WO2021015342A1 - Composition for preventing or treating spinal cord injury, comprising trpv4 antagonist - Google Patents

Abstract

The present invention relates to a composition for preventing or treating a spinal cord injury, comprising a transient receptor potential vanilloid 4 (TRPV4) antagonist. The pharmaceutical composition for preventing or treating a spinal cord injury, comprising a TRPV4 antagonist as an active ingredient, according to the present invention, may suppress neuropathic pain which is especially painful for a patient with respect to a spinal cord injury, suppress inflammatory responses and suppress glutamate toxicity, and thus has a high potential for being used as a spinal cord injury therapeutic agent.

Description

Composition for preventing or treating spinal cord injury comprising a TRPV4 antagonist

The present invention relates to a composition for preventing or treating spinal cord injury comprising a TRPV4 antagonist (transient receptor potential vanilloid 4 antagonist antagonist).

Spinal cord injury is one of the most common accidents in adults. According to US statistics, there are 2,500,000 patients worldwide, 130,000 cases each year, and the incidence rate is increasing every year. Korea is not an exception to these statistics, and according to the National Health Insurance Service, as a result of analyzing health insurance medical expenses payment data from 2013 to 2014, about 800 patients occurred, which showed a trend of increasing. In particular, the degree of loss to individuals and society is very serious because the diseased person occurs in relatively young patients in the form of trauma.

Spinal cord injury is a secondary degenerative process that causes mechanical damage and thus progressive loss of tissue. The pathological phenomena that occur after spinal cord injury are divided into two major stages: primary and secondary injuries over time. The primary injury is a phenomenon that occurs within minutes after trauma injury, and necrosis, which causes cells to die at the wound site, mainly occurs. The cause of this reaction is a mediated inflammatory response mainly induced by pro-inflammatory cytokines. Secondary damage following primary damage progresses slowly over several hours to several days, and not only the cells in the wound area die, but also the surrounding nerve cells gradually apoptosis. The inflammatory response that occurs in the primary injury is the main mechanism of this secondary injury and is involved in causing the neuropathology of chronic spinal cord injury. As the inflammatory reaction continues, apoptosis is gradually expanded, and the damaged area inside the spinal cord is widened, resulting in permanent loss of function.

The current direction of spinal cord injury treatment is to minimize secondary spinal cord injury and restore neurological function to the maximum. First, if spinal cord injury occurs, there is a method of removing bone fragments or dislocations that compress the spinal cord through surgical treatment, and using high-dose steroid drugs to prevent cell necrosis due to inflammatory reactions of nerve cells. However, administration of steroid drugs in high doses to patients causes enormous side effects such as sepsis, gastrointestinal bleeding, and pneumonia, so they are limited in actual clinical use. In addition, various treatment possibilities such as antioxidants, glutamate receptor inhibitors, ion channel inhibitors, antibodies to gangliosides or axon regeneration inhibitors, anti-inflammatory agents, neurotrophic factors, etc. have been tried, but there is no effective drug.

For the above reasons, there is virtually no stable treatment that can bring about the recovery of the damaged spinal nerve, and the development of a treatment for spinal cord injury is urgently needed. Spinal cord injury causes permanent disability, but the need for new drug development is very high as there are no available treatments. There is a need for a therapeutic agent capable of relieving pain without having side effects, having strong anti-inflammatory activity, and capable of treating spinal cord injury.

One aspect is to provide a composition for preventing or treating spinal cord injury comprising a TRPV4 antagonist (transient receptor potential vanilloid 4 antagonist) as an active ingredient.

Another aspect is to provide a health functional food for preventing or improving spinal cord injury comprising a TRPV4 antagonist (transient receptor potential vanilloid 4 antagonist) as an active ingredient.

Another aspect is to provide the use of a TRPV4 antagonist (transient receptor potential vanilloid 4 antagonist) for the manufacture of a medicament for preventing or treating spinal cord injury.

Another aspect is to provide a method of preventing or treating spinal cord injury comprising administering a pharmaceutically effective amount of a transient receptor potential vanilloid 4 antagonist to a subject in need thereof.

One aspect provides a composition for preventing or treating spinal cord injury comprising a TRPV4 antagonist (transient receptor potential vanilloid 4 antagonist) as an active ingredient.

Another aspect is to provide the use of a TRPV4 antagonist (transient receptor potential vanilloid 4 antagonist) for the manufacture of a medicament for preventing or treating spinal cord injury.

Another aspect is to provide a method of preventing or treating spinal cord injury comprising administering a pharmaceutically effective amount of a transient receptor potential vanilloid 4 antagonist to a subject in need thereof.

The term'TRPV4' used in the present invention may mean a transient receptor potential vanilloid 4 or a transient receptor potential cation channel subfamily V member 4, and , It may be an ion channel protein encoded by the TRPV4 gene in humans. The TRPV4 gene was co-discovered by W. Liedtke et al. (Strotmann R, Harteneck C, Nunnenmacher K, Schultz G, Plant TD (October 2000). "OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity". Nat. Cell Biol . 2 (10): 695-702).

The TRPV4 gene encodes TRPV4, initially named "vanilloid-receptor related osmotically activated channel" (VR-OAC) and "OSM9-like transient receptor potential channel, factor 4 (OTRPC4)", and the vanilloid superfamily in transient receptor potential, TRP). The TRPV4 protein can be a Ca2+ permeable, non-selective cationic channel that has been shown to be involved in several physiological functions, dysfunctions and diseases, and is responsible for brain, vascular function, liver, intestine, kidney and bladder function, skin barrier function and UV-B radiation. It can act in the regulation of systemic osmotic pressure for skin response, skeletal growth and structural integrity, and can be involved in joint function, airway function and lung function, retina and inner ear function, and pain. The TRPV4 channel also responds to osmotic pressure, mechanical and chemical changes, and thermal changes (warmth). Channel activation can be sensitized by inflammation and injury.

Transient receptor potential (TRP) pathway is a type of cation channel protein expressed in various tissues. When activated, the TRP pathway acts as a receptor that stimulates cells by causing the influx of cations into cells. I can. The influx of calcium ions into cells induces depolarization and activation of the cells. The types of stimuli that can activate the TRP channel are very diverse, including photo stimulation, mechanical stimulation such as vibration or contact, temperature stimulation, chemical stimulation, osmotic pressure, etc., in addition to the transporter material as an agent. It can respond to many types of stimuli. Among them, TRPV4 (transient receptor potential vanilloid 4) can act as an intracellular pathway for calcium ions, and can act as a mechanical sensor or a pressure sensor.

The term'TRPV4 antagonist' as used in the present invention is a competitive antagonist that inhibits the action of an agonist if it is a substance that attenuates the action of TRPV4, or non-competitive antagonists that irreversibly bind to the receptor. As an inclusive term, it may be used without limitation. In particular, it selectively antagonizes only TRPV4 without affecting the activity of three human transient receptor potential (TRP) channels, including capsaicin-activated TRPV1, camphor-activated TRPV3, and methanol-activated TRPM8. I can.

In one embodiment, the TRPV4 antagonist may be one or more selected from the group consisting of RN-1734, ruthenium red, HC-067047, RN-9893, capsazepine, GSK205, and mixtures thereof.

RN-1734, which is a specific example of a TRPV4 antagonist, has the chemical name 2,4-dichloro-N-isopropyl-N-(2-isopropylaminoethyl)benzenesulfonamide (2,4-Dichloro-N-isopropyl-N-( 2-isopropylaminoethyl)benzenesulfonamide), another term is also called TRPV4 antagonist I (Transient Receptor Potential Vanilloid-4 Antagonist I), and may be C ₁₄ H ₂₂ Cl ₂ N ₂ O ₂ S. RN1734 is a TRPV4 agonist, without affecting the activity of three human transient receptor potential (TRP) channels, including capsaicin-activated TRPV1, camphor-activated TRPV3 and methanol-activated TRPM8. It is a cell-permeable benzenesulfonamide compound that selectively antagonizes 4α-PDD, and is available from Sigma-Aldrich, Tocris, Santa Cruz biotechnology, Merch, Calbiochem, and is a compound of the following formula:

[Formula 1]

.

Ruthenium Red, an embodiment of the TRPV4 antagonist, is not highly selective, but can act as a general inhibitor of TRP. It may be a reddish brown flaky crystal obtained by dissolving ruthenium(III) chloride in concentrated ammonia water and heating at 40°C. Chemical name 2RuCl ₂ (OH)ㆍ7NH ₃ ㆍ3H ₂ O has a composition and the structure is similar to [Ru(NH ₃ ) ₄ (NO ₃ )Cl]NO ₃ [Ru(NH ₃ )4(OH) Cl]Clㆍ It is said to be 2H ₂ O, but considering color, magnetism, stability, etc., it is presumed that it is not mononuclear complex but binuclear complex.

HC-067047, a specific example of a TRPV4 antagonist, has the formula 2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(prefluoromethyl)phenyl]-1H -Pyrrole-3-carboxamide (2-Methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide) . As a potent and selective inhibitor of hTRPV4, the compound is capable of inhibiting human, rat and mouse TRPV4 with IC50 values of 48, 133 and 17 nM, respectively. HC-067047 does not inhibit other TRPV isoforms at 5 μM concentration. HC-067047 is capable of inhibiting hERG channel and menthol receptor TRPM8 with IC50 values of 370 and 780nM, respectively, and is a compound represented by Formula 2:

[Formula 2]

.

RN-9893, which is a specific example of the TRPV4 antagonist, is N-(4-(4-Isopropylpiperazin-1-ylsulfonyl)phenyl)-2-nitro-4-(trifluoromethyl)benzamide, and is a compound represented by the following Chemical Formula 3. RN-9893 is a potent and selective antagonist of the Transient Receptor Potential ion channel TRPV4 and has IC50 values of 420nM, 660nM and 320nM for human, rat and mouse TRPV4 receptors, respectively. RN-9893 shows excellent selectivity compared to common TRP receptors, with IC50> 30 μM for TRPV3 and about 30 μM for TRPM8:

[Formula 3]

.

Capsazepine, a specific example of the TRPV4 antagonist, has the chemical name N-[2-(4-Chlorophenyl)ethyl]-7,8-dihydroxy-1,3,4,5-tetrahydro-2H-2-benzazepine- As 2-carbothioamide has been reported to block nociception of capsaicin, which activates the TRPV1 ion channel, it may be considered a TRPV1 antagonist. In addition to the antagonistic effect on the TRPV1 channel, it may be reported that it inhibits the cold active TRPM8 channel.

GSK205, a specific example of the TRPV4 antagonist, may have the chemical name N-(4-(2-(Benzyl(methyl)amino)ethyl)phenyl)-5-(pyridin-3-yl)thiazol-2-amine, and TRPV4 It has been reported as antagonist III. It can be purchased from Calbiochem.

Inhibition of TRPV4 can suppress neuropathic pain, inhibit inflammatory reactions, and inhibit glutamate toxicity, thus blocking secondary injury that occurs after spinal cord injury. A protective effect can be obtained.

Since the present invention confirmed that the TRPV4 antagonist can effectively treat damaged spinal cord without side effects, the pharmaceutical composition according to the present invention includes acute transverse myelitis, acute disseminated myelitis, which are diseases related to spinal cord injury as well as spinal cord injury due to trauma. It can be used as a preventive or therapeutic agent for myelopathy, non-Hodgkin's lymphoma, hydrocephalus, hereditary ataxia, neurosyphilis, Minamata disease, Lou Gehrig's disease, and multiple conjunctivosis.

Secondary injuries such as inflammatory reaction, ischemia, and vascular disruption that appear after spinal cord injury can be a very important factor in determining the patient's prognosis. The best strategy to achieve neuropathic pain may be to suppress neuropathic pain, suppress the inflammatory response, and suppress glutamate toxicity. In this respect, the TRPV4 antagonist according to the present invention may be for suppressing neuropathic pain, suppressing an inflammatory response, or suppressing glutamate toxicity.

The TRPV4 antagonist may be included in the composition as a pharmaceutically acceptable salt thereof. The salt may be an acid addition salt or a base addition salt. The acid addition salt includes a salt with an inorganic acid or an organic acid. Examples of the inorganic acid salt include salts with hydrochloric acid, hydrobromic acid, phosphoric acid, or sulfuric acid, and the organic acid salts include acetic acid, trifluoroacetic acid, propionic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, and lactic acid. , Benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid or naphthalenedisulfonic acid, and the like, but is not limited thereto. As the base addition salt, an alkali metal salt (eg, sodium or potassium salt), an alkaline earth metal salt (eg, calcium or magnesium salt), or an ammonia salt or an organic amine salt such as diethylamine, triethylamine, ethyl Diisopropylamine, procaine, dibenzylamine, N-methylmorpholine, or salts with dihydroabiethylamine.

It is construed herein that the TRPV4 antagonist or a pharmaceutically acceptable salt thereof may exist in any crystalline and amorphous form thereof, and in any form selected from the group consisting of hydrates, solvates, and co-crystals thereof.

The pharmaceutical composition according to the present invention may be an oral or parenteral dosage form. Pharmaceutical preparations include preparations for oral administration such as tablets, hard or soft capsules, solutions, and suspensions, and these pharmaceutical preparations are conventional pharmaceutically acceptable carriers, for example, excipients and binders in the case of preparations for oral administration. , A disintegrant, a lubricant, a solubilizing agent, a suspending agent, a preservative or an extender, etc., can be used. In addition, the pharmaceutical composition may be used as a formulation for parenteral administration such as cream, gel, patch, spray, ointment, warning agent, lotion, and the like.

The pharmaceutical composition may further include commonly used excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc., and tablets, capsules, powders, granules, suspensions, emulsions, syrups, and Other solutions can be formulated.

For formulations such as tablets and capsules, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid Lubricating oils such as magnesium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax are contained. In the case of capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil is contained.

In addition, the pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection injection method may be selected. In order to formulate a formulation for parenteral administration, the bile acids or salts thereof of the present invention are mixed in water together with a stabilizer or buffer to prepare a suspension, which is prepared in a unit dosage form of an ampoule or vial.

As used herein, the term "administration" refers to introducing the pharmaceutical composition of the present invention to an individual suspected of inflammatory disease or spinal cord injury by any suitable method, and the route of administration is oral or parenteral as long as it can reach the target tissue. It can be administered through a variety of oral routes.

The pharmaceutical composition of the present specification can be administered in a pharmaceutically effective amount.

As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the individual, age, sex, and disease. It can be determined by the type, activity of the drug, sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and can be easily determined by a person skilled in the art. The dosage of the pharmaceutical composition of the present invention may be determined by an expert according to various factors such as the patient's condition, age, sex, and complications, but in general, 0.1 mg to 10 g per 1 kg of adults, or 10 mg to 5 g It can be administered in doses. In addition, a daily dose of the pharmaceutical composition or a dose of 1/2, 1/3 or 1/4 thereof is contained per unit dosage form, and may be administered 1 to 6 times a day. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

In another embodiment, the TRPV4 antagonist may improve motor function of a spinal cord injured individual or promote repair of a tissue of the spinal cord injured. Accordingly, the TRPV4 antagonist or a pharmaceutically acceptable salt thereof according to an embodiment may be usefully used in a composition for preventing, improving, or treating spinal cord injury.

Another aspect provides a health functional food for preventing or improving spinal cord injury comprising a TRPV4 antagonist or a pharmaceutically acceptable salt thereof as an active ingredient.

The food may be a health supplement food, a health functional food, a functional food, etc., but is not limited thereto, and the addition of the compound of the present invention to natural foods, processed foods, patient foods, and general food materials is also included.

The food composition according to the present invention may be added as it is the TRPV4 antagonist or a salt thereof of the present invention, or may be used with other foods or food compositions, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, improvement or therapeutic treatment).

When preparing food or beverage, 0.001 to 99.99 parts by weight, preferably 0.001 to 30 parts by weight, may be added based on 100 parts by weight of the raw material of the food or beverage. The effective dose of the matrix metalloproteinase-8 inhibitor or its salt may be used in accordance with the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, the above It may be less than the range, and since there is no problem in terms of safety, the component may be used in an amount greater than the range.

There is no particular limitation on the type of food. The food composition may be used in the form of preparations for oral administration such as tablets, hard or soft capsules, solutions, suspensions, etc., and these preparations are acceptable conventional carriers, for example, excipients in the case of preparations for oral administration, It can be prepared using a binder, disintegrant, lubricant, solubilizer, suspending agent, preservative or extender.

In addition, examples of foods include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes. , And other nutritional supplements, but are not limited to these types of food.

The pharmaceutical composition for spinal cord injury or treatment comprising a TRPV4 antagonist according to the present invention as an active ingredient can suppress neuropathic pain, which is particularly painful to patients in spinal cord injury, can suppress inflammatory reactions, and suppress glutamate toxicity. It is highly likely to be used as a treatment for spinal cord injury.

1 is a graph showing the expression of IL-6, IL-1beta, TNF-alpha, and iNOS by time after treatment with LPS (1 ug/ml) for inducing inflammation in a macrophage/microglial cell line. . In the case of treatment with the TRPV4 antagonist, the results were shown to suppress inflammation. In addition, when LPS was treated with neuronal cell lines (neuronal cell lines Neuro2A, PC12), the expression of TRPV4 in a concentration-dependent manner was measured by time and shown as a graph.

FIG. 2 shows nerve growth factor (NGF), retinoic acid (RA), in order to confirm cell growth after 12 hours, 24 hours, 48 hours, and 72 hours in neuronal cell lines Neuro2A, PC12, This is an immunohistostaining picture that confirmed the nerve regeneration effect by treatment with TRPV4 agonist (GSK1016790A 1 uM) and TRPV4 antagonist (RN-1734 10 μM; HC-067047 1 μM).

3A shows a picture of an operation of a rat model, which is a nerve injury model.

3B is a Western blot result confirming the expression level of TRPV4 from the 3rd hour to the 28th day after each injury.

Figure 3c is a graph of qRT-PCR results confirming the expression level of TRPV4 from the 3rd hour to the 28th day after each injury.

3D is a graph of the qRT-PCR results confirming the expression level of Pacsin-3 from the 3rd hour to the 28th day after each injury.

Figure 3e is an immunohistostaining photograph confirming the expression level of TRPV4 from the 3rd hour to the 28th day after each injury.

4A is a schematic diagram showing the role of TRPV4 as a protein located in the plasma membrane and functioning as a calcium channel.

Figure 4b is a recording of qRT-PCR results 3 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days after injury to Ca ²⁺ /calmodulin-dependent protein kinase (CDPK) It is a graph.

4C is a graph showing qRT-PCR results after 3 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days and 28 days after injury to Calmodulin.

4D is a graph of qRT-PCR results recorded at 3 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days after injury to Calpain.

Figure 4e is a photograph of recording immunohistochemical staining results for Calpain-1 3 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days after injury. .

Figure 4f is a photograph of the results of immunohistochemical staining for Calpain-2 after 3 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days after injury. .

Figure 5 shows Ca ²⁺ levels after spinal cord injury using two-photon microscopy (TPM) (1). These are pictures and graphs of Ca ²⁺ imaging taken 3 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days and 28 days after injury.

6A is a schematic diagram showing a graded spinal cord injury compression model (mild (20 gram), moderate (35 gram), severe (50-gram) injury model) and a photograph of the degree of damage according to the weight of the weight (in each injury model Luxol Fast Blue (LFB) staining).

6B is a photograph of the result of TRPV4 immunohistochemical staining. mild (20 gram), moderate (35 gram), severe (50-gram) injury.

6C is a graph showing qRT-PCR results for TRPV4.

6D is a graph showing qRT-PCR results for Pacsin-3.

6E is a Western blot result for TRPV4.

7 is a control group (Sham), and each grade-specific injury model (mild (20 gram), moderate (35 gram), severe (50-gram) injury model), Interleukin-6 (IL-6), Occludin, Arachidonate 15 -Lipoxygenase (ALOX15), Aquaporin-4 (Aqua-4), Aquaporin-5 (Aqua-5), Aquaporin-9 (Aqua-9), Tumor necrosis factor alpha (TNF-a), heme oxygenase-1 (HO- 1), Angiopoietin-1 (Ang-1), Nucleophosmin, Potassium Voltage-Gated Channel Subfamily J Member 10 (KCNJ10) is a graph showing the qRT-PCR results.

8 is a graph showing qRT-PCR results for TRPV4, Interleukin-6 (IL-6), Nitric oxide synthase (iNOS), Tumor necrosis factor alpha (TNF-α), and Occludin.

It will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of a pharmaceutical composition for treatment or spinal cord injury comprising a TRPV4 antagonist as an active ingredient

Example 1-1: Preparation of a pharmaceutical composition for treatment or spinal cord injury containing RN-1734 as an active ingredient

RN-1734 was purchased and prepared from Calbiochem. The composition according to the present invention was prepared by dissolving this in 1% dimethyl sulfoxide (DMSO) to prepare a concentration of 10 μM.

Example 1-2: Preparation of a pharmaceutical composition for treatment or spinal cord injury containing HC-067047 as an active ingredient

HC-067047 was purchased and prepared from Calbiochem. The composition according to the present invention was prepared by dissolving this in 1% dimethyl sulfoxide (DMSO) to prepare a concentration of 1 μM.

Thus, a composition for preventing or treating spinal cord injury comprising a TRPV4 antagonist according to the present invention was prepared, and the effect of treating spinal cord injury was confirmed in the following experimental examples using the pharmaceutical composition prepared in Examples 1-1 and 1-2. I did.

Experimental Example 1: Construction of an animal model of spinal cord injury and administration of drugs

1.1. Spinal Cord Trauma Compression Model Animal Breeds and Supplies

9-11 weeks of age 250-300g Sprague Dawley female rats, animals were purchased through Orient Bio for verification process breed and appearance. Afterwards, it was reared within the limits of the Animal Ethics Committee of Cha Medical University.

1.2. Animal anesthesia and sacrifice

SD rats (8-10 weeks old) were anesthetized by injecting 1 μl per g of zoletil (50 mg/kg, Virbac Laboratories, France) and lumpun (10 mg/kg, Bayer, Korea) in a 1:1 ratio. After the experiment is over, euthanize them by referring to the guidelines for GUIDE FOR THE CARE AND USE OF LABORATORY ANIMALS Eighth Edition.

1.3. Spinal Cord Compression Model

The device to induce spinal cord injury is applied to the weight drop method.It consists of weights of 20g, 35g, and 50g and a support rod.After laminectomy is performed at the T10 area, the vertebral ring plate (lamina) is removed and the diameter is 1mm. It induces spinal cord injury with an impactor that is additionally combined with the phosphorus support rod. The weights may use different weights, and a schematic diagram thereof is as shown in FIG. 1.

Specifically, first of all, the body weight was measured, and hair removal was performed in a circular shape with a radius of about 4 cm around the thoracic spine 10 of the rat using an epilator. The epilation area was disinfected with 70% alcohol. The surgical site was disinfected with povidone and 70% alcohol. After checking the location of thoracic spine No. 10 (T10), only the surgical site was covered with an exposed sterile surgical cloth, and the outer skin was incised about 5 cm using a surgical knife. The muscle layer exposed after the incision of the skin was also incised about 2 cm in the 10 th thoracic vertebrae using a surgical knife. When the 10th thoracic spinous process is exposed, the spinous process is removed using an Offset Bone Nipper (FST, Germany), and the vertebral ring plate (lamina) is removed from the back of the spinal cord without damage to the dura meter. Exposed. The spinous processes of the 8th and 11th thoracic vertebrae were fixed with Allis Forceps to expose the posterior spinal cord without damaging the dura. After that, the spinal cord was compressed at the 10 th thoracic position for 5 minutes using weights (20g, 35g, 50g) to prepare a spinal cord compression model for each grade.

1.4. Administration of pharmaceutical composition

The composition prepared in Example 1 above was 30mg/kg and administered by intraperitoneal injection twice a day for 14 days 30 minutes after the operation in 1.3. above.

1.5. All experimental groups

The entire experiment was performed using 30 rats, and each detailed experimental group is as follows.

Control 1: Positive control [sham] (spinal cord exposure: thoracic spine 9 thoracic arch was excised to expose 10 pleural fluid) = 10 mice

Experimental group 1: Negative control (no treatment, only injury) = 10 animals

Experimental group 2: Injury + Example 1-1, 1-2 each administration group = 5 animals each

Experimental Example 2: Confirmation of the inhibitory effect of TRPV4 antagonist on inflammation after induction of inflammatory response in various cells

In macrophage/microglial cell lines, LPS (1 μg/ml) was treated to induce inflammatory response, and then the inflammatory factor cytokines IL-6, IL-1beta, TNF-alpha, and iNOS were expressed over time. Measured by RT-PCR. In addition, after LPS treatment was performed on neuronal cell lines (neuronal cell lines Neuro2A, PC12), the expression level of TRPV4 was measured. After RNA was extracted from each cell line with Trizol, RT PCR was performed. Quantitative real-time PCR was performed using SYBR Green Master Mix, and mRNA expression was confirmed using ABI StepOne Real-time PCR System. In each step of PCR, the time and temperature conditions were 95°C for 10 minutes after initial denaturation, 95°C for 15 seconds, 60°C for 30 seconds, and a total of 40 rotations. Target mRNA and GAPDH were compared by real-time PCR, and the results were analyzed by the ΔΔCT method.

As shown in the graph of FIG. 1, when the TRPV4 antagonist was treated, inflammation was suppressed. In addition, when LPS was treated with neuronal cell lines (neuronal cell lines Neuro2A, PC12), it was confirmed that TRPV4 was expressed in a concentration-dependent manner.

Experimental Example 3: Confirmation of nerve regeneration effect of TRPV4 antagonist in nerve cells

Each nerve growth factor (NGF), retinoic acid (RA), and TRPV4 agonist in order to check the neurite growth after 12 hours, 24 hours, 48 hours, and 72 hours in neuronal cell lines Neuro2A, PC12. (GSK1016790A 1 μM), a TRPV4 antagonist (RN-1734 10uM; HC-067047 1uM) was treated to confirm the nerve regeneration effect. After spinal cord injury, the spine was placed in 4% PFA for each elapsed time and fixed for a week, and then 1 cm was cut from the spinal cord injury site to make a paraffin block as a longitudinal section. The resulting block was cut to a thickness of 5 μm and subjected to a deparaffining process to perform immunostaining.

As shown in the immunohistostaining picture of FIG. 2, it can be seen that the nerve regeneration effect is excellent when 10 μM of RN-1734 and 1 μM of HC-067047, which are TRPV4 antagonists, are treated. In other words, it can be said that inhibition of the TRPV4 antagonist TRPV4 ion channel is effective for nerve regeneration.

Experimental Example 4: Confirmation of the expression level of TRPV4 after spinal cord injury using a nerve injury model (mouse model) and

A nerve injury model was prepared using the method described in point 1.3 above. 3A shows a picture of an operation of a rat model, which is a nerve injury model. TRPV4, Pacsin-3, Ca ²⁺ /calmodulin-dependent protein kinase (CDPK), Calmodulin, Calpain-1,2 (Calpain-1) from 3 hours after each spinal cord injury afterwards. The expression level of ,2) was measured by each western blot and qRT-PCR method.

In addition, in the control (Sham), and each grade-specific injury model (mild (20 gram), moderate (35 gram), severe (50-gram) injury model), Interleukin-6 (IL-6), Occludin, Arachidonate 15-Lipoxygenase (ALOX15), Aquaporin-4 (Aqua-4), Aquaporin-5 (Aqua-5), Aquaporin-9 (Aqua-9), Tumor necrosis factor alpha (TNF-a), heme oxygenase-1 (HO-1) , Angiopoietin-1 (Ang-1), Nucleophosmin, Potassium Voltage-Gated Channel Subfamily J Member 10 (KCNJ10) expression levels were measured by qRT-PCR method.

After spinal cord injury, 1 cm was cut out from the spinal cord injury site by time, and after each RNA was extracted with Trizol, RT-PCR was performed.

In addition, after spinal cord injury, the vertebrae for each date were put in 4% PFA and fixed for a week, and then 1 cm was cut from the spinal cord injury site to make a paraffin block as a longitudinal section. The resulting block was cut to a thickness of 5 μm and subjected to a deparaffining process to perform immunostaining.

3B is a Western blot result confirming the expression level of TRPV4 from the 3rd hour to the 28th day after each injury.

Figure 3c is a graph of qRT-PCR results confirming the expression level of TRPV4 from the 3rd hour to the 28th day after each injury.

3D is a graph of the qRT-PCR results confirming the expression level of Pacsin-3 from the 3rd hour to the 28th day after each injury.

Figure 3e is an immunohistostaining photograph confirming the expression level of TRPV4 from the 3rd hour to the 28th day after each injury.

4A is a schematic diagram showing the role of TRPV4 as a protein located in the plasma membrane and functioning as a calcium channel.

Figure 4b is a recording of qRT-PCR results 3 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days after injury to Ca ²⁺ /calmodulin-dependent protein kinase (CDPK) It is a graph.

4C is a graph showing qRT-PCR results after 3 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days and 28 days after injury to Calmodulin.

4D is a graph of qRT-PCR results recorded at 3 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days after injury to Calpain.

Figure 4e is a photograph of recording immunohistochemical staining results for Calpain-1 3 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days after injury. .

Figure 4f is a photograph of the results of immunohistochemical staining for Calpain-2 after 3 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days after injury. .

6A is a schematic diagram showing a graded spinal cord injury compression model (mild (20 gram), moderate (35 gram), severe (50-gram) injury model) and a photograph of the degree of damage according to the weight of the weight (in each injury model Luxol Fast Blue (LFB) staining).

6B is a photograph of the result of TRPV4 immunohistochemical staining. mild (20 gram), moderate (35 gram), severe (50-gram) injury.

6C is a graph showing qRT-PCR results for TRPV4.

6D is a graph showing qRT-PCR results for Pacsin-3.

6E is a Western blot result for TRPV4.

7 is a control (Sham), and each grade-specific injury model (mild (20 gram), moderate (35 gram), severe (50-gram) injury model), Interleukin-6 (IL-6), Occludin, Arachidonate 15 -Lipoxygenase (ALOX15), Aquaporin-4 (Aqua-4), Aquaporin-5 (Aqua-5), Aquaporin-9 (Aqua-9), Tumor necrosis factor alpha (TNF-a), heme oxygenase-1 (HO- 1), Angiopoietin-1 (Ang-1), Nucleophosmin, Potassium Voltage-Gated Channel Subfamily J Member 10 (KCNJ10) is a graph showing the qRT-PCR results.

8 is a graph showing qRT-PCR results for TRPV4, Interleukin-6 (IL-6), Nitric oxide synthase (iNOS), Tumor necrosis factor alpha (TNF-a), and Occludin.

This confirmed the increase in the expression level of TRPV4 after spinal cord injury, and the anti-inflammatory and neuroprotective effects that would occur when TRPV4 antagonists were treated. According to these results, it was confirmed that the inhibition of the TRPV4 ion channel by the TRPV4 antagonist has an inhibitory effect on the inflammatory response and has a neuroprotective effect, and improvement of neurological function through an anti-inflammatory response after spinal cord injury can be achieved.

Experimental Example 5: 2-quantum microscopy experiment for comparison of calcium ion secretion after spinal cord injury

In a rat model of spinal cord injury using two-photon microscopy (TPM), Ca ²⁺ levels after spinal cord injury (1). Ca ²⁺ imaging was performed 3 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days and 28 days after injury.

The results are shown in FIG. 5. Fig. 5.1 shows the results of a longitudinal section and 5.2 shows a transverse section.

Claims (7)

1. A pharmaceutical composition for preventing or treating spinal cord injury comprising a TRPV4 antagonist (transient receptor potential vanilloid 4 antagonist) as an active ingredient.
2. The method according to claim 1, wherein the spinal cord injury is spinal cord injury due to trauma, acute transverse myelitis, acute disseminated myelitis, myelopathy, non-Hodgkin's lymphoma, hydrocephalus, hereditary ataxia, neurosyphilis, Minamata disease, Lou Gehrig's disease, and multiple sclerosis. Phosphorus pharmaceutical composition.
3. The pharmaceutical composition according to claim 1, wherein the TRPV4 antagonist inhibits neuropathic pain, inhibits glutamate toxicity, or prevents or treats spinal cord injury through anti-inflammatory activity.
4. The pharmaceutical according to claim 1, wherein the TRPV4 antagonist is at least one selected from the group consisting of RN-1734, ruthenium red, HC-067047, RN-9893, capsazepine, GSK205, and mixtures thereof. Ever composition.
5. A health functional food for preventing or improving spinal cord injury containing a TRPV4 antagonist (transient receptor potential vanilloid 4 antagonist) as an active ingredient.
6. The use of a TRPV4 antagonist (transient receptor potential vanilloid 4 antagonist) for the manufacture of a medicament for the prevention or treatment of spinal cord injury.
7. A method of preventing or treating spinal cord injury comprising administering a pharmaceutically effective amount of a TRPV4 antagonist (transient receptor potential vanilloid 4 antagonist) to a subject in need thereof.

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