Pharmaceutical composition for promoting angiogenesis

Abstract

To provide a useful drug with an angiogenesis promoting action in the treatment of tissue damage such as peripheral nerve injury.SOLUTION: The pharmaceutical composition containing vitamin B12 as an active ingredient is found to have a vascular endothelial cell migration action and an angiogenesis promoting action, and further have a PI3K-Akt-mTOR pathway activation action which exhibits a phosphorylation promoting action on PI3K, Akt, and mTOR, and is involved in angiogenesis. In addition, since the pharmaceutical composition has an Akt pathway activation action via actions of a methylation cycle and Icmt, a RAS protein expression activation action, a macrophage Ras activation action, an M-Ras activation action and the like, it is highly useful as an angiogenesis promoting agent in the early stage after receiving tissue damage such as peripheral nerve injury. Furthermore, since wound healing is delayed in diabetes, aging and the like, the pharmaceutical composition can also be used as the angiogenesis promoting agent to promote the healing of such tissue wounds.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for promoting angiogenesis, and more specifically to a pharmaceutical composition having a therapeutic effect against tissue damage (hereinafter, sometimes simply referred to as "the pharmaceutical composition"). [Background technology]

[0002] There are various types of tissue damage, for example, peripheral nerve damage occurs with severe limb trauma or iatrogenic damage, causing sensory disorders, residual pain, etc. Although peripheral nerves have the ability to regenerate themselves, even with currently available treatment methods, complete functional recovery is often not achieved, and treatment is often difficult. It has become clear that as part of the healing process after peripheral nerve injury, new blood vessels are formed to bridge the damaged area prior to the regeneration of nerve axons, and in recent years, there has been increasing attention on the relationship between nerves and blood vessels in the healing of peripheral nerve injuries and other conditions.

[0003] Angiogenesis is the formation of new blood vessels. They grow out of pre-existing blood vessels. It is a vital physiological process that enables growth, skeletal muscle hypertrophy, menstruation, pregnancy, and wound healing.

[0004] For example, peripheral nerve injury can be broadly divided into discontinuous injury, in which the continuity at the injury site is broken, and continuous nerve injury, such as entrapment neuropathy (carpal tunnel syndrome, etc.), in which the injury site is continuous. Direct suture, autologous nerve grafting, etc. are selected as treatments for discontinuous injury. On the other hand, nerve avulsion and conservative treatment are selected as treatments for continuous nerve injury. Until now, artificial nerves have been developed as devices with a regenerative effect on peripheral nerve injury, but these are only used for discontinuous nerve injury. In addition, they simply bridge the defects at the injury site and do not have the effect of promoting nerve axon regeneration. If a long period of time is required for recovery after peripheral nerve injury, irreversible changes occur in the muscle tissue, so promoting nerve axon regeneration is an important issue. Therefore, there is a demand in the medical field for drugs that can be applied to both continuous and discontinuous nerve injury and are effective in treating nerve injury.

[0005] Although peripheral nerves have the ability to regenerate after injury, recovery of nerve function is not sufficient. When a nerve is injured, the axon and myelin sheath are phagocytosed and removed by Wallerian degeneration. Next, in the regeneration process, the regenerated axon extends distally within the Bungner's band, which is formed by undifferentiated Schwann cells, and reinnervation of the target muscle occurs. Finally, a myelin sheath is formed by Schwann cells surrounding the regenerated axon. However, the extension speed of the regenerated nerve is very slow, and if the distance to the target muscle is long, muscle atrophy occurs and sufficient functional recovery cannot be expected. Meanwhile, it has been revealed in recent years that macrophages play an important role in each stage of the regeneration process mentioned above, and this has attracted attention. The inflammatory function of macrophages is well known, but on the other hand, they also have a phenotype that has an anti-inflammatory function. The former is called M1 type and the latter is called M2 type, and it is thought that there is a continuity between these phenotypes (a shift occurs between M1 and M2). In general, it is said that nerve regeneration is promoted by increasing M2 macrophages, which have an anti-inflammatory phenotype.

[0006] Vitamin B1 is one of the active ingredients of this pharmaceutical composition.12 Vitamin B 12 Vitamin B 12 Effective in treating deficiency of vitamin B 12 It is known that deficiency can also cause neurological changes such as peripheral neuritis and spinal cord changes (Patent Document 1).

[0007] Vitamin B 12 Vitamin B is important for the normal functioning of the nervous system, and its deficiency is known to cause a systemic neurological disorder called "subacute combined degeneration of the spinal cord" (Non-Patent Document 1). 12 It has been reported that methylcobalamin (hereinafter sometimes referred to as "MeCbl"), an analog of methylcobalamin, promotes neurite outgrowth and neuronal survival at concentrations of 100 nM or more, that these effects are mediated by the methylation cycle, which is a reaction related to methylation, that MeCbl increases the activity of ERK1 / 2 and Akt through the methylation cycle, and that continuous administration of high doses of MeCbl improves nerve regeneration and functional recovery in a rat sciatic nerve injury model (Non-Patent Document 2).

[0008] As reported previously, MeCbl is known to have the effect of promoting peripheral nerve regeneration. However, it has not been disclosed that MeCbl promotes angiogenesis in the early stage of injury as one of its nerve regeneration promoting effects. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Special Publication No. 2016-513694 [Non-patent literature]

[0010] [Non-Patent Document 1] Scalabrino et al., Laboratory Investigation, 62(1990), 297-304 [Non-Patent Document 2] Okada et al. Experimental Neurology, 222(2010), 191-203 Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide a pharmaceutical composition for promoting angiogenesis, etc. Preferably, an object of the present invention is to provide a pharmaceutical composition for promoting angiogenesis, which is applied to an injured site in an appropriate manner to promote angiogenesis in the early stage after injury, thereby enhancing the healing effect of the injury. [Means for solving the problem]

[0012] In view of the above problems, the present inventors have conducted intensive research and have found that vitamin B 12 We found that vitamin B12 promotes angiogenesis in the early stages after tissue injury, especially peripheral nerve injury. 12 It has also been found that has vascular endothelial cell migration activity, angiogenesis promotion activity, PI3K-Akt-mTOR pathway activation activity, Akt pathway activation activity, Ras protein expression activation activity, macrophage Ras activation activity, and M-Ras activation activity. Based on these findings, further research was conducted and the present invention was completed.

[0013] That is, the present invention includes the following aspects: (1) Vitamin B 12 A pharmaceutical composition for promoting angiogenesis comprising: (2) The pharmaceutical composition for promoting angiogenesis according to (1) above, wherein the promotion of angiogenesis is achieved by promoting the migration of vascular endothelial cells and / or promoting the formation of blood vessels. (3) The pharmaceutical composition for promoting angiogenesis according to (1) or (2) above, wherein the promotion of angiogenesis is due to activation of Ras in an intracellular signal transduction pathway. (4) The pharmaceutical composition for promoting angiogenesis according to (3) above, wherein Ras is M-Ras. (5) The pharmaceutical composition for promoting angiogenesis according to any one of the above (1) to (4), wherein the promotion of angiogenesis is further mediated by activation of Akt in an intracellular signal transduction pathway. (6) The pharmaceutical composition for promoting angiogenesis according to any one of the above (3) to (5), wherein the cells are vascular endothelial cells and / or macrophages. (7) The pharmaceutical composition for promoting angiogenesis according to any one of (1) to (6) above, which is used for treating tissue damage. (8) The pharmaceutical composition for promoting angiogenesis according to (7) above, which is used for treating peripheral nerve injury. (9) The pharmaceutical composition for promoting angiogenesis according to (7) above, which is used for promoting wound healing. (10) The pharmaceutical composition for promoting angiogenesis according to any one of (1) to (9) above, which is used by continuous administration. (11) Vitamin B 12 The pharmaceutical composition for promoting angiogenesis according to any one of the above (1) to (10), wherein (12) A method for producing a pharmaceutical composition for promoting angiogenesis according to any one of (1) to (10) above, comprising administering to the patient a vitamin B 12 Use of. (13) Vitamin B for use in treating tissue damage by promoting angiogenesis 12 Containing pharmaceutical composition. (14) The vitamin B according to (13) above, wherein the treatment of tissue damage is treatment of peripheral nerve damage. 12 Containing pharmaceutical composition. (15) The vitamin B according to (13) above, wherein the treatment of tissue damage is the promotion of wound healing. 12 Containing pharmaceutical composition. (16) The vitamin B compound according to any one of (13) to (15) above, wherein the promotion of angiogenesis is due to the promotion of vascular endothelial cell migration and / or the promotion of vasculogenesis. 12 Containing pharmaceutical composition. (17) Vitamin B according to any one of (13) to (16) above, wherein the promotion of angiogenesis is due to activation of Ras in an intracellular signal transduction pathway. 12 Containing pharmaceutical composition. (18) The vitamin B compound according to (17) above, wherein Ras is M-Ras. 12 Containing pharmaceutical composition. (19) Vitamin B according to any one of (13) to (18) above, wherein the promotion of angiogenesis is further due to activation of Akt in an intracellular signal transduction pathway. 12 Containing pharmaceutical composition. (20) The vitamin B12 compound according to any one of (17) to (19) above, wherein the cells are vascular endothelial cells and / or macrophages. 12 Containing pharmaceutical composition. (21) Vitamin B according to any one of (13) to (20) above, which is administered continuously. 12 Containing pharmaceutical composition. (22) Vitamin B 12 The vitamin B according to any one of the above (13) to (21), wherein the vitamin B 12 Containing pharmaceutical composition. [Brief description of the drawings]

[0014] [Figure 1] The initial appearance of the frozen section after transplantation is shown. A: The transplanted section is enclosed within the dashed line. B: Bright-field image of the section immediately after surgery. C: Immunostained image of the section immediately after surgery (merged image of Nestin and p75). [Diagram 2] The neovascular area is shown 3 to 7 days after surgery. A: Neovascular area. The area inside the dashed line is the graft section (Scale bar: 500 μm). B: Evaluation of Nestin-positive area 3 days after surgery (n=3). C: Evaluation of Nestin-positive area 5 days after surgery (n=3). D: Evaluation of Nestin-positive area 7 days after surgery (n=3). [Diagram 3] The undifferentiated Schwann cell area is shown 3 to 7 days after surgery. A: Undifferentiated Schwann cell area. The area inside the dashed line is the transplanted section (Scale bar: 500 μm). B: Evaluation of p75 positive area 3 days after surgery (n=3). C: Evaluation of p75 positive area 5 days after surgery (n=3). D: Evaluation of p75 positive area 7 days after surgery (n=3). [Figure 4] Evaluation results of migration, angiogenesis, and proliferation of HUVECs. A and B: Scratch assay. C and D: Tube formation analysis. E: Cell proliferation assay (BrdU assay). [Diagram 5] RTK assay and Ras protein measurement results A, B: RTK response to VEGF and MeCbl administration C: Ras protein measurement results [Figure 6] Western blot results after MeCbl administration at different times A; p-Akt / Akt Western blot results B; p-ERK / ERK Western blot results [Figure 7] Western blot results A; p-Akt / Akt evaluation results B; p-ERK / ERK evaluation results [Figure 8] Western blot results A; Evaluation results of p-PI3K / PI3K B; Evaluation results of p-mTOR / mTOR [Figure 9] Western blot results A; p-Akt / Akt evaluation results B; p-ERK / ERK evaluation results [Figure 10] Results of evaluation of RTK activation using the ECL Western blotting detection system [Figure 11] Measurement results of Ras activity using Ras activation ELISA kit [Figure 12] Western blot results showing the effects of Nbtb, SAM, and Cysmethynil on the phosphorylation of Akt, CREB, and ERK. [Figure 13] Western blot results investigating the involvement of various Ras superfamily members in phosphorylation of Akt and ERK. [Figure 14] The effect of M-Ras overexpression on the phosphorylation of Akt and ERK was measured by Western blot. [Figure 15] The effect of M-Ras overexpression on the expression of Arg1 and CD206 as measured by Western blot. [Figure 16] Western blot results A; Arg1 / GDPDH evaluation results B; CD206 / GDPDH evaluation results DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] 1. Active Ingredients Vitamin B, the active ingredient of this pharmaceutical composition 12 Vitamin B includes cobalamin, its derivatives, and their salts. 12 Specific examples of the cobalamin include cobalamin with a substituted cobalt ion, and derivatives thereof. More specific examples include methylcobalamin, cyanocobalamin, hydroxocobalamin, sulfitocobalamin, adenosylcobalamin, and salts thereof. Among these, methylcobalamin, cyanocobalamin, hydroxocobalamin, and salts thereof are preferred, and methylcobalamin and salts thereof are more preferred.

[0016] Salts of cobalamin and its derivatives are not particularly limited as long as they are pharma- ceutically acceptable salts, and can be either acidic or basic salts.For example, examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; amino acid salts such as aspartate and glutamate.In addition, examples of basic salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt.

[0017] Vitamin B 12 The solvent is not particularly limited as long as it is pharma- ceutically acceptable, and examples of the solvent include water, ethanol, glycerol, and acetic acid.

[0018] Vitamin B 12 may be one type alone or two or more types in combination.

[0019] 2.Applications Vitamin B 12 Vitamin B has an angiogenesis promoting effect in the early stage after tissue damage, such as peripheral nerve damage. 12can be used as an active ingredient of a pharmaceutical composition for promoting angiogenesis.

[0020] Vitamin B 12 has the effects of inhibiting apoptosis, inhibiting necrosis, promoting axonal extension, promoting the induction of M2 macrophages / microglia, inhibiting the induction of M1 macrophages / microglia, and promoting nerve regeneration. 12 can be used as an active ingredient in apoptosis inhibitors, necrosis inhibitors, axonal extension promoters, M2 macrophage / microglia induction promoters, M1 macrophage / microglia induction inhibitors, M1:M2 ratio (ratio of M1 macrophages / microglia to M2 macrophages / microglia), nerve regeneration promoters, etc.

[0021] In addition, vitamin B 12 can be used as an active ingredient of an angiogenesis promoter in the treatment of tissue damage such as peripheral nerve damage and in promoting the healing of tissue wounds, based on at least one selected from the group consisting of vascular endothelial cell migration action, angiogenesis promotion action, PI3K-Akt-mTOR pathway activation action, Akt pathway activation action, Ras protein expression activation action, macrophage Ras activation action, and M-Ras activation action.

[0022] Peripheral nerve injury causes various symptoms, including various degrees of motor disorders (motor paralysis, muscle weakness, etc.), sensory disorders (sensory paralysis, numbness, etc.), and autonomic nerve disorders (abnormal sweating, etc.). Peripheral nerve injury is often caused by trauma, and the main problems are motor paralysis and sensory paralysis. While central nerve injury causes spastic paralysis, peripheral nerve injury causes flaccid paralysis. Furthermore, peripheral nerve injury causes significant muscle atrophy and sensory receptor atrophy due to the lack of neurotropic factors.

[0023] The pharmaceutical composition of the present invention comprises vitamin B 12There is no particular limitation as long as it contains the above, and other components may be included as necessary. The other components are not particularly limited as long as they are pharma- ceutically acceptable. The other components include additives as well as components having pharmacological effects. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, flavors, chelating agents, etc.

[0024] In addition, vitamin B 12 can independently exhibit angiogenesis-promoting effect, tissue damage therapeutic effect, tissue wound healing-promoting effect, nervous system disease therapeutic effect, apoptosis-suppressing effect, necrosis-suppressing effect, axon extension-promoting effect, M2 macrophage / microglia induction-promoting effect, M1 macrophage / microglia induction-suppressing effect (it is not denied that this is an effect of shifting from M1 macrophages / microglia to M2 macrophages / microglia), nerve regeneration-promoting effect, etc. Therefore, the pharmaceutical composition can exhibit the desired effect even without containing other components having these effects and / or actions, but may contain other components having a pharmacological action.

[0025] The subject to which the pharmaceutical composition can be applied is not particularly limited, and examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.

[0026] The pharmaceutical composition may be in any dosage form, for example, oral formulations such as tablets (including oral disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jellies, or parenteral formulations such as injections (e.g., drip injections (e.g., intravenous drip preparations), intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topicals (e.g., sheets, sprays, ointments, poultices, and lotions), suppositories, inhalants, eye drops, eye ointments, nasal drops, ear drops, and liposomes. In particular, formulations that can be administered to the affected area continuously are preferred. It is possible.

[0027] The route of administration of the pharmaceutical composition is not particularly limited as long as the desired effect can be obtained, and examples of the route include enteral administration such as oral administration and enema administration, as well as intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and parenteral administration using topical preparations.

[0028] According to the present invention, a pharmaceutical composition for promoting angiogenesis for treating tissue damage such as peripheral nerve damage can be provided. EXAMPLES

[0029] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0030] [Test Example I] The angiogenic effect of MeCbl in the early stage after peripheral nerve injury was examined using vascular endothelial cells.

[0031] Example 1 <Example 1-1. Preparation of sciatic nerve> The experiment was carried out using Wistar rats (8 weeks old, male, 200g, Oriental Yeast). The left sciatic nerve of the rat was exposed under general anesthesia, and a 5mm length of the sciatic nerve was harvested. The harvested nerve slice was frozen in liquid nitrogen and allowed to thaw naturally, which was repeated five times to decellularize the harvested slice. The decellularized tissue slice was used to crosslink the sciatic nerve defect, and two groups were prepared: one group in which an osmotic pump containing MeCbl at a concentration adjusted to release 1mg / kg / day subcutaneously was placed (MeCbl administration group), and the other group in which an osmotic pump containing only saline was placed (saline administration group).

[0032] <Example 1-2. Tissue preparation and immunohistochemical test> The grafted nerve sections were harvested on the 3rd, 5th, and 7th days after surgery and soaked in 4% formalin solution overnight at room temperature. They were then transferred to 30% sucrose solution, left at room temperature for 2 days, and frozen using embedding medium. The sections frozen in a vacuum cryostat were cut into 10 μm thick slices and immunostained using Nestin (ab6142) and low affinity nerve growth factor receptor (p75) (Chemicon International, Inc. AB1554 / 100UL) as primary antibodies. The areas of the neovascular part stained with Nestin and the undifferentiated Schwann cell part stained with p75 were evaluated using public domain image processing software (Image Processing and Analysis in Java: Image J) and were evaluated as a percentage of the total area of ​​the grafted section.

[0033] As an example of the results of Example 1, FIG. 1 shows the initial appearance after transplantation of frozen sections, FIG. 2 shows the results of the neovascularized area of ​​the transplanted section, and FIG. 3 shows the results of the undifferentiated Schwann cell area of ​​the transplanted section. At day 0 after surgery, it was confirmed that neither Nestin nor p75 was stained (FIG. 1C). The vascular area indicated by the Nestin-positive area was not observed in the transplanted section, but was confirmed from day 3 after surgery onwards. At day 5 after surgery, bridging by blood vessels was observed in both the MeCbl-administered group and the saline-administered group. The area of ​​the vascular area was significantly increased in the MeCbl-administered group at days 3 and 5 after surgery (FIGS. 2A, B, C). From the above, it was demonstrated that the administration of MeCbl promotes the rate of angiogenesis. The Schwann cell bridge, indicated by the p75-positive area, was completed by postoperative day 7. The area of ​​the undifferentiated Schwann cell region tended to increase in the MeCbl-administered group on postoperative days 3 and 5 (Fig. 3B, C), and was significantly increased in the MeCbl-administered group on postoperative day 7 (Fig. 3D). These results demonstrate that administration of MeCbl promotes angiogenesis as well as the generation of Schwann cells that form and maintain the myelination sheath that surrounds nerve axons.

[0034] Example 2 Example 2-1. Culture of human umbilical vein endothelial cells Human umbilical vein endothelial cells (HUVEC) were obtained from the JCRB Cell Bank and cultured. MCDB107 medium was used as the medium, and was prepared by adding 10% FBS and 1% penicillin. The cultured HUVEC were used in Examples 2 and 3.

[0035] <Example 2-2. Cell proliferation measurement test> 5.0×10 HUVECs were placed in a 96-well plate. 3 The cells were seeded and MeCbl was administered at 0 to 1 mM. After 12 hours and 24 hours, the cells were reacted with bromodeoxyuridine (BrdU) using a cell proliferation assay kit (Bromodeoxyuridine Assay Kit: BrdU Assay Kit) according to the attached instructions, and cell proliferation was evaluated.

[0036] <Example 2-3. Wound healing assay (scratch assay)> HUVECs were placed in a 2-well insert plate at 2.0 × 10 4 The cells were seeded and cultured in MCDB107 medium supplemented with 0.5% FBS for 24 hours to standardize the cell cycle. After that, the insert was removed and MeCbl was administered at 0 to 1 mM. The area of ​​the gap was evaluated using Image J after 12 hours and 24 hours.

[0037] <Example 2-4. Tube formation image analysis> The surface of a 96-well plate was coated with 50 μl / well of Matrigel (extracellular matrix, BD Biosciences), and HUVECs were cultured at 1.0 × 10 3 After 12 hours, the number of vascular connections formed was counted and evaluated using Image J.

[0038] An example of the results of Example 2 is shown in Figure 4. In the cell proliferation assay (BrdU Assay), no significant difference was observed between each concentration group of MeCbl administration and the control group (Figure 4E). In other words, it was considered that abnormal cell proliferation due to the administration of MeCbl was not observed. On the other hand, in the scratch assay, when evaluated 24 hours after the start, significant differences were observed in the MeCbl administration groups at concentrations of 10 nM, 100 nM, and 1 μM compared with the control group (Figures 4A and 4B). In addition, in the tube formation image analysis, the number of vascular junctions was significantly increased in the MeCbl administration groups at concentrations of 10 nM, 100 nM, 1 μM, and 10 μM (Figures 4C and 4D). These results indicate that MeCbl has the effect of promoting vascular endothelial cell migration and angiogenesis at a certain concentration.

[0039] Example 3 <Example 3-1. Receptor tyrosine kinase (RTK) assay> HUVECs were treated with 100 nM MeCbl or 10 ng / ml vascular endothelial growth factor (VEGF) and incubated for 30 minutes. Proteins were extracted according to the protocol of the Receptor Tyrosine Kinase Assay Kit (RTK Assay Kit), and the reaction to the membrane prepared in advance was evaluated.

[0040] <Example 3-2. Measurement of activated Ras> HUVECs were treated with 100nM MeCbl, 10ng / ml VEGF, 1μM methionine synthase inhibitor (4-nitro-2,1,3-benzothiadiazole: Nbtd), or 360nM VEGF receptor inhibitor (VEGF Receptor Blocker: VEGFR blocker) and incubated for 30 minutes. Proteins were extracted according to the kit's instructions, and their reaction to a pre-prepared Ras antibody was evaluated.

[0041] <Example 3-3. Western blot analysis> HUVECs were treated with 100nM MeCbl, 10ng / ml VEGF, or 100nM MeCbl + 10ng / ml VEGF, incubated for 30 minutes, and then protein was collected. In addition, 360μM VEGFR blocker (Sigma-Aldrich 676481) was administered, incubated for 30 minutes, and then 100nM MeCbl or 10ng / ml VEGF was administered, and protein was collected. The collected proteins were electrophoresed and transferred to a membrane, and p-Akt / Akt, p-ERK / ERK, p-PI3K / PI3K, and p-mTOR / mTOR were evaluated.

[0042] As an example of the results of Example 3, FIG. 5 shows the results of receptor tyrosine kinase assay and activated Ras protein measurement, and FIGS. 6 to 9 show the results of Western blot analysis. The results of the receptor tyrosine kinase assay showed that the MeCbl-treated group did not respond to RTK on the cell membrane surface, unlike the VEGF-treated group (Fig. 5A, B). On the other hand, MeCbl activated Ras in the same way as VEGF did (Fig. 5C). In addition, Western blot analysis showed that MeCbl administration did not activate the extracellular signal-regulated kinase (ERK) pathway, but did activate the phosphatidylinositol 3-kinase / protein kinase B / mammalian target of rapamycin (PI3K-AKT-mTOR) pathway (Fig. 6A, B and Fig. 7A, B). Furthermore, simultaneous administration of MeCbl and VEGF did not enhance the effect, and activation of the PI3K-Akt-mTOR pathway by MeCbl administration was observed even when a VEGFR blocker was administered (Fig. 7A, B and Fig. 8A, B).

[0043] Furthermore, administration of Nbtd, an inhibitor of methionine synthase, reduced Akt activation by MeCbl administration, and the addition of S-Adenocyl Methionine (SAM) increased Akt activity again. However, administration of an isoprenylcysteine ​​carboxyl methyltransferase (Icmt)-inhibitor did not increase Akt activity even in the presence of MeCbl or SAM (Fig. 9A, B). These results suggest that MeCbl activates the Akt pathway through the methylation cycle and the action of Icmt.

[0044] [Test Example II] It has been revealed that systemic administration of MeCbl therapy shifts the phenotype of macrophages that gather around the injured area from inflammatory M1 to anti-inflammatory / tissue repair M2. Furthermore, from the above Test Example I, the mechanism of action of MeCbl in in vitro experiments was thought to be the promotion of phosphorylation of intracellular signaling molecules Akt and Ras. Furthermore, with the aim of clarifying the detailed mechanism, the subtypes of Ras proteins that are susceptible to the action of MeCbl were investigated using macrophage cells. Specifically, the following is done.

[0045] Example 4 RTKs, such as insulin receptor and VEGF receptor, have Ras downstream, which further leads to phosphorylation of Akt and ERK. To investigate whether this mechanism is the mechanism by which MeCbl promotes Akt phosphorylation, we evaluated RTK activation using the Proteome profiler mouse phospho-RTK array kit.

[0046] <Example 4-1. Preparation of macrophage cell line> The mouse-derived macrophage cell line J774A.1 (JCRB9108) was purchased from the JCRB Cell Bank (Culture Resource Research Laboratory) in Osaka Prefecture. Cultivation was carried out in Dulbecco’s modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. The cultured macrophage cells were used in Examples 4 to 7.

[0047] <Example 4-2. Phospho Receptor Tyrosine Kinase (RTK) Array> Based on preliminary experiments, MeCbl (Merck) was added to the cell culture medium of the mouse macrophage cell line J774A.1 to a concentration of 100 nM. After 30 minutes, proteins were extracted from the cells. Using the Proteome profiler mouse phospho RTK array kit (R&D systems), the detection of tyrosine-phosphorylated RTK was performed according to the procedure manual using an ECL Western blotting detection system.

[0048] An example of the results of Example 4 is shown in Figure 10. No difference in RTK phosphorylation was observed between the non-MeCbl-administered group and the administered group. From this, it was considered that MeCbl does not promote RTK phosphorylation.

[0049] Example 5 Although it was shown from the results of Example 4 above that MeCbl does not phosphorylate RTK, in order to investigate whether it activates Ras regardless, the Ras activity was measured using a Ras activation ELISA kit. <Measurement of Activated Ras by Ras activation ELISA kit> The activation of Ras by MeCbl administration was examined using a Ras activation ELISA kit (Cell Biolabs). 100 nM MeCbl alone or MeCbl and 1 μM 5-(3-methylphenyl)-1-octyl-1H-indole-3-acetamide (cysmethynil) (an inhibitor of isoprenylcysteine ​​carboxyl methyltransferase (Icmt) that activates Ras by methylating it) were added to macrophage cell culture medium, and proteins were collected from the cells 30 minutes later. The amount of activated Ras was measured by ELISA according to the manufacturer's instructions.

[0050] An example of the test results of Example 5 is shown in Figure 11. Significant activation of Ras was observed in the MeCbl-administered group compared to the control group, and activated Ras was significantly reduced in the group administered Cysmethynil in addition to MeCbl, reaching the same level as the control group. From these results, it was considered that MeCbl promotes phosphorylation of Ras via Icmt, not via RTK.

[0051] Example 6 MeCbl acts as a coenzyme for methionine synthesis and contributes to the methylation cycle, but SAM, a component of the cycle, donates a methyl group to inactive Ras through the action of Icmt, causing Ras to become active. The results of Example 5 above suggested the involvement of Icmt in Ras activation by MeCbl, but to confirm whether Ras is activated by the above-mentioned mechanism, studies were carried out using 1 μM of methionine synthase inhibitors Nbtd (Cayman), SAM, and Cysmethynil. The amount of each protein was measured by the following Western blot analysis.

[0052] <Western blot analysis> Macrophage cells were seeded in 6 cm dishes, and after 3-4 days, proteins were collected using cell lysis buffer containing protease inhibitor cocktail. Protein concentrations were measured using a BCA assay, and 50 μg of each sample was electrophoresed by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After blocking with blocking buffer for 1 hour, the cells were reacted with primary antibodies four times overnight. The secondary antibodies were reacted at room temperature for 1 hour, and detected using an ECL Western blotting detection system. The primary antibodies used were anti-Akt rabbit monoclonal antibody (CST), anti-p-Akt rabbit monoclonal antibody (CST), anti-CREB rabbit monoclonal antibody (CST), anti-p-CREB rabbit monoclonal antibody (CST), anti-ERK rabbit monoclonal antibody (CST), and anti-p-ERK rabbit monoclonal antibody (CST). The secondary antibody used was anti-rabbit IgG horseradish peroxidase linked whole antibody from donkey (GE Healthcare Life Sciences).

[0053] An example of the results of Example 6 is shown in Fig. 12. The promoting effect of MeCbl on Akt phosphorylation was suppressed by Nbtd, and when SAM downstream of it was added, Akt phosphorylation was rescued. When Cysmethynil was further added here, Akt phosphorylation was suppressed again. When Cysmethynil was added simultaneously with MeCbl, Akt phosphorylation was also suppressed. Similar changes to those in Akt phosphorylation were also seen in the phosphorylation of CREB, a transcription factor downstream of Akt. On the other hand, no particular change was observed regarding the phosphorylation of ERK. From these results, as described above, it was considered that MeCbl promotes Akt phosphorylation via Ras activation by Icmt through the methylation cycle.

[0054] Example 7 Activated Ras generally acts on the phosphorylation of both Akt and ERK. However, in this example, MeCbl was mainly involved in Akt phosphorylation and had no certain influence on ERK. From this, it was predicted that MeCbl might affect Ras that acts particularly on Akt phosphorylation among the Ras superfamily. M-Ras has such characteristics, and thus, knockdown of more common Ras such as H-Ras, K-Ras, and N-Ras was performed to examine the Ras that is strongly affected by MeCbl.

[0055] <Knockdown treatment of Ras gene> To examine the Ras superfamily that mainly acts in the Ras activation by MeCbl, siRNAs (Ambion) of H-Ras, K-Ras, N-Ras, and M-Ras were lipofected into macrophage cells using Lipofectamine RNAiMAX (Invitrogen) from 24 hours before MeCbl administration. Protein collection from the cells was performed 30 minutes after MeCbl administration. Each protein was detected by Western blot analysis in the same manner as in Example 6 above.

[0056] An example of the results of Example 7 is shown in FIG. 13. In cells with knockdown of H-Ras and N-Ras, the promoting effect of MeCbl administration on Akt phosphorylation was still observed. In cells with knockdown of K-Ras, a similar trend was observed, but no significant promotion was seen. On the other hand, in cells with knockdown of M-Ras, Akt phosphorylation by MeCbl was not observed at all. Regarding ERK phosphorylation, no significant change was seen in MeCbl administration with knockdown of any Ras. From the above results, it was considered that the Ras activated by MeCbl was mainly M-Ras among the superfamily.

[0057] Example 8 Since it was considered that the Ras activated by MeCbl was mainly M-Ras among the superfamily as described above, the following tests were conducted for confirmation.

[0058] <Effect of MeCbl in Overexpression of M-Ras Gene> Overexpression of M-Ras was transfected into macrophage J774A.1 cells using Lipofectamine 3000 (ThermoFisher) with pre-designed plasmid DNA purchased from Origene. For verification of intracellular signals, MeCbl was administered 48 hours after transfection, and protein collection was performed 30 minutes later. In the case of verification of M2 marker expression, IL-4 and MeCbl were administered simultaneously with transfection, and protein collection was performed 48 hours later.

[0059] An example of the results of Example 8 is shown in Figures 14 to 16. Akt phosphorylation was promoted even with only M-Ras overexpression without the addition of MeCbl, and ERK was not affected (Figure 14). When the expression of phenotype proteins of M2 macrophages was actually updated, the expression levels of M2 markers Arg1 and CD206 were significantly increased in the case of overexpressing M-Ras in addition to IL-4 administration compared to the case of administering IL-4 alone, and the degree of increase was equivalent to that in the case of administering IL-4 and MeCbl. Furthermore, when M-Ras was overexpressed in addition to administering IL-4 and MeCbl, no synergistic increase in the expression levels of Arg1 and CD206 was observed (Figures 15 and 16). From these results, as in the results of Example 7, it was considered that the effect of MeCbl in promoting Akt phosphorylation was mainly mediated by M-Ras of the Ras family. [Industrial Applicability]

[0060] From the above results, the vitamin B 12 The pharmaceutical composition containing as an active ingredient has a vascular endothelial cell migration effect, an angiogenesis promoting effect, and further exhibits a phosphorylation promoting effect of PI3K, Akt, and mTOR, and has been shown to have an activation effect of the PI3K-Akt-mTOR pathway involved in angiogenesis. In addition, since the pharmaceutical composition has an Akt pathway activation effect via the action of the methylation cycle and Icmt, an activation effect of RAS protein expression, an activation effect of Ras in macrophages, an activation effect of M-Ras, etc., it is highly useful as an angiogenesis promoting agent in the early stage after tissue injury such as peripheral nerve injury. Furthermore, since wound healing is delayed in diabetes, aging, etc., the pharmaceutical composition can also be used as an angiogenesis promoting agent to promote the healing of such tissue wounds.

Claims

1. Vitamin B 12 A pharmaceutical composition for promoting angiogenesis comprising:

2. 2. The pharmaceutical composition for promoting angiogenesis according to claim 1, wherein the promotion of angiogenesis is achieved by promoting the migration of vascular endothelial cells and / or promoting the formation of blood vessels.

3. The pharmaceutical composition for promoting angiogenesis according to claim 1, wherein the promotion of angiogenesis is due to activation of Ras in an intracellular signal transduction pathway.

4. The pharmaceutical composition for promoting angiogenesis according to claim 3, wherein Ras is M-Ras.

5. The pharmaceutical composition for promoting angiogenesis according to claim 3, wherein the cells are vascular endothelial cells and / or macrophages.

6. The pharmaceutical composition for promoting angiogenesis according to any one of claims 1 to 5, which is used for treating tissue damage.

7. The pharmaceutical composition for promoting angiogenesis according to claim 6, which is used for treating peripheral nerve injury.

8. The pharmaceutical composition for promoting angiogenesis according to claim 6 , which is used for promoting wound healing.

9. Vitamin B 12 The pharmaceutical composition for promoting angiogenesis according to any one of claims 1 to 5, wherein said compound is methylcobalamin.

10. A method for producing the pharmaceutical composition for promoting angiogenesis according to any one of claims 1 to 5, comprising administering to said patient a vitamin B 12 Use of.

11. Vitamin B for use in the treatment of tissue damage by promoting angiogenesis 12 Containing pharmaceutical composition.

12. 12. The vitamin B according to claim 11, wherein the treatment of tissue damage is treatment of peripheral nerve damage. 12 Containing pharmaceutical composition.

13. 12. The vitamin B according to claim 11, wherein the treatment of tissue damage is the promotion of wound healing. 12 Containing pharmaceutical composition.

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