Pharmaceutical composition for treating TRPV1 activity-mediated diseases

Abstract

The present invention relates to a novel pharmaceutical composition for treatment, and more particularly to a pharmaceutical composition for treatment of diseases mediated by TRPV1 activity. The composition for treating pain using TRPV1 activity inhibitors of the present invention effectively suppresses neuropathic pain caused by spinal nerve injury, and can therefore be used as a novel pain treatment for various pain conditions and diseases associated with the TRPV1 channel, such as arthritis and diabetic peripheral neuropathy. Furthermore, since the composition effectively inhibits TRPV1 activity, it can also be used as a treatment for various TRPV1 activity-mediated diseases.

Description

Technical Field

[0001] The present invention relates to a novel therapeutic pharmaceutical composition, and more particularly to a pharmaceutical composition for treating TRPV1 activation-mediated diseases or a method for treating TRPV1 activation-mediated diseases.

Background Art

[0002] Pain is "an unpleasant sensory and emotional experience associated with actual or potential tissue damage". Generally, pain is alleviated as the damaged tissue heals. However, if pain becomes chronic, even though the damaged site has clearly completely healed, pain occurs persistently without any stimulus, and generally, pain is caused by harmless stimuli that do not normally cause pain.

[0003] On the other hand, TRPV1 (transient receptor potential vanilloid type 1) is one of the important nociceptors in the cellular mechanism by which noxious stimuli cause pain and is a member of the TRP (transient receptor potential) family. In addition, TRPV1 is a non-selective cation channel activated by various stimuli such as noxious heat (above 42 degrees Celsius), capsaicin, resiniferatoxin (RTX), and protons. It is mainly expressed at the terminals of nociceptive fibers and is a core molecule involved in the development of peripheral and central sensitization that causes chronic pain. TRPV1 has been presented as a major target for the treatment of chronic pain. However, pharmacological inhibition of TRPV1 causes severe hyperthermia of unknown cause, and TRPV1 inhibitors are known to be difficult to develop as therapeutic agents.

[0004] GDF11 is a protein belonging to the TGF-β family, and its expression increases in young animals, and it is known to promote neurogenesis and angiogenesis. Regarding the chronic pain regulatory effect of TGF-β belonging to the same family, many studies have been conducted, but the part regarding the pain-relieving effect of GDF11 remains unknown.

[0005] Korean Patent Publication No. 10-2017-0093283 relates to a composition containing GDF11 and its uses, and provides the fibroblast proliferation effect of a human-derived adult stem cell culture solution containing GDF11. However, research or descriptions related to the chronic pain-relieving effect of GDF11 have not been disclosed.

Summary of the Invention

Problems to be Solved by the Invention

[0006] As a result of intensive efforts to provide a substance that can treat chronic pain by suppressing TRPV1 and solve side effects such as severe hyperthermia accompanied by the suppression of TRPV1, the inventors have found that GDF11 has the effect of alleviating pain caused by various causes, and thus completed the present invention.

[0007] Therefore, an object of the present invention is to provide a pharmaceutical composition for treating TRPV1-mediated diseases that can treat pain-associated diseases including chronic pain and TRPV1-mediated diseases without side effects. However, such problems are exemplary and do not limit the scope of the present invention.

Means for Solving the Problems

[0008] According to one aspect of the present invention, there is provided a pharmaceutical composition for treating TRPV1-mediated diseases, which contains, as an active ingredient, a GDF11 (growth differentiation factor 11) peptide containing the amino acid sequence shown in SEQ ID NO: 1, a polynucleotide encoding the peptide, or an expression vector containing the polynucleotide.

[0009] According to another aspect of the present invention, there is provided a method for treating a TRPV1 activity-mediated disease, comprising administering to a patient suffering from a TRPV1 activity-mediated disease a composition comprising a GDF11 (growth differentiation factor 11) peptide comprising the amino acid sequence shown in SEQ ID NO: 1, a polynucleotide encoding the peptide, or an expression vector comprising the polynucleotide.

[0010] Definition of Terms: As used herein, the term "GDF11 (growth differentiation factor 11)" also refers to BMP11 (bone morphogenetic protein 11) and means a protein expressed by the GDF11 gene present on human chromosome 12.

[0011] As used herein, the term "TRPV1 (Transient receptor potential channel, Vanilloid subfamily member 1)" belongs to a large family of TRP channels consisting of non voltage gated cation channels including the regions of heat and vision, taste, smell, and touch. TRPV1 is activated by heat, protons, and endogenous substances that interact synergistically to cause pain signals.

[0012] As used in the present invention, the term "vector" refers to a tool used for gene transfer and is regarded as a concept corresponding to a drug carrier. Therefore, not only a DNA vector composed of the same nucleic acid molecule as the transgene to be introduced, such as a general plasmid vector, but also nanoparticles composed of silica or gold, liposomes, extracellular vesicles, and phospholipid membrane structures such as exosomes, and cationic polymers such as chitosan, polyethyleneimine, and polylysine, and reagents used for DNA transfection such as calcium phosphate are included in the broad sense of the vector.

[0013] Detailed Description of the Invention: According to one aspect of the present invention, there is provided a pharmaceutical composition for treating TRPV1 activity-mediated diseases, which comprises, as an active ingredient, a GDF11 (growth differentiation factor 11) peptide containing the amino acid sequence shown in SEQ ID NO: 1, a polynucleotide encoding the peptide, or an expression vector containing the polynucleotide.

[0014] In the pharmaceutical composition, the expression vector may be a viral vector or a non-viral vector. The viral vector may be an adeno-associated virus vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector, a vaccinia virus vector, a Sendai virus vector, a flavivirus vector, a rhabdovirus vector, a retrovirus vector, or a lentivirus vector. The serotype of the adeno-associated virus (AAV) vector may be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, or AAV16.

[0015] In the pharmaceutical composition, the non-viral vector may be a DNA vector, a nanoparticle, a cationic polymer, an exosome, an extracellular vesicle, or a liposome. The DNA vector may be a plasmid vector, a cosmid vector, a phagemid vector, or an artificial human chromosome.

[0016] In the pharmaceutical composition, the TRPV1 activity-mediated disease may be selected from the group consisting of pain, hypertension, stroke, myocardial ischaemia, urinary incontinence, urinary bladder hypersensitiveness, irritable bowel syndrome, fecal urgency, stomach-duodenal ulcer, gastro-esophageal reflux disease (GERD), Crohn’s disease, haemorrhoid, asthma, chronic obstructive pulmonary disease, pruritus, psoriasis, hearing loss, tinnitus, cough, hypertrichosis, and alopecia.

[0017] In the pharmaceutical composition, the pain may be nociceptive pain, psychogenic pain, inflammatory pain, or pathological pain, and the pathological pain may be neuropathic pain, cancer pain, anti-cancer agent-induced pain, postoperative pain, trigeminal neuralgia pain, idiopathic pain, diabetic neuropathic pain, or migraine.

[0018] According to another aspect of the present invention, there is provided a method for relieving or treating pain, comprising the step of administering the pharmaceutical composition.

[0019] The pain according to the present invention includes nociceptive pain, psychogenic pain, inflammatory pain associated with tissue damage and infiltration of immune cells, and pathological pain (such as fibromyalgia, irritable bowel syndrome, and dysfunctional pain such as tension headache) which is a disease state caused by damage or abnormal function of the nervous system. The pain may also include anatomically distinct back pain: neck pain, middle back pain, lower back pain or tailbone pain. The pain may also include neuropathic pain, pain such as migraine, etc. Neuropathic pain is a chronic neurological disorder caused when the nervous system is damaged by various causes such as trauma, inflammation, ischemic injury, or metabolites, and is caused by damage or disease that affects the somatosensory system. Generally, it is a type of non-malignant chronic pain caused by abnormalities in the nerves, spinal cord, and brain and is estimated to be experienced by more than 1% of the population.

[0020] Neuropathic pain can be associated with abnormal sensations called dysesthesia, allodynia where pain is felt in response to harmless stimuli that do not normally cause pain, and hyperalgesia where there is an increased and prolonged pain response to harmful stimuli such as heat. Neuropathic pain can also have continuous and / or intermittent (episodic) components, the latter being likened to an electric shock. Common characteristics include heat, cold, pins and needles, numbness, and itching. Depending on whether the peripheral or central nervous system is affected, it can be divided into peripheral neuropathic pain and central neuropathic pain. In contrast, nociceptive pain is usually expressed as an ache. Migraine is also associated with a number of autonomic symptoms and is a chronic disorder that causes headaches ranging from mild to severe. The exact mechanisms of these migraines have not been clarified to date. The basic theory is related to increased excitability of the cerebral cortex and abnormal regulation of pain neurons in the trigeminal nucleus of the brainstem. For example, the pain may be at least one selected from the group consisting of neuropathic pain, cancer pain, postoperative pain, trigeminal neuralgia pain, idiopathic pain, diabetic neuropathic pain, or migraine, etc.

[0021] In the pharmaceutical composition of the present invention, the effective amount of the said compound can vary depending on the type of the patient's affected part, the application site, the number of treatments, the treatment time, the dosage form, the patient's condition, the type of adjuvant, etc. The dosage is not particularly limited, but it may be 0.01 μg / kg / day to 10 mg / kg / day. The daily dose may be administered once a day, or divided into 2 - 3 times a day at appropriate intervals, or may be administered intermittently at several-day intervals.

[0022] In the pharmaceutical composition of the present invention, the compound may be contained in an amount of 0.01 to 100% by weight based on the total weight of the composition. The pharmaceutical composition of the present invention may further contain suitable carriers, excipients, and diluents commonly used in the production of pharmaceutical compositions. In addition, solid or liquid formulation additives may be used in the production of the pharmaceutical composition. The formulation additives may be either organic or inorganic. Examples of excipients include lactose, sucrose, white sugar, glucose, cornstarch, starch, talc, sorbitol, crystalline cellulose, dextrin, kaolin, calcium carbonate, and silicon dioxide. Examples of binders include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, dextrin, and pectin. Examples of lubricants include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. As coloring agents, any agents that are usually permitted to be added to pharmaceuticals can be used. These tablets and granules can be appropriately coated with sugar coating, gelatin coating, or other coatings as necessary. In addition, preservatives, antioxidants, etc. may be added as necessary.

[0023] The pharmaceutical composition of the present invention may be produced in any dosage form commonly produced in the art, and the form of the formulation is not particularly limited.

[0024] The pharmaceutical composition of the present invention may be administered orally or parenterally. Preferably, as parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intracerebroventricular injection, intracerebrospinal fluid injection, intrathecal injection, transforaminal injection, intramuscular injection, intraperitoneal injection, etc.

[0025] The present invention provides a method for relieving pain and / or treatment, which includes the step of administering a therapeutically effective amount of the pharmaceutical composition to a patient in need of pain relief and / or treatment. The method may further include the step of confirming whether the patient is a patient in need of pain relief and / or treatment before the step of administration. The term "therapeutically effective amount" can depend on the amount of the active ingredient that provides the desired effect, pain relief and / or therapeutic effect.

[0026] The pharmaceutical composition of the present invention is useful for the prevention and treatment of diseases associated with the activity of TRPV1, including but not limited to pain such as acute pain, chronic pain, neuropathic pain, postoperative pain, rheumatoid arthritis pain, arthritic pain, postherpetic neuralgia, neuralgia, headache, toothache, pelvic pain, migraine, bone cancer pain, anticancer agent-induced pain, breast pain, and visceral pain; nerve-related diseases such as neuropathy, HIV-related neuropathy, nerve injury, nerve degeneration, and stroke; diabetic peripheral neuropathy; fecal urgency; irritable bowel syndrome; inflammatory bowel disease; gastrointestinal diseases such as gastroesophageal reflux disease (GERD), gastric and duodenal ulcers, and Crohn's disease; respiratory diseases such as asthma, chronic obstructive pulmonary disease, and cough; urinary incontinence; urinary bladder hypersensitivity; neurotic / allergic / inflammatory skin diseases such as psoriasis, pruritus, prurigo, and dermatitis; hyperacusis; hearing loss; tinnitus; vestibular hypersensitiveness; heart diseases such as myocardial ischemia; hemorrhagic shock; hair growth-related diseases such as hirsutism, effluvium, and alopecia; rhinitis; pancreatitis; cystitis; vulvodynia; mental diseases such as tension or fear; obesity; type 1 diabetes and type 2 diabetes may be included, but are not limited thereto.

[0027] GDF11 of the present invention is a protein encoded by the 12th gene of growth differentiation factor. GDF11 plays a role as a cytokine, and the molecular structures of human and young mouse are identical. The bone morphogenetic protein group is characterized by a polybasic proteolytic processing site that cleaves to produce a protein containing 7 conserved cysteine residues. Treatment with systemic GDF11 improved the vascular structures of the hippocampus and cortex in aged mice, improved neurogenesis, and systemic replenishment of GDF11 improved β-cell survival and morphology and glucose metabolism in all non-genetic and genetic mouse models of type 2 diabetes.

[0028] GDF11 does not affect appetite or GDF15 levels in the blood, induces phenotypes such as calorie restriction, restores the insulin / IGF-1 signaling pathway, acts directly on adipocytes to stimulate the secretion of adiponectin from white adipose tissue, and restores neurogenesis in the aging brain. It is also a regulator of skin biology, has an important impact on the production of procollagen I and hyaluronic acid, activates the Smad2 / 3 phosphorylation pathway in skin endothelial cells, and improves the skin vasculature. Increased GDF11 levels in aged mice further improved muscle structure and functional characteristics and increased exercise ability of strength and endurance.

[0029] In addition, GDF11 has been shown to reduce oxidative stress, decrease the levels of AGE, protein oxidation, and lipid peroxidation, delay the accumulation of age-related histological markers, and greatly suppress the decrease in CAT, GPX, and SOD activities. Members of the GDF11 family are regulators of cell growth and differentiation in all embryonic and adult tissues, and studies in zebrafish and Xenopus have reported that the protein is involved in mesoderm formation and neurogenesis during embryonic development.

[0030] The human GDF11 represented by SEQ ID NO: 2 and GDF11 derived from other mammals having at least 96% or more homology therewith may be used. The GDF11 derived from other mammals may be Mus musculus (SEQ ID NO: 3), Pan troglodytes (SEQ ID NO: 4), Gorilla beringei graueri (SEQ ID NO: 5), Rhinopithecus roxellana (SEQ ID NO: 6), Nomascus leucogenys (SEQ ID NO: 7), Macaca mulatta (SEQ ID NO: 8), Pan paniscus (SEQ ID NO: 9), or Sus scrofa domesticus (SEQ ID NO: 10).

[0031] The GDF11 protein derived from humans is a protein with extremely high homology to GDF11 derived from other primates, and all or part of the full-length peptide or mature peptide derived from other animals may be used. Among them, the mature peptide is present at amino acid positions 299 to 407 (SEQ ID NO: 1), and amino acid positions 1 to 298 are the parts that are cleaved among the signal peptide and the precursor protein. In the case of the mature peptide, the homology with other mammals is shown to be almost 100%, so genes or proteins derived from other animals can be utilized. In addition, the polynucleotide encoding the mature peptide may be represented by SEQ ID NO: 11.

[0032] The present invention may be realized by gene therapy utilizing a gene instead of a protein or a peptide. For such gene therapy, a gene construct in which a polynucleotide encoding GDF11 is operably linked to a regulatory sequence so as to facilitate gene amplification and manipulation is incorporated into various expression vectors, and a recombinant vector is used. Such expression vectors are classified into viral vectors and non-viral vectors. Examples of the viral vectors include adeno-associated virus vectors, adenovirus vectors, alphavirus vectors, herpes simplex virus vectors, vaccinia virus vectors, Sendai virus vectors, flavivirus vectors, rhabdovirus vectors, retrovirus vectors, and lentivirus vectors. The viral vectors used in gene therapy are clearly summarized in the literature, which is incorporated herein by reference. However, among these viral vectors, in the case of retroviruses and lentiviruses, the transgene is randomly integrated into the host genome, which may cause unexpected side effects such as the development of cancer. Therefore, adeno-associated virus, which is integrated only at a specific position on the genome of host cells, has attracted attention.

[0033] The adeno-associated virus (AAV) is a single-stranded DNA virus and a helper-dependent human parvovirus. Its genome size is approximately 4.7 kbp. The N-terminal part of the genome encodes the rep gene involved in viral replication and the expression of viral genes, and the C-terminal part encodes the cap gene that encrypts the viral capsid protein. It is composed of inverted terminal repeats (ITRs) with approximately 145 bases incorporated at both terminal sites. Four proteins are translated from the rep region, which are classified as rep78, rep68, rep52, and rep40 according to their molecular weights and play important functions in AAV DNA replication. Three proteins, namely VP1, VP2, and VP3, are translated from the cap region, and these are the structural proteins required for AAV virus assembly.

[0034] The AAV vector of the present invention may be used to carry a foreign gene sequence into cells according to various virus infection methods known in the art, and the method is not particularly limited.

[0035] On the other hand, the expression vector for gene therapy according to an embodiment of the present invention may be incorporated into a non-viral expression vector, particularly a DNA vector such as a plasmid vector, containing a gene construct in which a polynucleotide encoding GDF11 is operably linked to a regulatory sequence and used.

[0036] The term "operably linked to" as used in the present invention means that the nucleic acid sequence of interest is linked to the regulatory sequence in such a way that it is expressed in the living body into which the transgene is to be introduced.

[0037] The term "regulatory sequence" is meant to include promoters, enhancers, and other regulatory elements (e.g., polyadenylation signals). Regulatory sequences can direct the nucleic acid of interest to be expressed always in many host cells, direct the nucleic acid of interest to be expressed only in cells of a particular tissue (e.g., tissue-specific regulatory sequences), or direct expression to be induced by a specific signal (e.g., inducible regulatory sequences). Those skilled in the art will understand that the design of expression vectors used in gene therapy can vary depending on factors such as the choice of host into which the transgene is introduced and the level of desired protein expression. The expression vectors of the present invention can be introduced into a host and express the GDF11 protein. Thus, an expression vector according to an embodiment of the present invention must have regulatory sequences of eukaryotic cells, particularly eukaryotic cells capable of expressing a foreign gene in mammals. Regulatory sequences that enable the expression of foreign genes in such eukaryotic cells are well known to those skilled in the art. As described above, these include regulatory sequences responsible for normal transcription initiation and poly-A signals that selectively are responsible for transcription termination and stabilization of transcripts. Additional regulatory sequences may include translation promoting factors and / or native-combinatorial or heterologous promoter regions in addition to transcription regulatory factors. For example, regulatory sequences that enable expression in cells within mammals include the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous sarcoma virus), human elongation factor 1α-promoter, glucocorticoid-inducible MMTV-promoter (Moloney murine tumor virus), metallothionein-inducible or tetracycline-inducible promoters, or amplifiers such as CMV amplifiers or SV40-amplifiers. For expression within nerve cells, it is contemplated that a neurofilament-promoter, PGDF-promoter, NSE-promoter, PrP-promoter, or thy-1-promoter, etc. may be used. The promoters, etc. are known in the art and are described in the literature.The regulatory sequence may include a transcription termination signal such as an SV40-poly-A site or a TK-poly-A site downstream of the polynucleotide according to an embodiment of the present invention. In the present invention, suitable expression vectors are known in the art, and examples thereof include the Okayama-Berg cDNA expression vector pcDV1 (Parmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogene), pSPORT1 (GIBCO BRL), pGX-27, pX, yeast two-hybrid vectors such as pEG202 and dpJG4-5, and the like. In addition to the nucleic acid molecule of the present invention, the vector may further include a polynucleotide encoding a secretion signal. The secretion signal is well known to those skilled in the art. Further, depending on the expression system used, a leader sequence capable of directing GDF11 to an intracellular compartment may be combined with the coding sequence of the polynucleotide according to an embodiment of the present invention, and preferably, it is a leader sequence capable of directly secreting the translated protein or the protein into the periplasm or extracellular matrix of the cell.

[0038] In addition, the expression vector of the present invention may be produced, for example, by standard recombinant DNA techniques, which include, for example, blunt-end and sticky-end ligation, restriction enzyme treatment to provide appropriate ends, removal of phosphate groups by alkaline phosphatase treatment to prevent inappropriate ligation, and enzymatic ligation by T4 DNA ligase. The vector of the present invention can be produced by recombining DNA encoding a signal peptide obtained by chemical synthesis or genetic recombination techniques and DNA encoding the bispecific fusion protein of the present invention into a vector containing appropriate regulatory sequences. The vector containing the regulatory sequences can be commercially purchased or manufactured.

[0039] The expression vector may further include a polynucleotide encoding a secretion signal sequence, and the secretion signal sequence induces extracellular secretion of a recombinant protein expressed intracellularly, and may be a tPA (tissue plasminogen activator) signal sequence, an HSV gDs (herpes simplex virus glycoprotein Ds) signal sequence, or a growth hormone signal sequence.

[0040] The non-viral expression vector for gene therapy according to an embodiment of the present invention may be a DNA vector capable of expressing the GDF11 in a host cell, and the DNA vector may be in any form such as a plasmid vector, a cosmid vector, a phagemid vector, an artificial human chromosome, etc.

[0041] In addition, as a gene transfer method used for gene therapy using the DNA expression vector as described above, electroporation, gene gun, ultrasonic microbubble therapy, magnetofection, and methods of transfection using vectors such as gold and silica nanoparticles, cationic polymers, liposomes, nanoparticles, and exosomes may be used. These gene transfer methods are well known in the industry. Recently, a method of directly injecting a DNA vaccine composition into human muscle using a small electroporation device and applying irreversible electroporation has been used.

[0042] The activity of TRPV1, which is one of the permeable non-selective cation channel families, induces the influx of Ca2+ and is inhibited by specific antagonists such as capsazepine. It is also directly activated by capsaicin, heat, low pH, bradykinin, PGE2, or ATP, etc. Such activation conditions mean that TRPV1 is a primary biological sensor for thermo-chemical stimuli and tissue damage. TRPV1 has been reported to be present in various tissues such as the brain, kidneys, bronchial epithelial cells, and epidermal keratinocytes. Capsaicin stimulation increases the concentration of intracellular Ca2+ in keratinocytes, which is inhibited by capsazepine. Although the TRPV1 antagonist has been reported as a therapeutic agent for chronic pain, since pharmacological inhibition of TRPV1 causes high fever, the development of an analgesic without the effect of high fever has been required. Therefore, the present inventor has conducted research on a composition for treating pain that can effectively inhibit TRPV1, found that GDF11 becomes a novel antagonist molecule for pain treatment, and developed a composition for treating pain using TRPV1 activity inhibition of the present invention. The present inventor observed that recombinant GDF11 inhibits the function of TRPV1 channels associated in a concentration-dependent manner in primary small sensory neurons (DRG) of mice using intracellular calcium (Ca2+) imaging and the whole-cell patch-clamp method. Also, in TRPV1-transfected HEK293 cell lines, results similar to those in DRG neurons were confirmed. Therefore, the above results suggest that GDF11 of the present invention can be utilized as a novel therapeutic agent for antagonizing TRPV1 channels in chronic pain and as a therapeutic agent capable of treating various mediated diseases associated with TRPV1 activity.

[0043] According to another aspect of the present invention, there is provided a method for treating a TRPV1 activity-mediated disease, comprising the step of administering to a patient with a TRPV1 activity-mediated disease a composition comprising a GDF11 (growth differentiation factor 11) peptide comprising the amino acid sequence shown in SEQ ID NO: 1, a polynucleotide encoding the peptide, or an expression vector comprising the polynucleotide.

[0044] Since the above-described expression vector or TRPV1 activity-mediated disease is the same concept as that used in the above-described pharmaceutical composition, the description is replaced by that description.

Advantages of the Invention

[0045] The composition for treating pain using TRPV1 activity inhibition of the present invention as described above shows the effect of efficiently suppressing neuropathic pain caused by spinal nerve injury, and thus can be utilized as a novel therapeutic agent for various pain conditions and diseases related to the TRPV1 channel, such as arthritis and diabetic peripheral neuropathy. Also, since it effectively inhibits TRPV1 activity, it can also be utilized as a therapeutic agent for treating various TRPV1 activity-mediated diseases. Of course, such effects do not limit the scope of the present invention.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0047] General Method Establishment of a Mouse Model of Spinal Cord Nerve Injury with Neuropathic Pain The present inventor produced a spinal nerve transection (SNT) animal model. Specifically, 6-week-old male C57BL / 6 mice were acclimated for 1 week in the breeding environment of the animal laboratory, and then, once, radiant heat stimulation was applied to the plantar surface of the right hind limb using an infrared heat stimulation device, and the time until the avoidance response was measured to set a reference value. Thereafter, the experimental groups were randomly separated (n = 5 - 6 mice / group), and the spinal nerve injury surgery was performed. First, under anesthesia by intraperitoneal injection of pentobarbital sodium (60 mg / kg), the skin on the back of the mouse was incised, the 5th lumbar spinal nerve (L5 spinal nerve) was transected, and the incised muscle and skin were sutured with a surgical stapler.

[0048] Establishment of an Animal Model of Peripheral Neuropathy The present inventor produced a chemotherapy-induced peripheral neuropathy (CIPN) animal model.

[0049] Specifically, 6-week-old male C57BL / 6 mice were acclimated for 1 week in the breeding environment of the animal laboratory. 10 mg of paclitaxel (Sigma aldrich, USA) was dissolved in 1.66 ml of a solution prepared by mixing Cremophor EL and absolute ethanol at a ratio of 1:1 to make a stock solution at 6 mg / ml. 100 μl aliquots were placed in microtubes and stored at -20°C. Immediately before administration, after thawing the paclitaxel stock, it was diluted 1 / 30 in physiological saline and administered intraperitoneally at a dose of 2 mg / kg once a day for 5 days (cumulative dose 10 mg / kg) to induce anticancer drug-induced pain. Seven days after the last administration, the heat pain response was evaluated, and individuals with an avoidance reaction time of less than 6 seconds to radiant heat stimulation were considered to have developed anticancer drug-induced pain.

[0050] Measurement of Thermal Hyperalgesia Place an acrylic chamber without a bottom on the glass plate of the plantar test analgesia meters, put the mice in the chamber for 2 hours each day for 3 days, and let them go through the stabilization process. Before creating the spinal cord injury mouse model, measure the basic pain response value to radiant heat stimulation. Set the infrared intensity to 25 or 30 to obtain a basic pain avoidance reaction time of 8 - 10 seconds. Apply radiant heat stimulation to the plantar surface of the right hind limb of the mouse 3 times at intervals of at least 1 minute per individual, measure the avoidance reaction time, and present the average value as the heat pain sensation reaction value. When evaluating the heat pain sensation reaction, limit the duration of the heat stimulation to 20 seconds to prevent thermal damage to the plantar tissue caused by the radiant heat stimulation. After measuring the basic pain reaction value, calculate the change rate of the reaction value of each individual based on the average value of the whole population, and select individuals belonging to the range of the overall average value ± 50%. Only the selected individuals were used to create a neuropathic pain model. After nerve injury, on the 5th day, measure the heat hyperalgesia reaction under the above - mentioned conditions. When measuring the heat pain sensation reaction, every 2 hours, put the experimental animals into the acrylic chamber to stabilize them, then apply radiant heat stimulation and measure the heat pain sensation reaction between 4 pm and 6 pm. Subsequently, randomly divide the neuropathic pain animal model into 2 groups, administer human recombinant GDF11 or physiological saline intrathecally, and then evaluate the analgesic effect of the heat pain sensation reaction (Figure 2a). The researcher in a blind state measured the heat pain sensation reaction for the experimental group. After the planned heat pain sensation reaction evaluation test was completed, the results were analyzed using the information of the drug - administered group and statistical processing was performed.

[0051] Intrathecal Drug Administration Under isoflurane inhalation anesthesia, use a syringe to directly administer the drug intrathecally. Connect a 30 - gauge injection needle to a Hamilton syringe, fill it with GDF11 or physiological saline, and then inject it into the space between the 5th and 6th vertebrae to administer GDF11 at a concentration of 10 ng / 5 μl.

[0052] Culture of Mouse Sensory Nerve Cells The inventor of the present invention performed primary culture of sensory neurons from dorsal root ganglion (DRG) cells in mice. Specifically, C57BL / 6 mice at 6-9 weeks after birth were anesthetized with isoflurane for 5 minutes, disinfected with 70% ethanol, and then the back was incised. Thereafter, the spinal DRG at all sites were dissected and placed in a solution mixed with HBSS (10-fold) and 10 mM HEPES (wood et al, J Neurosi. 8, pp3208-3220, 1988). Thereafter, the dorsal root ganglion was cultured in a solution mixed with 0.2 mg / ml collagenase A and 3 mg / ml dispase II, washed once with DMEM medium containing 10% FBS, and then disrupted using a Pasteur pipette. Next, an average of 100-300 cells were dispensed onto a coverslip coated with poly-d-lyrine, cultured for 1 hour, and then 2 ml of Neurobasal medium containing 10% FBS, 1% penicillin-streptomycin, and 1X B27 supplement was added for further culture.

[0053] Intracellular Ca2+ Influx Experiment For the calcium influx measurement experiment, the primary cultured sensory neurons were exposed to DMEM medium containing 2 μM Fura-2AM for 40 minutes, and then the degree of intracellular influx of calcium ions was measured based on the ratio of F340 / F380, and the calcium influx inhibition rate by GDF11 pretreatment was measured for the increased F340 / F380 caused by 200 nM capsaicin. Specifically, the measurement of intracellular calcium ion concentration was performed using a microscope equipped with an imaging camera and a fluorescence measurement device connected thereto. The ratio of fluorescence wavelengths (F340 / F380) shown during fluorescence measurement reflects the intracellular calcium ion concentration. The perfusion fluid containing the drug was perfused at a rate of 2-3 ml / min. The composition of the perfusion fluid was 140 mM NaCl, 5 mM KCl, 1 mM CaCl 2 2, 1 mM MgCl 2It was 10 mM glucose and 10 mM HEPES, and NaOH was added to adjust the pH to 7.4 (Figure 1b).

[0054] Single-Cell Patch-Clamp Experiment By patch-clamp experiment, the inhibitory rate of capsaicin-induced inward current by GDF11 was measured. The composition of the solution in the patch pipette was 126 mM K-gluconate, 10 mM NaCl, 1 mM MgCl 2 、0.1 mM Na 2 GTP, 2 mM Na 2 ATP, 10 mM HEPES, 10 mM EGTA, and KOH was added to adjust the pH to 7.4. The composition of the extracellular perfusion fluid was 140 mM NaCl, 2 mM EGTA, 1 MgCl 2 、10 mM glucose, 5 mM KCl, 10 mM HEPES, and NaOH was added to adjust the pH to 7.4. The extracellular perfusion fluid containing the drug was perfused at a rate of 2 - 3 ml / min by gravity, and through the extracellular perfusion fluid, capsaicin and GDF11 were perfused into the nociceptive neurons to measure the inhibitory rate of capsaicin-induced inward current by GDF11.

[0055] Transformation of pcDNA TRPV1 Expression Vector in HEK293 Cells After the present inventors simultaneously transformed the HEK293 cells with the TRPV1 gene and the GFP (green fluorescence protein) gene, calcium influx and inward current were measured by the intracellular Ca2+ influx measurement experiment and the single-cell patch-clamp experiment described above. Specifically, lipofectamine TM 2000 (Invitrogen, USA) was used to transform the pcDNA TRPV1 expression vector using the following method. First, after removing the medium of HEK293 cells cultured in a 35 mm cell culture dish, only DMEM medium was added, and 5% CO 2, Wet-cultured at 37°C. pcDNA-TRPV1 (1 μg) and Lipofectamine (10 μl) were dissolved in their respective DMEM media. After waiting for 5 minutes, the two media were mixed and further waited for 20 minutes. Then, after removing the DMEM medium from the dish to which HEK293 cells had adhered, the medium containing the pcDNA prepared above was dispensed and cultured for 4 hours in 5% CO 2 , Wet-cultured at 37°C. Exchanged with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, cultured for 12 hours, and calcium imaging or patch clamp experiments were performed within 24 hours.

[0056] Production of Viral Vector The inventor produced a viral vector containing the GDF11 gene.

[0057] Specifically, to induce the constitutive expression of GDF11 in infected cells upon infection of nerve cells, the serotype of AAV5 and the CMV promoter were selected. Also, an IRES (Internal ribosome entry site) and an eGFP (enhanced green fluorescence protein) nucleotide sequence were added after the GDF11 nucleotide sequence so that GDF11 and GFP proteins were expressed independently of each other in the infected cells, and through the GFP fluorescent protein signal, the distribution of the infected cells and the expression of GDF11 in the tissues of experimental animals could be evaluated. After loading the GDF11 gene onto the AAV5 vector to produce a viral vector, nucleotide sequencing was used to verify the integration of the normal GDF11 gene, and the titer of the tested GDF11-expressing virus was 1.22×10 13 GC / ml.

[0058] Tissue Immunostaining After injecting the viral vector containing the gene into the spinal canal, the inventor performed tissue immunostaining to confirm the expression of GDF11 in sensory nerve cells.

[0059] Specifically, the experimental animals were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg / kg). After filling a 30-ml syringe with 30 ml of 0.9% NaCl solution, the right atrial side of the heart was incised, and the syringe needle was inserted into the left ventricle to remove the blood. Subsequently, 30 ml of 10% formalin solution was administered into the left ventricle. After fixation, the DRG tissues of L4 and L5 were sampled. The removed DRG tissues were immersed in 10% formalin solution for 16 hours to further proceed with the fixation work. Thereafter, the tissues were transferred to 30% sucrose solution and the dehydration process was carried out for 2 days. Subsequently, the DRG tissues were frozen at -20°C and then tissue sections with a thickness of 12 μm were made. The tubulin antibody (Santa Cruz, USA) was diluted 1 / 100 in 0.1M PBS solution and the DRG tissue sections were treated for 24 hours. After washing with 0.1M PBS, fluorescence images were captured using a confocal microscope.

[0060] Experimental Statistics Statistical analysis was performed using Prism (Graphpad, version 501) software. Specifically, after confirming the normality by normality test, parametric or non-parametric test methods were used. For the parametric and non-parametric test methods, when there were two or more groups, t-test was performed, and when there were three or more groups, one-way ANOVA test or two-way ANOVA test was used for analysis. Error bars visualized SEM (standard error of measurement). If the experimental results were significant, * or # was marked [1 for P < 0.05, 2 for P < 0.005, 3 for P < 0.001].

[0061] [Example 1] Evaluation of thermal pain hypersensitivity <1-1> Mouse Model of Chronic Pain Caused by Spinal Cord Nerve Injury The inventor produced a chronic pain mouse experimental model of spinal nerve injury (SNT). After administering GDF11 (10 ng) into the spinal canal, the heat pain sensation response was evaluated using a Hargreaves apparatus. As a result, it was revealed that, compared with the control group, by administering the GDF11 of the present invention to the chronic pain mouse model, the phenomenon of heat pain hypersensitization was suppressed and the avoidance reaction latency of the hind paw plantar surface increased (Figure 2a).

[0062] <1-2> Mouse Model of Peripheral Neuropathy The inventor produced an experimental model of chemotherapy-induced peripheral neuropathy (CIPN) mice. After intraperitoneally administering 0.1 mg / kg or 0.2 mg / kg of GDF11, the heat pain sensation response was evaluated using a Hargreaves apparatus.

[0063] As a result, as shown in Figures 2b and 2c, it was confirmed that the administration of GDF11 suppressed the phenomenon of heat pain hypersensitization and increased the avoidance reaction latency. In particular, compared with gabapentin (Gabapentin; Sigma aldrich, USA), which is one of the representative pain therapeutic agents, it was revealed that GDF11 suppressed the phenomenon of heat pain hypersensitization with 650 times less amount and for a longer period (Figure 2b).

[0064] [Example 2] Analysis of calcium influx into mouse sensory neurons and HEK293 cells In primary cultured mouse sensory neurons and HEK293 cells transformed using the pcDNA-TRPV1 vector, the calcium influx by the treatment with the GDF11 (10 nM) and capsaicin (200 nM) of the present invention, and the calcium influx by the treatment according to different GDF11 concentrations (0.1 nM, 1 nM, or 10 nM) were comparatively analyzed.

[0065] As a result, it was revealed that, compared with the control group not treated with GDF11, the treatment with the GDF11 of the present invention decreased the TRPV1-mediated Ca2+ influx (Ca2+ transient) phenomenon in a concentration-dependent manner (Figures 3a, 3b and Figures 4a, 4b). The degree of calcium influx was shown by normalizing based on the size of the first calcium reaction by capsaicin.

[0066] [Example 3] Analysis of the size of inward current in mouse sensory neurons and HEK293 cells In primary cultured mouse sensory neurons and HEK293 cells transformed with the pcDNA-TRPV1 vector, the size of the inward current induced by the treatment with GDF11 (10 nM) and capsaicin (200 nM) of the present invention, and the size of the inward current by treatment according to different GDF11 concentrations (0.1 nM, 1 nM, or 10 nM) were compared and analyzed. As a result, it was revealed that compared with the control group not treated with GDF11, GDF11 of the present invention concentration-dependently reduced the capsaicin-induced TRPV1-mediated inward current in mouse sensory neurons (Figs. 5a, 5b and Figs. 6a, 6b). The measurement of the inward current was shown after being standardized based on the size of the first response to capsaicin.

[0067] The results of Examples 1 to 3 above suggest that since the treatment with GDF11 effectively suppresses TRPV1 ion channel activity at low concentrations, it can be utilized as an effective antagonist of TRPV1.

[0068] [Example 4] Confirmation of side effects of GDF11 Existing TRPV1 antagonists are known to be accompanied by side effects such as abnormal fever or hypothermia under the administration conditions showing analgesic efficacy. The applicant of the present application confirmed whether such side effects occur due to the administration of GDF11.

[0069] Specifically, in a cancer pain model mouse with a part of the back hair shaved to expose the skin, BCTC (30 mg / kg) or GDF11 (0.1 mg / kg or 0.2 mg / kg) at a concentration that similarly suppresses TRPV1 activity by capsaicin was intraperitoneally administered, and then the temperature of the exposed skin was measured by time zone using an infrared thermal imaging camera.

[0070] As a result, as shown in Fig. 7, it was revealed that even when GDF11 was administered, there was no significant change in the body temperature of the mice, and it was confirmed that administration of GDF11 did not cause side effects of abnormal fever or hypothermia. On the other hand, when BCTC, a representative TRPV1 antagonist, was intraperitoneally administered at a dose of 30 mg / kg, side effects of abnormal fever were shown.

[0071] [Example 5] Confirmation of GDF11 expression of viral vector When administering the produced viral vector, the present inventor confirmed whether the expression of GDF11 in the body was induced.

[0072] Specifically, for a neuropathic pain model caused by peripheral spinal nerve injury, 5 μl of a GDF11 viral vector at a concentration of 1.22×10 11 GC / ml was injected intrathecally. Five weeks later, the mice were sacrificed, and the DRG tissues of L4 and L5 were sampled to prepare freeze-dried sections. The tissues were immunostained with a tubulin antibody, and the GFP fluorescence signal was regarded as the expression level of GDF11, and imaging work was performed with a confocal microscope.

[0073] As a result, as shown in Fig. 8, it was confirmed that GDF11 was expressed in the DRG neurons of L4 or L5 of the mice.

[0074] [Example 6] Confirmation of pain-relieving effect of viral vector The present inventor confirmed whether the produced viral vector AAV5-GDF11 showed an analgesic effect in a neuropathic pain animal model.

[0075] Specifically, after measuring the basic pain response value before making the pain model, the peripheral spinal nerve was transected to make a neuropathic pain model. Three days after the surgery of the pain model, the heat pain sensation response was evaluated, and individuals with an avoidance reaction threshold of 6 seconds or less were regarded as the pain model. For this pain model, 1.22×10 115 μl of the GDF11 viral vector at a concentration of GC / ml was injected into the intrathecal space, and the thermal pain response was evaluated once a week using a Hargreaves apparatus.

[0076] As a result, as shown in Figure 9, it was confirmed that administration of AAV5-GDF11 suppressed the phenomenon of thermal hyperalgesia and increased the avoidance reaction latency.

[0077] In summary, GDF11, a pain treatment composition using TRPV1 activity inhibition of the present invention, was clearly shown to rapidly and effectively inhibit the TRPV1 channel. Therefore, it can be utilized as a novel therapeutic agent for various pain conditions and diseases related to the TRPV1 channel, such as arthritis and diabetic peripheral neuropathy.

[0078] The present invention has been described with reference to the above-described embodiments, but these are merely exemplary, and those having ordinary knowledge in the technical field will understand that various modifications and equivalent other embodiments are possible based thereon. Therefore, the true technical protection scope of the present invention should be determined by the technical idea of the appended claims.

Industrial Applicability

[0079] The pain treatment composition using TRPV1 activity inhibition of the present invention shows an effect of efficiently suppressing neuropathic pain caused by spinal cord nerve injury. Therefore, it can be utilized as a novel pain treatment agent for various pain conditions and diseases related to the TRPV1 channel, such as arthritis and diabetic peripheral neuropathy. In addition, since it effectively inhibits TRPV1 activity, it can also be utilized as a therapeutic agent for treating various TRPV1 activity-mediated diseases, and thus has high industrial applicability.

Sequence Listing Free-Text

[0080] SEQ ID NO: 1 shows the amino acid sequence of the mature peptide of GDF11 (growth differentiation factor 11). SEQ ID NO: 2 shows the full-length amino acid sequence of human GDF11 peptide. SEQ ID NO: 3 shows the amino acid sequence of mouse GDF11. SEQ ID NO: 4 shows the amino acid sequence of chimpanzee GDF11. SEQ ID NO: 5 shows the amino acid sequence of gorilla GDF11. SEQ ID NO: 6 shows the amino acid sequence of rhesus macaque GDF11. SEQ ID NO: 7 shows the amino acid sequence of crab-eating macaque GDF11. SEQ ID NO: 8 shows the amino acid sequence of cynomolgus macaque GDF11. SEQ ID NO: 9 shows the amino acid sequence of bonobo GDF11. SEQ ID NO: 10 shows the amino acid sequence of porcine GDF11. SEQ ID NO: 11 shows the polynucleotide sequence encoding the GDF 11 peptide (SEQ ID NO: 1).

Claims

1. A pharmaceutical composition for treating TRPV1 activity-mediated diseases, comprising, as an active ingredient, a GDF11 (growth differentiation factor 11) peptide containing the amino acid sequence shown in SEQ ID NO: 1, a polynucleotide encoding the peptide, or an expression vector containing the polynucleotide, wherein the diseases are selected from the group consisting of neuropathic pain, cancer pain, pain caused by anti-cancer agents, postoperative pain, trigeminal neuralgia pain, diabetic neuropathic pain, migraine, arthralgia, and rheumatoid arthritis pain.

2. The pharmaceutical composition according to claim 1, wherein the expression vector is a viral vector or a non-viral vector.

3. The pharmaceutical composition according to claim 2, wherein the viral vector is an adeno-associated virus (AAV) vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector, a vaccinia virus vector, a Sendai virus vector, a flavivirus vector, a rhabdovirus vector, a retrovirus vector, or a lentivirus vector.

4. The pharmaceutical composition according to claim 3, wherein the serotype of the adeno-associated virus (AAV) vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, or AAV16.

5. The pharmaceutical composition according to claim 2, wherein the non-viral vector is a DNA vector, a nanoparticle, a cationic polymer, an exosome, an extracellular vesicle, or a liposome.

6. The pharmaceutical composition according to claim 5, wherein the DNA vector is a plasmid vector, a cosmid vector, a phagemid vector, or an artificial human chromosome.

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