Protein nitration inhibitor and prophylactic or therapeutic agent for disease or symptom caused by protein nitration

Abstract

This protein nitration inhibitor or a prophylactic or therapeutic agent for diseases or symptoms caused by protein nitration contains glycerol or a prodrug thereof. This method for preventing or treating diseases or symptoms caused by protein nitration in a subject includes a step for administering glycerol or a prodrug thereof to the subject.

Description

Protein nitration inhibitor and preventive or therapeutic agent for diseases or symptoms caused by protein nitration

[0001] The present invention relates to a protein nitration inhibitor and a preventive or therapeutic agent for diseases or symptoms caused by protein nitration.

[0002] Proteins may be nitrated by peroxynitrite (a reactive nitrogen species rapidly generated by the reaction of nitric oxide radicals and reactive oxygen species) generated in vivo, and a nitro group is added to the benzene ring of tyrosine residues and tryptophan residues (especially tyrosine residues) among the amino acids constituting the protein, resulting in protein nitration.

[0003] In recent years, it has been reported that protein nitration is related to various diseases and has attracted attention as a new factor in various diseases. For example, in Non-Patent Document 1, nitrated tyrosine (3-nitrotyrosine) occurs before the symptoms of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and prion disease appear, and it is described that nitrated tyrosine becomes a target for early diagnosis of these diseases. Also, for example, in Non-Patent Documents 2 to 3, it has been reported that when tyrosine residues in the protein of hepatocyte growth factor (HGF) are nitrated, the physiological activity of HGF disappears.

[0004] Regarding the inhibition of protein nitration, for example, Patent Document 1 discloses that an extract of jaboticaba (Plinia cauliflora) has an effect of inhibiting protein nitration.

[0005] Japanese Unexamined Patent Application Publication No. 2023-128847

[0006] M. Bandookwala et al., “3-Nitrotyrosine: a versatile oxidative stress biomarker for major neurodegenerative diseases”, International Journal of Neuroscience, 2020Oct;130(10):1047-1062.A. Elgaabari et al., “A pilot study on nitration / dysfunction of NK1segment of myogenic stem cell activator HGF”, Biochemistry and Biophysics Reports Vol. 31, e101295 (2022).A. Elgaabari et al., “Age-related nitration / dysfunction of myogenic stem cell activator HGF”, Aging Cell Vol. 23, e14041 (2024).

[0007] The present invention aims to provide a novel protein nitration inhibitor.

[0008] The inventors of this invention discovered that glycerol can suppress the nitration of hepatocyte growth factor (HGF), leading to the completion of this invention.

[0009] In other words, the present invention relates to, for example, the following inventions: [1] A protein nitration inhibitor containing glycerol or a prodrug thereof. [2] The nitration inhibitor according to [1], wherein the protein contains hepatocyte growth factor. [3] The nitration inhibitor according to [1] or [2], which is administered orally, sublingually, intramuscularly, enterally, nasally, intravenously, subcutaneously, or intraperitoneally. [4] A preventive or therapeutic agent for diseases or symptoms caused by protein nitration, containing glycerol or a prodrug thereof. [5] The preventive or therapeutic agent according to [4], wherein the disease is selected from the group consisting of age-related sarcopenia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, prion disease, atherosclerosis, and colorectal cancer. [6] The preventive or therapeutic agent according to [4] or [5], wherein the disease is age-related sarcopenia. [7] A preventive or therapeutic agent according to any one of [4] to [6], wherein the above symptoms are muscle atrophy and / or muscle regeneration failure. [8] A preventive or therapeutic agent according to any one of [4] to [7], wherein the above protein comprises hepatocyte growth factor. [9] A preventive or therapeutic agent according to any one of [4] to [8], administered orally, sublingually, intramuscularly, enterally, nasally, intravenously, subcutaneously, or intraperitoneally.

[10] A nitration inhibitor according to any one of [1] to [3], wherein the nitration of the above protein comprises in vivo and / or in vitro nitration.

[11] A method for preventing or treating a disease or symptom caused by protein nitration in a subject, comprising the step of administering glycerol or a prodrug thereof to the subject.

[12] Glycerol or a prodrug thereof for use in the prevention or treatment of a disease or symptom caused by protein nitration.

[13] Use of glycerol or a prodrug thereof in the manufacture of a preventive or therapeutic agent for a disease or symptom caused by protein nitration.

[14] Use of glycerol or its prodrug for inhibiting protein nitration in vitro.

[15] Use of a composition comprising glycerol or its prodrug for inhibiting protein nitration in vitro.

[0010] According to the present invention, a novel protein nitration inhibitor can be provided.

[0011] This figure shows the results of evaluating the effect of glycerol on the nitration of HGF by Western blotting. This figure shows the results of evaluating the physiological activity of HGF by bromodeoxyuridine (BrdU) uptake assay. This figure shows the results of evaluating the binding ability of HGF to the cell membrane receptor c-met by c-met binding assay. This figure shows the results of comparing the food intake of mice in each group when glycerol was administered to the drinking water of a disuse muscle atrophy model mouse. This figure shows the results of comparing the muscle weight of mice in each group when glycerol was administered to the drinking water of a disuse muscle atrophy model mouse. This figure shows the results of immunostaining of frozen sections of calf muscle (Gas + Sol + Pla) from mice in each group when glycerol was administered to the drinking water of a disuse muscle atrophy model mouse with anti-nitrated Y250 monoclonal antibody.

[0012] The following describes in detail embodiments for carrying out the present invention. However, the present invention is not limited to the following embodiments.

[0013] The protein nitration inhibitor according to this embodiment (hereinafter also referred to as "nitration inhibitor according to this embodiment") contains glycerol or its prodrug.

[0014] Glycerol is a compound also known as glycerin or propane-1,2,3-triol, and is a type of trihydric alcohol.

[0015] Glycerol prodrugs are substances that are metabolized in the body to become glycerol and exert their effects. Examples include nitroglycerin, glycerol trinitrate, glycerol esters (e.g., glycerol monolaurate), and glycerol salts (e.g., sodium glycerolate).

[0016] The protein can be any protein that can be nitrated, and may contain tyrosine residues and / or tryptophan residues, but it is preferable that the protein contains tyrosine residues. If the protein contains tyrosine residues, the side chains of the tyrosine residues of the protein must be exposed on the surface (solvent-exposed surface area; Accessible Surface Area (ASA) value of 65 Å). ２ Preferably, the protein has tyrosine residues located near glutamate residues and / or tyrosine residues not located near cysteine ​​residues, and it is preferable that the protein has tyrosine residues located near glutamate residues and not near cysteine ​​residues. Here, "nearby" means that when the protein forms a three-dimensional structure, the two amino acid residues are located at a distance of 100 Å or less, 90 Å or less, 80 Å or less, 70 Å or less, 60 Å or less, 50 Å or less, 40 Å or less, 30 Å or less, 20 Å or less, or 10 Å or less.

[0017] It has been reported that tyrosine residues adjacent to glutamic acid residues show a high tendency to nitrate (JY. Ng et al., Open Biology, 2013 Feb 6;3(2):120148.), while cysteine ​​residues undergo peroxynitrite (ONOO － It has been reported that it acts as an intramolecular scavenger, thus moving peroxynitrite away from tyrosine residues (N. Abello et al., Ph.D. Thesis University Groningen / UMCG researchdatabase (2009), A. Petruk et al., Archives of Biochemistry and Biophysics, 525(1), 82-91).

[0018] Specific examples of proteins include hepatocyte growth factor (HGF), alpha-synuclein, tau, and various albumins (e.g., serum albumin), with hepatocyte growth factor (HGF) being preferred.

[0019] It is known that when HGF binds to the cell membrane receptor c-met, for example, muscle stem cells (satellite cells) are activated. Furthermore, HGF dysfunction leads to inhibition of muscle growth, hypertrophy, regeneration, or maintenance, causing muscle atrophy or muscle regeneration failure. In HGF, it is known that tyrosine residues corresponding to the 198th and 250th tyrosine residues in human HGF can be nitrated. In avian HGF, it is known that tyrosine residues corresponding to the 250th tyrosine residue in human HGF can be nitrated. It is known that nitration of the 198th and / or 250th tyrosine residues in human HGF, as well as the corresponding tyrosine residues in non-human HGF, causes the loss of HGF activity. The protein nitration inhibitor according to this embodiment can suppress the nitration of the tyrosine residues at the specific positions described above, when the protein is HGF.

[0020] The nitration inhibitor according to this embodiment can suppress the nitration of HGF, and can also suppress the decrease in HGF's ability to bind to c-met, as well as the decrease in its downstream effects (such as the activation of muscle stem cells (satellite cells) by HGF). In other words, when the protein is HGF, the nitration inhibitor according to this embodiment can be used to suppress the decrease in HGF's ability to bind to c-met, and to suppress the decrease in the ability of HGF to activate muscle stem cells (satellite cells).

[0021] The nitration inhibitor according to this embodiment contains glycerol or its prodrug, and therefore, the presence of the protein nitration inhibitor according to this embodiment reduces the nitration level of the protein compared to the case where the nitration inhibitor is not present. Here, protein nitration includes in vivo and / or in vitro nitration. The reduction in the protein nitration level may be, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 60% or more, or 70% or more compared to the case where the protein nitration inhibitor according to this embodiment is not present. Since glycerol or its prodrug does not react specifically with specific proteins such as antibodies, it is considered that it can inhibit nitration for all proteins that can be nitrated.

[0022] The inhibitory effect on protein nitration can be evaluated, for example, by the method used in the examples described below. Specifically, for example, protein and peroxynitrite (ONOO － By mixing the two substances and performing a protein nitration treatment, the inhibition of protein nitration can be evaluated by Western blotting or other methods using an antibody that can specifically detect nitrated proteins (proteins containing nitrated amino acid residues (tyrosine residues or tryptophan residues)).

[0023] The glycerol or its prodrug content in the nitration inhibitor according to this embodiment may be, for example, 1% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, 95% by mass or more, 96% by mass or more, 97% by mass or more, 98% by mass or more, 99% by mass or more, or 100% by mass, based on the total mass of the nitration inhibitor.

[0024] The nitration inhibitor according to this embodiment may further contain, in addition to glycerol or its prodrug, additives such as excipients, buffers, stabilizers, antioxidants, binders, disintegrants, fillers, and emulsifiers. Furthermore, these additives are preferably pharmaceutically acceptable.

[0025] In suppressing protein nitration in vivo, the target of administration of the nitration inhibitor according to this embodiment may be a mammal or a bird, and may be a human or a non-human mammal. Furthermore, the target of administration of the nitration inhibitor according to this embodiment may be livestock or poultry, and is preferably poultry. Examples of livestock include cattle, horses, pigs, donkeys, sheep, and goats, and examples of poultry include chickens, ducks, geese, ostriches, turkeys, waterfowl, and quail.

[0026] The dosage of the nitration inhibitor according to this embodiment for inhibiting protein nitration in vivo is not particularly limited as long as an amount that can be obtained to inhibit protein nitration is obtained, and may vary depending on the symptoms, disease, age, route of administration, dosage form, etc., and can be appropriately determined by those skilled in the art. The dosage of the nitration inhibitor according to this embodiment can be administered to the subject as glycerol or its prodrug, for example, at a dose of 0.1 to 500 mg / kg per day, and 0.1 to 500 mg / kg may be administered once or in multiple doses per day.

[0027] The nitration inhibitor according to this embodiment may be administered, for example, orally, sublingually, intramuscularly, enterally, nasally, intravenously, subcutaneously, or intraperitoneally, and is preferably administered orally. For example, antibodies and the like cannot be administered orally because they are subjected to protein degradation by digestive enzymes in the body, and therefore need to be administered intravenously or intraperitoneally. In contrast, the nitration inhibitor according to this embodiment is not affected by digestive enzymes in the body, and therefore has the advantage of being able to exert its effect even by administration routes other than intravenous or intraperitoneal administration.

[0028] The dosage form of the nitration inhibitor according to this embodiment may be any of the following: solid, liquid, paste, etc., and may be tablets (including uncoated tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, lozenges, etc.), capsules, pills, powders, fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, or injections (including cases where the solution is prepared by mixing it with distilled water or an infusion solution such as an amino acid solution or electrolyte solution at the time of use).

[0029] The nitration inhibitor according to this embodiment can suppress protein nitration through the action of glycerol or its prodrug. Furthermore, as mentioned above, protein nitration has been reported to be related to various diseases. Therefore, by suppressing protein nitration, it is expected that diseases that develop or progress due to protein nitration can be prevented or treated.

[0030] Therefore, the nitration inhibitor according to this embodiment can also be configured as a preventive or therapeutic agent for diseases or symptoms caused by protein nitration.

[0031] Diseases caused by protein nitration may be selected from the group consisting of, for example, age-related sarcopenia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, prion disease, atherosclerosis, and colorectal cancer, with age-related sarcopenia being preferred. Age-related sarcopenia is a disease in which muscle mass or muscle strength decreases with age.

[0032] Symptoms resulting from protein nitration include, for example, muscle atrophy and / or muscle regeneration failure, and may also include muscle atrophy and / or muscle regeneration failure. Muscle atrophy and muscle regeneration failure may result from prolonged disuse of muscles or a decrease in the frequency or intensity of use due to aging, being bedridden, trauma, postoperative immobilization, weightlessness, or suffering from some disease.

[0033] Muscle atrophy and / or muscle regeneration failure may be muscle atrophy and / or muscle regeneration failure of the entire leg, muscle atrophy and / or muscle regeneration failure of the calf, or muscle atrophy and / or muscle regeneration failure of the soleus muscle.

[0034] Other embodiments of the preventive or therapeutic agent for diseases or symptoms caused by protein nitration according to this embodiment are the same as embodiments of the nitration inhibitor according to this embodiment.

[0035] The present invention also provides a method for preventing or treating a disease or symptom caused by protein nitration in a subject, comprising the step of administering glycerol or a prodrug thereof to the subject. In this embodiment, the subject may be administered an effective amount of glycerol or a prodrug thereof, which can be appropriately determined by those skilled in the art, and may be, for example, the dose of the nitration inhibitor described above. The subject may be a subject that has or is likely to have a disease or symptom caused by protein nitration, and the disease or symptom caused by protein nitration is as described above. The route of administration is not particularly limited and may be, for example, the same as the route of administration of the nitration inhibitor described above.

[0036] The present invention also provides glycerol or its prodrug for use in the prevention or treatment of diseases or symptoms caused by protein nitration. Here, the amount of glycerol or its prodrug used can be appropriately determined by those skilled in the art, and may be, for example, the dosage of the nitration inhibitor described above. The prevention or treatment of diseases or symptoms caused by protein nitration is as described above.

[0037] The present invention also provides the use of glycerol or its prodrugs in the manufacture of preventive or therapeutic agents for diseases or symptoms caused by protein nitration. The prevention or treatment of diseases or symptoms caused by protein nitration is as described above.

[0038] The present invention further provides the use of glycerol or its prodrug for inhibiting protein nitration in vitro, or the use of a composition containing glycerol or its prodrug for inhibiting protein nitration in vitro. The content of glycerol or its prodrug in the composition is not particularly limited and may be, for example, the content of glycerol or its prodrug in the nitration inhibitor described above.

[0039] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0040] [Example 1: Evaluation of the effect of glycerol on the nitration of HGF] Glycerol was added to a mouse recombinant HGF solution dissolved in PBS to concentrations of 0 v / v%, 0.03 v / v%, 0.17 v / v%, or 0.50 v / v%, and then immediately nitrated with peroxynitrite (ONOO － Nitration treatment was performed by adding () and the mixture was held for 30 minutes (pH 7.4, 25°C). The molar ratio of HGF to peroxynitrite was adjusted to HGF:peroxynitrite = 1:500.

[0041] Nitration of tyrosine in HGF (nitrated Y), nitration of the 250th tyrosine residue of HGF (nitrated Y250), and nitration of the 198th tyrosine residue of HGF (nitrated Y198) were detected by Western blotting. In Western blotting, an anti-nitrated Y monoclonal antibody, an anti-HGFα chain monoclonal antibody, an antibody that specifically binds to nitrated Y198 of HGF (anti-nitrated Y198-HGF monoclonal antibody), and an antibody that specifically binds to nitrated Y250 of HGF (anti-nitrated Y250-HGF monoclonal antibody) were used. The anti-HGFα chain monoclonal antibody, anti-nitrated Y198-HGF monoclonal antibody, and anti-nitrated Y250-HGF monoclonal antibody were each used to detect their respective targets on the membrane after removing the antibodies bound to the membrane using an SDS-β-mercaptoethanol solution for the membrane used when performing Western blotting with any of the antibodies.

[0042] A commercially available product (manufactured by Santa Cruz Biotechnology) was used as the anti-nitrated Y monoclonal antibody. A commercially available product (manufactured by Santa Cruz Biotechnology) was used as the anti-HGFα chain monoclonal antibody. The anti-nitrated Y198-HGF monoclonal antibody and anti-nitrated Y250-HGF monoclonal antibody were produced by the inventors (A. Elgaabari et al., Aging Cell. 2024 Feb;23(2):e14041. (The clone numbers are 1C6 and 2C3, respectively.)). The results are shown in Figure 1.

[0043] As shown in Figure 1, the detection levels of nitration Y, and the nitration of tyrosine residues 198 (Y198) and 250 (Y250) of HGF decreased in a glycerol concentration-dependent manner (first panel from the top and third-fourth panels in Figure 1). The detection level of the anti-HGFα chain remained almost the same regardless of the glycerol concentration, indicating that the amount of HGF on the transfer membrane was roughly the same. In other words, the decrease in the detection level of nitration described above indicates a decrease in the nitration level of the tyrosine residue (Y) of HGF (second panel from the top in Figure 1). These results indicate that the nitration of tyrosine residues 198 (Y198) and 250 (Y250) of HGF was suppressed in a glycerol concentration-dependent manner.

[0044] [Example 2: Evaluation of the effect of glycerol on the physiological activity of HGF] HGF is known to be an activator of muscle stem cells (satellite cells) essential for muscle hypertrophy and regeneration (R. Tatsumi et al., Developmental Biology 194(1), 114-128 (1998), R. Tatsumi et al., Experimental Cell Research 267(1), 107-114 (2001), R. Tatsumi et al., Molecular Biology of the Cell 13(8), 2909-2918 (2002), Tatsumi et al., Muscle & Nerve 30(5), 654-658 (2004), Tatsumi et al., American Journal of Physiology-Cell Physiology 290(6), C1487-C1494 (2006), Tatsumi et al., American Journal (ofPhysiology-Cell Physiology 296(4), C922-C929 (2009)). Therefore, the physiological activity of HGF was evaluated using muscle stem cells (satellite cells) isolated from rats by a bromodeoxyuridine (BrdU) uptake assay as follows.

[0045] Skeletal muscle stem cells (satellite cells) isolated from the skeletal muscle of adult rats according to a non-patent document (R. E. Allen et al., Methods Cell Biology, 1997; 52: 155-76.) were seeded and cultured in DMEM medium (pH 7.2) containing 10% normal horse serum for 24 hours. As samples to be tested, PBS (negative control), a sample containing HGF stored at 4°C until immediately before use (positive control 1 (HGF)), a sample containing HGF stored at room temperature (positive control 2 (HGF)), a sample containing nitrated HGF (nitrated HGF), a sample containing the nitrated HGF prepared in Example 1, a sample containing the nitrated HGF and glycerol at various concentrations, a sample obtained by adding 0.5 v / v% glycerol to the sample of positive control 1 (0.5% glycerol + HGF1), and a sample obtained by adding 0.5 v / v% glycerol to the sample of positive control 2 (0.5% glycerol + HGF2) were prepared.

[0046] The prepared samples were added to the medium and the muscle stem cells were cultured for an additional 24 hours. For the samples containing HGF, they were added to the medium such that the final concentration of HGF was 5 ng / mL. Thereafter, bromodeoxyuridine (BrdU, manufactured by Sigma-Aldrich) was added to a final concentration of 10 μM. Two hours after the addition of BrdU, the incorporation activity of BrdU was visualized by immunostaining using an anti-BrdU monoclonal antibody (clone number G3G4; obtained from the Developmental Studies Hybridoma Bank) and an HRP-labeled secondary antibody (manufactured by Sigma-Aldrich), and the activation rate of muscle stem cells was measured by calculating the ratio of BrdU-positive cells to all cells. The results are shown in Figure 2.

[0047] As shown in Figure 2, compared to the case where only PBS was added instead of HGF (negative control in Figure 2), the proportion of BrdU-positive cells was significantly higher (P-value less than 1%) when samples containing only HGF were added (positive controls 1 and 2 in Figure 2), indicating that muscle stem cells were activated by the addition of HGF. On the other hand, when samples containing nitrated HGF (nitrated HGF in Figure 2) were added, the proportion of BrdU-positive cells was similar to that when only PBS was added (negative control in Figure 2), indicating that the physiological activity of HGF is reduced when it is nitrated.

[0048] However, when samples containing nitrated HGF and glycerol of various concentrations (0%, 0.03%, 0.17%, and 0.5% in Figure 2) were added, the proportion of BrdU-positive cells increased significantly compared to when a sample containing nitrated HGF (nitrated HGF in Figure 2) was added (the P-value for the 0.03% glycerol-added group was 5% or less, and the P-values ​​for the 0.17% and 0.5% glycerol-added groups were 1% or less).

[0049] The results from Examples 1 and 2 indicate that the presence of glycerol suppresses the nitration of HGF and maintains the physiological activity of HGF (activation of muscle stem cells).

[0050] [Example 3: Evaluation of the effect of glycerol on the c-met binding ability of HGF] HGF is a cell growth factor and is known to express intracellular signaling activity by binding to c-met, a cell membrane receptor. Therefore, the ability of HGF to bind to c-met was evaluated as follows using a c-met binding assay (ELISA-like assay).

[0051] The following samples were prepared for testing: PBS (a, negative control), a sample containing only HGF stored at 4°C until immediately before use (b, positive control), a sample containing HGF after nitration treatment prepared in Example 1 (c), samples containing HGF nitrated in the presence of various concentrations of glycerol (d, e, f), and an HGF sample containing 0.5 v / v% glycerol (g, HGF: no nitration treatment).

[0052] Recombinant c-met Fc chimeras (50 ng / well) were coated onto 96-well plates and blocked with PBS containing 1% BSA, 5% sucrose, and 0.05% sodium azide. The samples to be tested were then added to the wells and incubated at 37°C for 2 hours. Subsequently, the plates were washed with 0.1% polyethylene sorbitan monolaurate (Tween 20)-Tris-buffered saline (TTBS), fixed with ice-cold PBS containing 3.7% paraformaldehyde, and retreated overnight at 4°C with the blocking solution. Recombinant c-met Fc chimeras (manufactured by R&D Systems) are recombinant preparations in which the Fc region of immunoglobulin is ligated to the extracellular domain of mouse c-met (the domain with a binding site for HGF).

[0053] Subsequently, biotinylated anti-HGF polyclonal antibody, streptavidin-HRP conjugate (manufactured by R&D Systems), and TMB substrate solution (manufactured by R&D Systems) were added to the wells, and absorbance was measured at wavelengths of 450 and 545 nm to detect HGF bound to c-met. The results are shown in Figure 3.

[0054] As shown in Figure 3, when the sample containing only nitrated HGF prepared in Example 1 was added (bar graph c in Figure 3), the absorbance decreased significantly compared to when the sample containing only HGF stored at 4°C until immediately beforehand (positive control, bar graph b in Figure 3) was added (bar graph b in Figure 3), and fell to almost the same level as the negative control (bar graph a). On the other hand, when the HGF samples prepared in Example 1, which were nitrated in the presence of 0.17% and 0.50 v / v% glycerol, were added (bar graphs e and f in Figure 3), the absorbance was significantly higher than when the sample containing only nitrated HGF prepared in Example 1 was added (bar graph c in Figure 3) (P-value less than 1%).

[0055] The results from Examples 1 to 3 indicate that the presence of glycerol suppresses the nitration of HGF and maintains HGF's ability to bind to c-met, thereby maintaining the physiological activity of HGF (activation of muscle stem cells).

[0056] [Example 4: Experiment on administering glycerol to drinking water in a disuse muscle atrophy model mouse] A disuse muscle atrophy model mouse was created using the tail suspension method, and the effect of administering glycerol to drinking water on disuse muscle atrophy was investigated. It is known that HGF nitration occurs in vivo in disuse muscle atrophy model mice created using the tail suspension method.

[0057] In Example 4, as shown in the schedule diagram in Figure 4, three groups were established: a control group (normal rearing, no glycerol; top of the schedule diagram in Figure 4), a disintegration group (no glycerol; middle of the schedule diagram in Figure 4) which underwent 3 days of pre-re

[0058] Furthermore, Gas (gastrocnemius muscle), Sol (soleus muscle), Pla (plantar muscle), EDL (extensor digitorum longus muscle), and TA (tibialis anterior muscle) were extracted from mice in the three groups, and their wet weights were compared. The results are shown in Figure 5.

[0059] As shown in Figure 5, a significant decrease in muscle weight due to disuse was observed, particularly in the soleus (Sol) and gastrocnemius (Gas) muscles, which are representative antigravity muscles, and the administration of glycerol-containing water suppressed this decrease in muscle weight (P-value ≤ 5%). A significant decrease in muscle weight due to disuse was also observed in the plantar muscle (Pla), another antigravity muscle (P-value ≤ 5%), and the administration of glycerol-containing water tended to suppress this decrease. On the other hand, no significant decrease in muscle weight due to disuse was observed in the shin muscles (EDL and TA), which are not antigravity muscles, suggesting that the muscle disuse experiment using tail suspension (including feeding and drinking) was well conducted.

[0060] Frozen sections of calf muscle (Gas + Sol + Pla) were immunofluoresced using an anti-nitrated Y250-HGF monoclonal antibody to visualize nitrated HGF. The results are shown in Figure 6. In Figure 6, the control group is labeled "Control," the disused group is labeled "Inactive (Disused)," and the glycerol + disused group is labeled "Glycerol + Inactive (Disused)."

[0061] In the soleus muscle (Sol) of mice in the disused group, a significant increase in fluorescence intensity indicating HGF nitration was observed, whereas in the glycerol + disused group, the HGF nitration level was similar to that of the control group. In other words, it was confirmed in vivo that HGF nitration is suppressed by the administration of glycerol in drinking water.

Claims

1. A protein nitration inhibitor containing glycerol or its prodrug.

2. The nitration inhibitor according to claim 1, wherein the protein comprises hepatocyte growth factor.

3. The nitration inhibitor according to claim 1 or 2, which is administered orally, sublingually, intramuscularly, enterally, nasally, intravenously, subcutaneously, or intraperitoneally.

4. A preventive or therapeutic agent for diseases or symptoms caused by protein nitration, containing glycerol or its prodrugs.

5. The preventive or therapeutic agent according to claim 4, wherein the disease is selected from the group consisting of age-related sarcopenia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, prion disease, atherosclerosis, and colorectal cancer.

6. The preventive or therapeutic agent according to claim 5, wherein the disease is age-related sarcopenia.

7. The preventive or therapeutic agent according to any one of claims 4 to 6, wherein the symptom is muscle atrophy and / or muscle regeneration failure.

8. The preventive or therapeutic agent according to claim 7, wherein the protein comprises hepatocyte growth factor.

9. A preventive agent or therapeutic agent according to any one of claims 4 to 6, which is administered orally, sublingually, intramuscularly, enterally, nasally, intravenously, subcutaneously, or intraperitoneally.

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