Nerve regeneration agent and nerve regeneration method

Abstract

The present invention addresses the problem of providing a novel method for regenerating nerves using a drug. As a means for solving the problem, the present invention provides a nerve regeneration agent containing an iron chelating agent, and a nerve regeneration method that includes a step in which an iron chelating agent is applied ex vivo to tissue requiring nerve regeneration.

Description

Nerve Regeneration Agent and Nerve Regeneration Method

[0001] The present invention relates to a nerve regeneration agent and a nerve regeneration method.

[0002] It is known that the central nervous system functions decline when it is damaged by trauma, inflammation, tumors, etc., or degenerates due to aging. As methods for regenerating the central nervous system, the following methods are known.

[0003] As a method for promoting nerve regeneration using electrical stimulation, electrostimulation therapy is known. Electrostimulation therapy is a method of activating the activities of nerve cells and promoting regeneration by applying electrical stimulation to the damaged site of the spinal cord or brain (Patent Document 1). Specifically, spinal cord stimulation devices, deep brain stimulation devices, etc. have been reported as those using this method.

[0004] A method of promoting the survival of nerve cells and the growth of axons by administering neurotrophic factors such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) to the damaged site is also known. Thereby, the regeneration of the damaged nerve circuit is expected.

[0005] A method of providing a scaffold to the damaged site using biomaterials is also known. The aim is to support the movement of nerve cells and the growth of new axons (Patent Document 2). Hydrogels, polymers, etc. are used, and those with designs that support the local release of drugs and growth factors have been proposed.

[0006] Also, a method of regenerating damaged nerve tissue using stem cells is known. In particular, mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPS cells) are used, and it is expected that these cells will differentiate into nerve cells and supporting cells and promote regeneration by transplanting them to the damaged site.

[0007] Furthermore, a method of promoting the regeneration of nerve cells using gene therapy technology is known. Methods of introducing genes for the purpose of enhancing the expression of nerve growth factor at the damaged site and methods of suppressing the expression of factors that inhibit regeneration (for example, inhibitory molecules such as Nogo-A) have been proposed.

[0008] Japanese Patent Application Laid-Open No. 2021-501027

[0009] Japanese Patent Publication No. 2023-099367

[0010] As mentioned above, various methods for central nervous system regeneration have been reported, but no method for regenerating nerves using drugs has yet been reported. Furthermore, these known methods have the following problems.

[0011] In stem cell therapy and gene therapy, the patient's body recognizes the transplanted cells or genes as foreign, triggering an immune response. This rejection not only hinders the effectiveness of the treatment but also carries the risk of causing inflammation and worsening of damage. Immunosuppressants may be necessary, but this carries concerns about side effects such as an increased risk of infection.

[0012] Central nervous system regeneration is extremely complex, requiring regenerated nerves to grow in the correct locations and establish functional connections. However, controlling stem cell differentiation and ensuring that regenerated axons accurately reach their intended targets is difficult, leading to misconnections. This can result in failure to achieve the expected functional recovery, or conversely, the occurrence of abnormal neural activity.

[0013] Many regenerative technologies are still in the clinical trial stage, and there is a lack of data on their long-term safety and efficacy. In particular, there are concerns about the risk of stem cells becoming cancerous and the unexpected occurrence of genetic mutations due to gene therapy. Furthermore, the effects of treatment may be temporary, and regenerated nerves may lose their function again over time, so long-term trials are needed to confirm sustained effects.

[0014] Therefore, the object of the present invention is to provide a novel method for regenerating nerves using a drug.

[0015] In the course of their research on iron, the inventors had obtained the finding that controlling iron levels in the body has a significant impact on the body, including immunity. For example, various effects have been confirmed, such as the occurrence of various diseases when iron is excessive, and conversely, the control of stem cell differentiation when iron is removed. Among these, it has also been confirmed that strong iron removal stimulates enhanced biological functions. Inspired by this discovery, the inventors arrived at the hypothesis that iron removal might also be beneficial for nerve regeneration. Based on this unique hypothesis, the inventors conducted further intensive studies and found that nerve regeneration can be achieved by administering an iron chelating agent. The present invention was completed by further studies based on this finding and includes the following embodiments.

[0016] [Item 1] A nerve regeneration agent comprising an iron chelating agent. [Item 2] The nerve regeneration agent according to Item 1, which is a central nervous system regeneration agent. [Item 3] The nerve regeneration agent according to Item 1 or 2, which is for improving cognitive function. [Item 4] The nerve regeneration agent according to any one of Items 1 to 3, which is for the treatment of at least one disease selected from the group consisting of trauma, inflammation, tumor, degenerative disease, and dementia. [Item 5] The nerve regeneration agent according to any one of Items 1 to 4, wherein the iron chelating agent is a solid, water-soluble, or colloidally dispersible iron chelating agent. [Item 6] The nerve regeneration agent according to any one of Items 1 to 4, wherein the iron chelating agent is roxadustat and / or a super polyphenol. [Item 7] The nerve regeneration agent according to Item 6, wherein the super polyphenol is SP10. [Item 8] The nerve regeneration agent according to any one of Items 1 to 7, which is for oral administration. [Item 9] A method for nerve regeneration, comprising the step of applying an iron chelating agent ex vivo to tissue requiring nerve regeneration. [Clause 10] The nerve regeneration method according to Claim 9, wherein the iron chelating agent is a solid, water-soluble, or colloidally dispersible iron chelating agent. [Clause 11] The nerve regeneration method according to Claim 9, wherein the iron chelating agent is roxadustat and / or a super polyphenol. [Clause 12] The nerve regeneration method according to Claim 11, wherein the super polyphenol is SP10.

[0017] According to the present invention, a novel method for regenerating nerves using a drug can be provided.

[0018] These are photographs of pathological sections from Test Example 1. "CTRL" indicates the control group, "Rox" indicates the Roxadustat-treated group, and "SP10" indicates the SP10-treated group (the same applies hereafter).

[0019] This graph compares the hippocampal area in Test Example 1. The vertical axis shows the relative area of ​​each treatment group, with the area of ​​"CTRL" set to 1.

[0020] The graph below shows the results of the hot plate test in Test Example 2. The vertical axis represents the escape reaction time, which is the time it takes to jump or lift one's feet.

[0021] This graph shows the difference in escape reaction time before and after drug administration (for each mouse) in the hot plate test of Test Example 2. The vertical axis represents the difference in escape reaction time before and after drug administration.

[0022] This graph shows the results of the light-dark transition test in Test Example 3. The vertical axis of the graph on the left shows the time it took to enter the dark box, and the vertical axis of the graph on the right shows the number of times the subject moved between the light and dark areas.

[0023] This graph shows the results of the light-dark transition test in Test Example 3. The vertical axis of the graph on the left shows the difference in time taken to enter the dark box before and after drug administration (for each mouse), and the vertical axis of the graph on the right shows the difference in the number of times the mice moved between the light and dark areas before and after drug administration (for each mouse).

[0024] This graph shows the blood test results for Test Example 4. It shows the changes in blood cells (WBC), hemoglobin (Hb), and platelets (PLT), respectively.

[0025] The analysis results for hippocampal area in Test Example 4 are shown.

[0026] The results of analyzing various signals using Western blotting on hippocampal proteins recovered in Test Example 4 are shown below.

[0027] The nerve regeneration agent of the present invention contains an iron chelating agent.

[0028] The iron chelating agent is not particularly limited. In the present invention, it has already been confirmed that when multiple iron chelating agents with different structures are administered, a similar nerve regeneration effect is observed in all cases. Therefore, it is obvious that such a nerve regeneration effect is caused by iron removal in the body, and for this reason, the structure of the iron chelating agent that can be used in the present invention is not particularly limited.

[0029] Therefore, in the present invention, any iron chelating agent can be used as an active ingredient. In particular, an iron chelating agent that does not pose a safety problem when administered in vivo is preferred. A wide range of known iron chelating agents can be used as such. The iron chelating agent may be solid, water-soluble, or colloidally dispersible. When the nerve regeneration agent of the present invention is administered in vivo, a water-soluble or colloidally dispersible iron chelating agent is preferred.

[0030] Roxadustat and / or super polyphenols are preferred as iron chelating agents.

[0031] Roxadustat is an iron chelating agent represented by the following structural formula. Roxadustat is available for purchase under the brand name "Evrenzo" (Astellas Pharma Inc.).

[0032]

[0033] Super polyphenols are used as iron removers, and for example, SP6, SP10, etc. can be used.

[0034] The structural formula of SP6 is as follows:

[0035]

[0036] The structural formula of SP10 is as follows:

[0037]

[0038] As iron chelating agents, in addition to the known ones exemplified above, these modified forms can also be used. Examples of the modified forms include structural modified forms with maintained or improved functions.

[0039] The nerve regenerating agent of the present invention contains at least one iron chelating agent. The nerve regenerating agent of the present invention may contain a combination of multiple kinds of iron chelating agents.

[0040] The content of the iron chelating agent in the nerve regenerating agent of the present invention can be appropriately set according to the administration purpose, administration subject, administration form, etc. Also, the blending amount can be appropriately adjusted according to the iron-removing ability of the iron chelating agent. For example, when using an iron chelating agent with extremely high iron-removing ability, the blending amount can be relatively small.

[0041] When the nerve regenerating agent of the present invention is administered to humans, the single dose per body weight of the iron chelating agent can be administered so that it is, for example, 0.1 mg / kg to 500 mg / kg, preferably 1 mg / kg to 100 mg / kg. In this case, the administration interval can be, for example, three times a week.

[0042] The nerve regenerating agent of the present invention may further contain other components in addition to the iron chelating agent as an active ingredient. As other components, additives such as pH adjusters, antiseptics, preservatives, antioxidants, stabilizers, proteins, and peptides can be appropriately blended.

[0043] The nerve regenerating agent of the present invention is preferably a central nerve regenerating agent.

[0044] In one aspect, the nerve regenerating agent of the present invention can be used for improving cognitive function.

[0045] In one aspect, the nerve regenerating agent of the present invention can be used for treating at least one disease selected from the group consisting of trauma, inflammation, tumor, degenerative diseases, and dementia. Specifically, it may be effective against nerve disorders caused by trauma, inflammation, or tumor. Alternatively, it may also be effective against nerves whose functions have declined due to age-related advanced degeneration.

[0046] The administration form of the nerve regenerative agent of the present invention is not particularly limited. When the nerve regenerative agent of the present invention is directly administered into the body, it is preferably for oral administration. Further, the nerve regenerative agent of the present invention can be included in an implant device and used so as to be applied to a site that requires nerve regeneration after implantation. Furthermore, the nerve regenerative agent of the present invention can also be administered into the body by hemodialysis.

[0047] The administration target of the nerve regenerative agent of the present invention is not particularly limited. The administration target can be a mammal. Alternatively, the administration target may be an experimental nerve tissue model. In this case, the nerve regenerative agent of the present invention can be used for the production of a nerve tissue model. The administration target is preferably a human.

[0048] Nerve regeneration can also be performed by applying the nerve regenerative agent of the present invention ex vivo to a tissue that requires nerve regeneration. The tissue that requires nerve regeneration may be a biological tissue taken out of the body, or may be an artificial tissue produced from pluripotent stem cells or the like. The nerve tissue regenerated in this way may then be transplanted into the body, or used as an experimental nerve tissue model.

[0049] The dosage form of the nerve regenerative agent of the present invention is not particularly limited. When it is for oral administration, it may be a tablet, capsule, powder, granule, fine granule, pill, suspension, emulsion, solution or syrup, etc. The nerve regenerative agent of the present invention may further contain additives such as necessary excipients according to the dosage form. For example, it can also be made into a capsule or the like coated with a coating layer together with additives such as an excipient, disintegrant, fluidizing agent, binder, etc. Widely known substances can be used as additives. For example, flavoring agents, perfuming agents, binders, lubricants, etc. can be mentioned.

[0050] Examples of excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, crystalline cellulose, dibasic calcium phosphate anhydrous, pregelatinized starch, corn starch and alginic acid, etc.

[0051] Examples of binders include simple syrup, glucose solution, starch solution, gelatin solution, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, carboxymethylcellulose, shellac, methylcellulose, ethylcellulose, sodium alginate, acacia gum, hydroxypropylmethylcellulose, hydroxypropylcellulose, water, and ethanol.

[0052] Examples of disintegrants include dried starch, alginic acid, agar powder, starch, cross-linked polyvinylpyrrolidone, cross-linked carboxymethylcellulose sodium, carboxymethylcellulose calcium, and sodium starch glycolate.

[0053] Examples of disintegration inhibitors include stearyl alcohol, stearic acid, cocoa butter, and hydrogenated oil.

[0054] Examples of anti-caking and anti-adhesion agents include aluminum silicate, calcium hydrogen phosphate, magnesium oxide, talc, and anhydrous silicic acid.

[0055] Examples of lubricants include carnauba wax, light anhydrous silicic acid, aluminum silicate, magnesium silicate, hydrogenated oil, hydrogenated vegetable oil derivatives, sesame oil, bleached beeswax, titanium dioxide, dried aluminum hydroxide gel, stearic acid, calcium stearate, magnesium stearate, talc, calcium hydrogen phosphate, sodium lauryl sulfate, and polyethylene glycol.

[0056] Examples of absorption enhancers include quaternary ammonium salts, sodium lauryl sulfate, urea, and enzymes.

[0057] Examples of carriers and adsorbents include starch, lactose, kaolin, bentonite, anhydrous silicic acid, hydrated silicon dioxide, magnesium aluminometasilicate, and colloidal silicic acid.

[0058] The nerve regeneration agent of the present invention can be formulated into dosage forms according to conventional methods, using the above-mentioned additives as appropriate.

[0059] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0060] Experiments were conducted in which aged mice were given two types of iron chelating agents (Roxadusstat and Super Polyphenol 10 (SP10)). Aged mice aged 45-48 weeks were prepared, and a hot plate test was performed. The mice were then divided into three groups to ensure even distribution of results. The groups were set up as follows: CTrl group (physiological saline), Roxadusstat group (50 mg / kg), and SP10 group (50 mg / kg). Each drug was administered orally three times a week for three weeks. After three weeks, cognitive function was tested, brains were collected from the mice, and the regenerative capacity of the central nervous system was examined.

[0061] <Example of study 1: Regeneration of the central nervous system> When pathological sections were prepared and the hippocampus was compared, the hippocampus was larger in the iron-removed group (Figure 1), and when comparing the area of ​​the hippocampus, the area of ​​the hippocampus was significantly larger in the drug-treated group (Rox, SP10) compared with the control group (Figure 2).

[0062] We investigated cognitive function. The hot plate test (pain sensitivity test) and the light-dark transition test were each administered twice on separate days to examine their effects on cognitive function.

[0063] <Test Example 2: Cognitive Function of the Brain (1) Hot Plate Test (Pain Sensitivity Test)> In the hot plate test (pain sensitivity test), both the Roxadustat group and the SP10 group showed a significant decrease in escape reaction time to jump or lift their feet (Figure 3).

[0064] Analysis of the difference in escape reaction time before and after drug administration (for each mouse) in the hot plate test showed that the CTRL group showed an increase in escape reaction time after 3 weeks, but both the Roxadustat and SP10 groups showed a decrease. This suggests that iron removal regenerated the central nervous system and improved cognitive function (Figure 4).

[0065] <Test Example 3: Cognitive Function of the Brain (2) Light-Darkness Test> In the light-darkness test, the Roxadustat group showed a decrease in the time it took to enter the dark box and a significant increase in the number of times they moved between the light and dark areas, suggesting an improvement in mental disorder symptoms. The SP10 group also showed a significant increase in the number of times they moved between the light and dark areas (Figure 5).

[0066] Furthermore, in the light-dark transition test, we analyzed the difference (for each mouse) in the time it took to enter the dark box and the number of times the mice moved between the light and dark areas before and after drug administration. Regarding the time it took to enter the dark box, an increase was observed in the CTRL group after 3 weeks, but there was no statistically significant difference in the Roxadustat group and the SP10 group, although both groups showed a decrease. In addition, the number of times the mice moved between the light and dark areas was significantly suppressed in the Roxadustat group and the SP10 group, suggesting that cognitive function had improved (Figure 6).

[0067] <Test Example 4: Blood sampling, head MRI, and hippocampal protein analysis> A control group and an iron chelating agent (SP10; 50 mg / kg, 3 times / week, Roxadustat; 50 mg / kg, 3 times / week) group were established for aged mice aged 43-48 weeks. Oral administration was performed for 8 weeks, and behavioral tests (Y-maze test, light-dark test), blood sampling, head MRI, and hippocampal protein collection were performed. Signal analysis was performed using Western blotting.

[0068] Eight weeks of administration of an iron chelating agent suppressed the decline in working memory in aged mice. No induction of anxiety behavior or differences in activity levels were observed.

[0069] When two types of iron chelating agents were administered, an increase in white blood cells was observed (Figure 7), and the hippocampal area was also enlarged (Figure 9).

[0070] Furthermore, while hippocampal nerve regeneration-related markers showed an upward trend in the SP10 administration group, HIF-1a and inflammatory markers did not increase (Figure 9).

[0071] These results suggest that inflammation is not being induced by iron chelating agents, but rather that an indirect immunological mechanism may be at work.

Claims

1. A nerve regeneration agent containing an iron chelating agent.

2. The nerve regeneration agent according to claim 1, which is a central nervous system regeneration agent.

3. The nerve regeneration agent according to claim 2, which is for improving cognitive function.

4. The nerve regeneration agent according to claim 1, for the treatment of at least one disease selected from the group consisting of trauma, inflammation, tumor, degenerative disease, and dementia.

5. The nerve regeneration agent according to claim 1, wherein the iron chelating agent is a solid, water-soluble, or colloidally dispersible iron chelating agent.

6. The nerve regeneration agent according to claim 1, wherein the iron chelating agent is a solid, water-soluble, or colloidally dispersible iron chelating agent.

7. The nerve regeneration agent according to claim 6, wherein the super polyphenol is SP10.

8. A nerve regeneration agent according to any one of claims 1 to 7, for oral administration.

9. A method for nerve regeneration, comprising the step of applying an iron chelating agent ex vivo to tissue requiring nerve regeneration.

10. The nerve regeneration method according to claim 9, wherein the iron chelating agent is a solid, water-soluble, or colloidally dispersible iron chelating agent.

11. The nerve regeneration method according to claim 9, wherein the iron chelating agent is roxadustat and / or super polyphenol.

12. The nerve regeneration method according to claim 11, wherein the super polyphenol is SP10.

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