot>

LPS (100 ng/ml) and anti-inflammatory cytokine IL-4 (20 ng/ml) were added to the microglial cell line (6-3 cells). Methylcobalamin solutions, each at different concentrations ranging from 1 nM to 1 mM, were added to the mixture, and proteins were isolated from each mixture at 1 and 3 days after addition. Electrophoresis and transfer to membrane were performed, the membrane was blocked, and the cells were reacted with each of the primary antibodies against M1 markers (iNOS and IL-1β) and M2 markers (Arg1 and CD206) at 4°C overnight. The cells were reacted with the secondary antibody at room temperature for 1 hour, and the bands were detected using a detector.

[0124]

Anti-iNOS, anti-IL-1β, anti-Arg1 and anti-CD206 (mannose receptor) antibodies were used as primary antibodies. Anti-rabbit IgG, HRP-linked whole sheep Ab were used as secondary antibodies.

[0125]

<Example 10-2. Results>

The results of Western blotting are shown in Fig. 14-15. The values corresponding to the data in the graphs in Fig. 14 are shown in Table 14, and the values corresponding to the data in the graphs in Fig. 15 are shown in Table 15. The amounts of proinflammatory (M1) markers of microglia were as follows: compared with LPS alone, the amount of IL-1β protein was significantly decreased when at least 1 μM methylcobalamin was added in combination, and the amount of iNOS protein was significantly decreased when at least 10 nM methylcobalamin was added in combination. The amounts of anti-inflammatory (M2) markers were as follows: compared with IL-4 alone, the amount of Arg1 protein was significantly increased when 1 nM to 10 μM methylcobalamin was added in combination. The amount of CD206 was significantly increased when 10 nM to 100 nM methylcobalamin was added in combination.

[0126]

[Table 14]

[0127]

[Table 15]

[0128]

Example 11. M2 macrophage induction promoting action and M1 macrophage induction inhibiting action

The promoting action of M2 macrophage induction and the inhibiting action of M1 macrophage induction were investigated. In particular, the study was conducted as follows.

[0129]

<Example 11-1. Immunofluorescent staining>

The rat spinal cord injury model from Example 9-1 was used. At 7, 14, and 28 days after surgery, the rats were anesthetized with an anesthetic agent and perfusion-fixed with 4% PFA. The spinal cord including the injury site was then collected and fixed with 20% sucrose for 24 hours, and the fixed spinal cord was frozen. The embedded tissue was cut into a 5-μm-thick section in the direction of the short axis of the nerve and placed on a glass slide. The sections were dried for 1 hour and fixed with 100% methanol for 30 minutes. The fixed sections were blocked, and the cells were allowed to react with primary antibodies at 4°C overnight. The cells were then allowed to react with secondary antibody at room temperature for 1 hour so that the nuclei were labeled with DAPI.

[0130]

Using transverse sections of the spinal cord on the affected side at a distance of 2 and 1 mm on the head side and 1 and 2 mm on the tail side from the injury site, the number of macrophages, the number of M1 macrophages (pro-inflammatory type), the proportion of M1 macrophages, the number of M2 macrophages (anti-inflammatory type), and the proportion of M2 macrophages per unit area, as well as the M1/M2 ratio were measured and calculated.

[0131]

Anti-CD68, anti-iNOS, and anti-Arg1 antibodies were used as primary antibodies. Alexa 488-labeled goat anti-rabbit IgG and Alexa 568-labeled goat anti-mouse IgG were used as secondary antibodies.

[0132]

<Example 11-2. Results>

The results of immunofluorescence staining are shown in Figs. 16-22. Figs. 16-18 show the differences in the number of macrophages, the number of M1 macrophages (proinflammatory type), the proportion of M1 macrophages, the number of M2 macrophages (antiinflammatory type), and the proportion of M2 macrophages per unit area for each position. Figs. 19-20 show the changes over time in days after surgery. Fig. 21 shows the changes in the M1/M2 ratio over time in days after surgery. Fig. 22 shows the changes at each position. The values corresponding to the data in the graphs in Figs. 16-18 are shown in Tables 16-18, respectively, and the values corresponding to the data in the graphs in Fig. 22 are shown in Table 19.

[0133]

In the methylcobalamin-treated group, a trend toward fewer accumulated macrophages per unit area was observed compared with the untreated group. In addition, considering the macrophage phenotypes, there were trends toward fewer M1 macrophages and more M2 macrophages. There were significant differences in some of the results.

[0134]

[Table 16]

[0135]

[Table 17]

[0136]

[Table 18]

[0137]

[Table 19]

[0138]

Example 12. Functional recovery-promoting effect of methylcobalamin in a photocoagulation-induced cerebral infarction model

<Example 12-1. Subject and Method>

Male C57BL/6J mice aged 8 to 10 weeks (about 24 g) were used. A photocoagulation-induced cerebral infarction model was established for laser irradiation after injection of rose bengal. In this model, the skull of each mouse was drilled to open a hole having a center 2 mm from the anterior fontanelle in an outward direction. Five minutes after injection of the photosensitive dye rose bengal, the right motor cortex as a center was irradiated with laser irradiation to create a cerebral infarction in the right motor cortex. An osmotic pump (ALZET osmotic pumps, for two weeks) was inserted immediately after creating a cerebral infarction. Mice were divided into methylcobalamin-treated group (N=3) and untreated group (N=4) and rotarod tests (velocity acceleration method) were performed at 2, 4, 7, 9, 11 and 14 days after cerebral infarction surgery. The time until the mouse fell from the rotarod was measured and the ratio to the maximum of 300 seconds was calculated as the baseline.

[0139]

<Example 12-2. Results>

The results are shown in Fig. 23. The corresponding values to the data in the graph of Fig. 23 are shown in Table 20. At 2, 4, and 9 days after the operation leading to cerebral infarction, the brain function examined in the rotarod test was significantly improved in the methylcobalamin-treated group compared with the untreated group.

[0140]

[Table 20]

Claims (5)

1. A method for treating a disease of the nervous system, said method comprising administering an agent consisting of methylcobalamin as an active ingredient by continuous intravenous drip infusion, wherein the disease of the nervous system is cerebral infarction or spinal cord injury. 2. The method according to claim 1, wherein the treatment of a disease of the nervous system is provided by promoting the induction of M2 macrophage/microglia. 3. The method according to claim 1 or 2, wherein the treatment of a disease of the nervous system is achieved by inhibiting the induction of macrophage/microglia M1. 4. The method according to any one of claims 1-3, wherein the treatment of a disease of the nervous system is achieved by reducing the ratio of macrophage/microglia M1 to macrophage/microglia M2. 5. The method according to any one of claims 1-4, wherein the treatment of a disease of the nervous system is provided by promoting nerve regeneration.

Images