Pharmaceutical composition for treating TRPV1 activity-mediated diseases

Abstract

Disclosed herein is a novel pharmaceutical composition for treating pain, wherein the pharmaceutical composition targets a transient receptor potential vanilloid 1 (TRPV1) receptor and acts as an antagonist.

Description

Pharmaceutical composition for treating diseases mediated by TRPV1 activity

[0001] The present invention relates to a novel pharmaceutical composition for treating pain that acts as an antagonist by targeting the TRPV1 (Transient receptor potential vanilloid 1) receptor.

[0002] The present invention relates to a novel therapeutic pharmaceutical composition, and more particularly, to a pharmaceutical composition for treating a disease mediated by TRPV1 target activity and a method for treating a disease mediated by TRPV1 activity.

[0003] According to the International Association for the Study of Pain (IASP), pain is defined as "an unpleasant sensory and emotional experience associated with actual or potential tissue damage." Pain can generally be categorized into nociceptive, inflammatory, and neuropathic pain.

[0004] Pain perception is crucial for the protection and survival of an organism, as it provides immediate warning from a harmful environment and allows for appropriate action.

[0005] Inflammatory pain results from tissue damage caused by various factors, including infection, tumors, and autoimmune conditions. When tissue is damaged, various chemical mediators are released by the damaged tissue and inflammatory cells, creating an environment rich in cytokines, growth factors, and various ions. Some inflammatory mediators sensitize the somatosensory nervous system, activating pain pathways. This reduces contact and movement in the inflamed area until the tissue damage heals, protecting the individual from further damage and promoting tissue recovery.

[0006] Neuropathic pain is caused by damage to the peripheral nervous system, such as diabetic polyneuropathy or postherpetic neuralgia, or to the central nervous system, such as stroke or spinal cord injury. Neuropathic pain manifests as allodynia, a painful response to stimuli that normally do not cause pain, and hyperalgesia, an excessively intense response to painful stimuli.

[0007] The TRPV1 ion channel is responsible for pain in the dorsal root ganglia and trigeminal ganglia and is specifically expressed in small-diameter sensory neurons. TRPV1, also known as the capsaicin receptor, is a non-selective cation channel with high permeability to cations, particularly Ca 2+ It has good permeability, and the in vitro activity of the compound is measured based on this. TRPV1 is mainly expressed in the primary sensory neurons of the dorsal root ganglia (DRG) and the synaptic neurons of the spinal cord dorsal horn, which are involved in pain transmission and neuroinflammation, and when activated (or excited) by various external or internal stimuli, pain information is transmitted to the central nervous system by action potentials induced by the influx of cations. TRPV1 is known to transmit the sensation of noxious heat by being activated by factors such as heat stimuli (≥40°C) as well as chemical ligands such as capsaicin, acid, and alcohol.

[0008] GLP-1 is an incretin hormone secreted by intestinal L cells. It has the property of regulating insulin secretion in response to stimulation of blood sugar levels, and is known as an antidiabetic agent. However, because the blood half-life of GLP-1 is very short, about 2 minutes, GLP analogs (GLP-1(7-36), exenatide, liraglutide, exendin 9-39, etc.) that can maintain the blood half-life for a longer period have been developed. However, the relationship between exendin 9-39 peptide, a GLP-1 receptor antagonist with the N-terminus truncated from exendin-4 peptide, a GLP-1 receptor agonist, and pain or TRPV1 is unknown.

[0009] The present invention relates to a novel pain-treating peptide, exendin 9-39, which directly targets the TRPV1 ion channel and acts as a TRPV1 antagonist. Furthermore, in order to exclude the potential effect of exendin 9-39 on the GLP-1 receptor, we discovered a core sequence, Exendin 20-29, which acts on TRPV1, and confirmed that Exendin 20-29 does not affect the function of the GLP-1 receptor. Therefore, the present invention relates to a novel exendin 20-29 peptide and its use for pain treatment.

[0010] The present invention provides a peptide for treating diseases mediated by TRPV1, which does not exhibit the side effects observed in conventional TRPV1-targeting therapeutics. Furthermore, the present invention relates to a pharmaceutical composition for treating pain comprising the novel peptide, and more particularly, to a pharmaceutical composition for treating diseases mediated by TRPV1 target activity and a method for treating diseases mediated by TRPV1 activity.

[0011] The present invention relates to a pharmaceutical composition for treating a disease mediated by TRPV1 target activity and a method for treating a disease mediated by TRPV1 activity.

[0012] Specifically, the present invention relates to a pharmaceutical composition comprising a GLP-1 analog peptide; a polynucleotide encoding the peptide; or an expression vector comprising the polynucleotide as an active ingredient, and for treating a disease mediated by TRPV1 activity by inhibiting TRPV1 activity.

[0013] In the present invention, the TRPV1 activity-mediated disease may be pain, and may be, for example, neuropathic pain, cancer pain, chemotherapy-induced peripheral neuropathy, postoperative pain, trigeminal neuralgia pain, diabetic neuropathic pain, migraine, arthralgia, rheumatoid arthritis pain, etc.

[0014] In the present invention, the GLP-1 analog peptide may be an exendin 9-39 peptide represented by the amino acid sequence of SEQ ID NO: 1 or an exendin 20-29 peptide represented by the amino acid sequence of SEQ ID NO: 2.

[0015] Accordingly, the present invention relates to a pharmaceutical composition comprising, as an active ingredient, an exendin 9-39 peptide represented by the amino acid sequence of SEQ ID NO: 1; a polynucleotide encoding the peptide; or an expression vector comprising the polynucleotide, for treating a disease mediated by TRPV1 activity by inhibiting pain or TRPV1 activity.

[0016] In addition, the present invention relates to a pharmaceutical composition comprising, as an active ingredient, an exendin 20-29 peptide represented by the amino acid sequence of SEQ ID NO: 2; a polynucleotide encoding the peptide; or an expression vector comprising the polynucleotide, for treating a disease mediated by TRPV1 activity by inhibiting pain or TRPV1 activity.

[0017] In addition, the present invention relates to an exendin 20-29 peptide represented by the amino acid sequence of SEQ ID NO: 2; a polynucleotide encoding the peptide; or an expression vector comprising the polynucleotide.

[0018] According to the present invention, a pharmaceutical composition for treating a disease that inhibits TRPV1 activity is provided, comprising exendin 9-39 and exendin 20-29 peptides comprising amino acid sequences of SEQ ID NOs: 1 and 2, and the sequences of the two peptides as active ingredients.

[0019] In addition, the present invention relates to a method for treating pain or a disease mediated by TRPV1 activity, comprising administering the exendin 9-39 and exendin 20-29 peptides; a polynucleotide encoding the exendin 9-39 peptide and a polynucleotide encoding the exendin 20-29 peptide; or an expression vector comprising the polynucleotide encoding the exendin 9-39 peptide and an expression vector comprising the polynucleotide encoding the exendin 20-29 peptide.

[0020] In addition, the present invention relates to a method for treating pain or a disease mediated by TRPV1 activity, comprising administering the exendin 9-39 peptide; a polynucleotide encoding the peptide; or an expression vector comprising the polynucleotide.

[0021] In addition, the present invention relates to a method for treating pain or a disease mediated by TRPV1 activity, comprising administering the exendin 20-29 peptide; a polynucleotide encoding the peptide; or an expression vector comprising the polynucleotide.

[0022] The present invention can treat, alleviate, or prevent various pain and / or TRPV1-related diseases without side effects. In particular, because the present invention has little or no effect on the GLP-1 receptor, administration of the present invention can reduce side effects such as hypoglycemia. Furthermore, administration of the pharmaceutical composition of the present invention does not cause side effects such as abnormal fever or hypothermia.

[0023] The pain treatment composition of the present invention utilizing TRPV1 activity inhibition, as described above, exhibits an excellent effect of alleviating pain behavioral responses sensitized to capsaicin without causing fever side effects, unlike existing known TRPV1 antagonists.

[0024] Therefore, it can 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. It also effectively inhibits TRPV1 activity, so it can be used to treat various TRPV1-mediated diseases (e.g., hypertension, stroke, myocardial ischemia, urinary incontinence, urinary bladder hypersensitivity, irritable bowel syndrome, fecal urgency, stomach-duodenal ulcer, gastroesophageal reflux disease (GERD), Crohn's disease, hemorrhoids, asthma, chronic obstructive pulmonary disease, pruritus, psoriasis, tinnitus, cough, hypertrichosis, alopecia, etc.).

[0025] Figure 1 is a graph analyzing the inward current curve and inward current size ratio by exendin 9-39 and capsaicin treatment of the present invention in mouse peripheral sensory neurons.

[0026] Figure 2 is a graph analyzing the graph and size ratio of calcium influx inhibition compared to the control group by exendin 9-39 and capsaicin treatment of the present invention in mouse peripheral sensory neurons.

[0027] Figure 3 is a graph analyzing the ratio of calcium influx compared to the control group by exendin 9-39 and capsaicin treatment of the present invention in HEK293T (Human Embryonic Kidney 293T) cells transformed with pcDNA-rat TRPV1 vector.

[0028] Figure 4 is a graph analyzing the ratio of calcium influx by concentration compared to the control group by treatment with exendin 9-39 and capsaicin of the present invention in a CHO K1 cell line overexpressing human TRPV1, and a table deriving the IC50 value.

[0029] Figure 5 shows the results of a pull-down assay in which a His protein was attached to the N-terminus of the exendin 9-39 peptide of the present invention and reacted with cell lysates of a CHO K1 cell line and a Naive-CHO K1 cell line in which human TRPV1 is overexpressed, to confirm that exendin 9-39 directly interacts with TRPV1, and then a binding protein was detected using Western blot.

[0030] Figure 6 is a fluorescence microscope photograph showing that exendin 9-39 directly binds to TRPV1 by attaching FITC fluorescence to the N-terminus of the exendin 9-39 peptide of the present invention and treating the CHO K1 cell line and Naive-CHO K1 cell line, in which human TRPV1 is overexpressed, with a human-TRPV1 antibody, respectively.

[0031] Figure 7 is a graph showing an inward current curve by treatment with the exendin 9-39 peptide of the present invention, the conventional TRPV1 antagonist BCTC, and low pH (pH 5.5) in a CHO K1 cell line in which human TRPV1 is overexpressed.

[0032] Figure 8 is a graph analyzing the ratio of inward current size compared to the control group by treatment with the exendin 9-39 peptide of the present invention, the existing TRPV1 antagonist BCTC, and low pH (pH 5.5) in a CHO K1 cell line in which human TRPV1 is overexpressed.

[0033] Figure 9 is a graph showing a calcium influx curve by treatment with the exendin 9-39 peptide of the present invention, the conventional TRPV1 antagonist BCTC (Bis-chloro-ethyl-thio-cyanate), and low pH (pH 5.5) in a CHO K1 cell line overexpressing human TRPV1.

[0034] Figure 10 is a graph analyzing the ratio of calcium influx compared to the control group by treatment with the exendin 9-39 peptide of the present invention, the conventional TRPV1 antagonist BCTC (Bis-chloro-ethyl-thio-cyanate), and low pH (pH 5.5) in a CHO K1 cell line overexpressing human TRPV1.

[0035] Figure 11 is a graph analyzing the pain behavioral response (licking) time by concentration when the exendin 9-39 peptide (5 μg or 10 μg) of the present invention is injected into the sole of the foot into a capsaicin-induced spontaneous pain mouse model.

[0036] The left drawing of Fig. 12 is a graph analyzing the latency for starting to show a pain behavioral response when a radiant heat stimulus is applied by injection of the exendin 9-39 peptide (5 or 10 μg) of the present invention into a capsaicin-induced spontaneous pain mouse model by plantar injection, by concentration.

[0037] The right-hand drawing of Fig. 12 is a graph analyzing the threshold value at which a pain behavioral response begins to be shown when a mechanical stimulus is applied by injecting the exendin 9-39 peptide (5 or 10 μg) of the present invention into a capsaicin-induced spontaneous pain mouse model, by concentration.

[0038] Figure 13 is a graph analyzing changes in body temperature over time after administration of the exendin 9-39 peptide (50 mg / kg) of the present invention to mice. It was confirmed that even when exendin 9-39 was administered at ten times the effective concentration, no changes in body temperature were observed in the mice.

[0039] Figure 14 is a graph analyzing the ratio of calcium influx compared to the control group by treatment with exendin 20-29, a core sequence that acts on TRPV1 among the exendin 9-39 sequences of the present invention, and capsaicin in a CHO K1 cell line in which human TRPV1 is overexpressed.

[0040] Figure 15 is a graph analyzing the ratio of inward current size compared to the control group by treatment with exendin 20-29, a core sequence that acts on TRPV1 among the exendin 9-39 sequences of the present invention, and capsaicin in a CHO K1 cell line in which human TRPV1 is overexpressed.

[0041] Figure 16 is a graph analyzing the pain behavioral response (licking) time by concentration when the exendin 20-29 peptide (20 μg) of the present invention is injected into the sole of the foot into a capsaicin-induced spontaneous pain mouse model.

[0042] The left drawing of Fig. 17 is a graph analyzing the latency for starting to show a pain behavioral response when a radiant heat stimulus is applied to a capsaicin-induced spontaneous pain mouse model by injecting the exendin 20-29 peptide (20 μg) of the present invention into the sole of the foot by concentration.

[0043] The right-hand drawing of Fig. 17 is a graph (right) analyzing the threshold value at which a pain behavioral response begins to be shown when a mechanical stimulus is given by injecting the exendin 20-29 peptide (20 μg) of the present invention into a capsaicin-induced spontaneous pain mouse model, by concentration.

[0044] Figures 18 and 19 are graphs showing the inward current curves by treatment with the exendin 20-29 peptide of the present invention, the conventional TRPV1 antagonist BCTC, and low pH (pH 5.5) in a CHO K1 cell line in which human TRPV1 is overexpressed, and graphs analyzing the ratio of the inward current size compared to the control group.

[0045] Figure 20 is a graph showing a calcium influx curve by treatment with the exendin 20-29 peptide of the present invention, the conventional TRPV1 antagonist BCTC, and low pH (pH 5.5) in a CHO K1 cell line in which human TRPV1 is overexpressed, and a graph analyzing the ratio of calcium influx compared to the control group.

[0046] Figure 21 shows the results of measuring changes in blood glucose levels by intraperitoneal injection of 200 μL of vehicle (saline), 10 μg / kg exendin 20-29, and 10 μg / kg exendin-4, a GLP-1 receptor agonist, for comparison, followed by intraperitoneal injection of 2 g / kg glucose 15 minutes later using the same method. Compared to exendin-4, which lowered blood glucose levels, exendin 20-29 did not affect blood glucose control.

[0047] Figure 22 is a schematic diagram of a daily experiment using a CFA-induced inflammatory pain mouse model.

[0048] Figure 23 is a graph analyzing the latency for starting to show a pain behavioral response when a radiant heat stimulus is applied to a CFA-induced inflammatory chronic pain mouse model by injecting the exendin 9-39 peptide (5 or 10 μg) of the present invention into the foot after CFA was injected into the foot in the same manner, according to the concentration.

[0049] Figure 24 is a graph analyzing the threshold value at which a pain behavioral response begins to be shown when a mechanical stimulus is applied to a CFA-induced inflammatory chronic pain mouse model by injecting the exendin 9-39 peptide (5 or 10 μg) of the present invention into the foot after CFA was injected into the foot in the same manner, by concentration.

[0050] Figure 25 shows the results of measuring the size of the sole swelling due to the inflammatory reaction using a digital caliper on days 1, 2, 4, 6, and 7.

[0051] Figure 26 is a graph analyzing the latency for starting to show a pain behavioral response when a radiant heat stimulus is applied to a CFA-induced inflammatory chronic pain mouse model by injecting the exendin 9-39 peptide (5 or 10 μg) of the present invention into the plantar after CFA was injected into the plantar, according to the concentration.

[0052] Figure 27 is a graph analyzing the threshold value at which a pain behavioral response begins to be shown when a mechanical stimulus is applied to a CFA-induced inflammatory chronic pain mouse model by injecting the exendin 9-39 peptide (5 or 10 μg) of the present invention into the foot after CFA was injected into the foot in the same manner, by concentration.

[0053] Figure 28 shows the results of measuring the size of the sole swelling due to the inflammatory reaction using a digital caliper on days 1, 2, 4, 6, and 7.

[0054] Figure 29 is a schematic diagram of a daily experiment using a mouse model of neuropathic pain induced by SNI.

[0055] Figure 30 is a graph analyzing the latency at which a pain behavioral response begins to be shown when a radiant heat stimulus is applied by injection of the exendin 9-39 peptide (10 μg) of the present invention into the sole after inducing neuropathic pain through SNI, by concentration.

[0056] Figure 31 is a graph analyzing the latency for starting to show a pain behavioral response when a radiant heat stimulus is applied by intraperitoneal injection of the exendin 9-39 peptide (10 μg) of the present invention after inducing neuropathic pain through SNI, by concentration.

[0057] Figure 32 is a graph analyzing the latency for the onset of pain behavioral response by concentration when radiant heat stimulation is given by intraperitoneal injection of the exendin 20-29 peptide (10 μg) of the present invention after inducing neuropathic pain through SNI.

[0058] Figure 33 schematically illustrates an outline of the present invention.

[0059]

[0060] Definition of terms:

[0061] The term "GLP-1 (glucagon-like peptide-1)" used herein, also called "glucagon-like peptide-1," is an incretin hormone secreted from gastrointestinal L cells when food is ingested. GLP-1 promotes insulin secretion and inhibits glucagon secretion in a glucose concentration-dependent manner. However, GLP-1 produced in the body is rapidly degraded by dipeptidyl peptidase-4 (DPP-4) within minutes and loses its activity. GLP-1 receptor agonists (liraglutide, exenatide, etc.) are peptide preparations made from GLP-1 analogs that are not hydrolyzed by DPP-4. The N-terminus of the GLP-1 analog exenatide peptide was truncated to develop the GLP-1 receptor antagonist exendin 9-39 peptide.

[0062] As used herein, "TRPV1 (Transient receptor potential channel, Vanilloid subfamily member 1)" belongs to a large family of TRP channels consisting of non-voltage gated cation channels that include the areas of heat, vision, taste, olfactory, and touch. TRPV1 is activated by heat, protons, and endogenous substances that interact through a synergistic effect, thereby inducing pain signals.

[0063] According to an aspect of the present invention, a pharmaceutical composition for treating a disease that inhibits TRPV1 activity is provided, comprising exendin 9-39 peptide and exendin 20-29, and the sequences of the two peptides as active ingredients.

[0064] In the pharmaceutical composition, the TRPV1 activity-mediated disease may be selected from the group consisting of pain, hypertension, stroke, myocardial ischemia, urinary incontinence, urinary bladder hypersensitiveness, irritable bowel syndrome, fecal urgency, stomach-duodenal ulcer, gastroesophageal reflux disease (GERD), Crohn's disease, hemorrhoids, asthma, chronic obstructive pulmonary disease, pruritus, psoriasis, tinnitus, cough, hypertrichosis, and alopecia.

[0065] 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, anticancer drug-induced pain, postoperative pain, trigeminal neuralgia pain, idiopathic pain, diabetic neuropathic pain, or migraine.

[0066] According to another aspect of the present invention, a method for alleviating or treating pain is provided, comprising a step of administering a pharmaceutical composition comprising the sequence of the four peptides as an active ingredient.

[0067] Pain according to the present invention includes nociceptive pain, psychogenic pain, inflammatory pain associated with tissue damage and immune cell infiltration, and pathological pain (functional pain such as fibromyalgia, irritable bowel syndrome, and tension headaches) caused by damage to the nervous system or its abnormal function. Pain may also include anatomically distinct pain such as neck pain, middle back pain, lower back pain, or tailbone pain. Pain may also include neuropathic pain, migraine, and the like. Neuropathic pain is a chronic neurological disorder caused by damage to the nervous system due to various causes such as trauma, inflammation, ischemic damage, or metabolic waste products, and may result from damage or disease affecting the somatosensory system. It is a type of non-malignant chronic pain that is generally caused by abnormalities in the nerves, spinal cord, and brain and is estimated to affect more than 1% of the population.

[0068] Neuropathic pain may be associated with abnormal sensations called dysesthesia, allodynia (painful sensations even to innocuous stimuli), and hyperalgesia (increased and prolonged pain in response to noxious stimuli, such as heat). Furthermore, neuropathic pain may be continuous and / or intermittent (paroxysmal), the latter being likened to an electric shock. Common characteristics include burning, cold, pins and needles sensations in the legs, numbness, and itching. Depending on whether the peripheral or central nervous system is affected, neuropathic pain can be classified as peripheral or central.

[0069] In contrast, nociceptive pain is often described as ache. Furthermore, migraine is a chronic disorder associated with multiple autonomic symptoms, causing headaches ranging from mild to severe. The precise mechanism of migraine remains unknown. The underlying theory involves increased excitability of the cerebral cortex and abnormal regulation of pain neurons in the trigeminal nucleus of the brainstem.

[0070] For example, the pain may be one or more selected from the group consisting of neuropathic pain, cancer pain, postoperative pain, trigeminal neuralgia pain, idiopathic pain, diabetic neuropathic pain, migraine, etc.

[0071] In the pharmaceutical composition of the present invention, the effective amount of the compound may vary depending on the type of the patient's affected area, application site, number of treatments, treatment time, formulation, patient's condition, type of adjuvant, etc. The amount used is not particularly limited, but may be 0.01 μg / kg / day to 10 mg / kg / day. The daily amount may be administered once a day, or divided into 2 to 3 times a day at appropriate intervals, or intermittently at intervals of several days.

[0072] In the pharmaceutical composition of the present invention, the compound may be contained in an amount of 0.1 to 100 wt% based on the total weight of the composition. The pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. In addition, solid or liquid formulation additives may be used in the manufacture of the pharmaceutical composition. The formulation additives may be either organic or inorganic. Examples of the excipients include lactose, sucrose, sucrose, 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. Any colorant that is generally approved for addition to pharmaceuticals can be used. These tablets and granules can be appropriately coated with sugar, gelatin, or other coatings as needed. In addition, preservatives, antioxidants, etc. can be added as needed.

[0073] The pharmaceutical composition of the present invention can be prepared in any formulation commonly manufactured in the art, and the form of the preparation is not particularly limited.

[0074] The pharmaceutical composition of the present invention can be administered orally or parenterally, and preferably parenterally, by intravenous injection, subcutaneous injection, intracerebroventricular injection, intracerebrospinal fluid injection, intrathecal injection, transforaminal injection, intramuscular injection, and intraperitoneal injection.

[0075] The present invention provides a method for alleviating and / or treating pain, comprising administering a therapeutically effective amount of the pharmaceutical composition to a patient in need of pain alleviation and / or treatment. The method may further comprise, prior to the administering step, a step of identifying the patient as a patient in need of pain alleviation and / or treatment. The term "therapeutically effective amount" may be based on the amount of active gradient that can achieve the desired effect, pain alleviation and / or treatment.

[0076] The pharmaceutical composition of the present invention is useful for the prevention and treatment of diseases associated with the activity of TRPV1, and the diseases associated with the activity of TRPV1 may be pain, and the pain may be acute pain, chronic pain, neuropathic pain, post-surgical pain, rheumatoid arthritis pain, arthritis pain, post-herpetic neuralgia, neuropathic pain, neuralgia, headache, toothache, pelvic pain, migraine, bone cancer pain, chemotherapy-induced pain, breast pain, or visceral pain. In addition, when the pain is pain, the pain may be nociceptive pain, neuropathic pain, visceral pain, somatic pain, acute pain, chronic pain, or pain due to heat injury by acid or heat.

[0077] Additionally, diseases associated with the activity of TRPV1 may include metabolic diseases, mental diseases, neurodegenerative diseases, gastrointestinal diseases, respiratory diseases, skin diseases, inflammatory diseases, cardiovascular diseases, urinary diseases, ear diseases, or hair-related diseases. At this time, the metabolic disease may be obesity, type 1 diabetes, or type 2 diabetes (Brito et al., Cells 3(2): 517-545, 2014), the mental disease may be epilepsy (Naziro┒lu et al., Curr. Neurolpharmacol., 13(2): 239-247, 2015), depression, anxiety disorder, or fear (Naziro┒lu et al., Curr. Neurolpharmacol., 13(2): 248-257, 2015), and the neurodegenerative disease may be Parkinson's disease, Alzheimer's disease (Duitama et al., Front. Neurosci. 2020; 14: 782, 2020), Huntington's disease (Ho et al., Am. J. Neurodegener. Dis. 2012; 1(1): 1-14, 2012), spinal cord injury (Wu et al., Pain 154(10):2130-2141, 2013), multiple sclerosis (Bassi et al., Front. Neurol. 2019; 10: 30, 2019), encephalomyelitis (Parenti et al., Br. J. Pharmacol., 173: 953-969, 2016), nerve damage (Ren et al., Neural. Regen. Res., 10(8): 1324-1331, 2015), diabetic neuropathy (Hong et al., J. Neurochem. 105(4): 1212-1222, 2008), or stroke (Miyanohara et al., Biochem. Biophys. Res. Commun. 467(3):478-483, 2015). and the above gastrointestinal diseases include gastroduodenal ulcer, functional dyspepsia, irritable bowel syndrome, and pancreatitis (Du et al., Front. Physiol.10: 1064, 2019), gastroesophageal reflux disease, inflammatory bowel disease (Storozhuk et al., BioMed Res. Int. 2019 Article ID 5806321, 2019), gastritis (Oliveira et al., Nutrients 11(1): 208, 2019), the respiratory disease may be asthma or chronic obstructive pulmonary disease (Kim, Allergy Asthma Immunol. Res. 10(3): 187-188, 2018), and the cardiovascular disease may be hemorrhagic shock (Akabori et al., Surgery, S0039-6060(20)30610-3, 2020), cardiac hypertrophy, heart failure (Horton et al., Channels (Austin) 7(1): 17-22, 2013) and the hair-related disease may be alopecia (Toth et al., Molecules 24: 918, 2019); The urinary disease may be urinary incontinence or bladder hypersensitivity (Round et al., Br. J. Clin. Pharmacol. 72(6): 921-931, 2011), the ear disease may be hyperacusis, hearing loss, tinnitus or vestibular hypersensitivity (Bauer et al., Int. Tinnitus J. 13(1):21-28, 2007), the skin disease may be prurigo ndularis (Toth et al., Br. J. Pharmacol. 171: 2568-2581, 2014), and the inflammation-related disease may be vulvodynia (Tympanidis et al., Eur. J. Pain 8(2):129-133, 2004). Rhinitis (Holleand et al., Br. J. Clin. Pharmacol. 77(5):777-788, 2014), allergic conjunctivitis (Kwon et al., Ocul. Immunol.Inflamm. 26(3): 440-448, 2018), pancreatitis (Schwartz et al., J. Neurosci. 33(13): 5603-5611, 2013), cystitis (Dornelles et al., Br. J. Pharmacol., 171(2): 452-467, 2014), rheumatoid arthritis, osteoarthritis (Galindo et al., Pharmaceuticals (Basel). 11(4): 105, 2018), gout (Hoffmeister et al., Rheumatol. (Oxford) 53(2): 240-249, 2014), psoriasis (Gauin et al., Protein Cell. 8(9): 644-661, 2017), pruritus (Gibson et al., PLoS One, 9(7): e100610, 2014), pruritus (Zeidler et al., Dermatol. Ther. (Heidelb). 9(4): 613-622, 2019), skin irritation (Li et al., Int. J. Cosmet. Sci. 39(1): 11-16, 2017), or atopic dermatitis (Toth et al., Br. J. Pharmacol. 171: 2568-2581, 2014).

[0078] GLP-1 acts on the pancreas to increase insulin secretion and decrease glucagon secretion, resulting in a blood sugar lowering effect. It also delays the passage of food from the stomach and acts on the brain to suppress appetite, thereby comprehensively controlling blood sugar levels and helping with weight loss. Furthermore, it improves the function of pancreatic beta cells, which has a positive effect on insulin sensitivity (Zander et al., Lancet 359:824-830, 2002). It was expected that blood sugar levels would improve by restoring the initial insulin secretion abnormality. However, the active form of GLP-1 had a very short half-life of approximately 2 minutes, limiting its use as a therapeutic agent. To overcome these limitations, efforts have been made to maintain the concentration of active GLP-1 by inhibiting DPP-4, the hydrolase that inactivates GLP-1, and to find peptides with a similar structure to GLP-1 that are not hydrolyzed by DPP-4. Currently, DPP-4 inhibitors (sitagliptin, vildagliptin, saxagliptin, linagliptin, alogliptin, gemigliptin, etc.) are being developed and used as drugs corresponding to the former, and GLP-1 analogues (exenatide, liraglutide, lixisenatide, dulaglutide, etc.) are being developed and used as drugs corresponding to the latter.

[0079] The present invention can be implemented by gene therapy (a therapy that inserts a polynucleotide encoding a peptide sequence into an expression vector to deliver a therapeutic gene into a cell) that utilizes genes instead of proteins or peptides. For such gene therapy, in order to facilitate amplification and manipulation of the genes, a recombinant vector is used in which a gene construct operably linked to a regulatory sequence is inserted into various expression vectors, wherein the polynucleotide encoding the exendin 9-39 and exendin 20-29 peptides, or the sequence of either of the two peptides, or the sequence of both peptides is included. However, in this case, since the gene construct is manufactured in a form that includes a signal sequence and a portion that is removed by cleavage, and a mature peptide can be produced through post-translational processing in the body, a polynucleotide encoding a full-length protein can also be used. Such expression vectors are divided into viral and non-viral vectors. The viral vectors used above include adeno-associated virus vectors, adenovirus vectors, alphavirus vectors, herpes simplex virus vectors, vaccinia vectors, Sendai virus vectors, flavivirus vectors, radobovirus vectors, retrovirus vectors, and lentivirus vectors. Viral vectors used in gene therapy are well summarized in the literature (K. Lundstrom, Diseases, 6(2): 42, 2018). The literature is incorporated herein by reference. However, among these viral vectors, in the case of retroviruses or lentiviruses, the transgene is inserted into the host genome, and since the insertion is random, there are concerns about unexpected side effects such as the occurrence of cancer. Accordingly, adeno-associated viruses, which insert only into a specific location in the genome of the host cell, are attracting attention.

[0080] The above adeno-associated virus (AAV) is a single-stranded DNA virus and a helper-dependent human parvovirus. The genome size is approximately 4.7 kbp, and the N-terminal portion of the genome encodes the rep gene involved in viral replication and viral gene expression, and the C-terminal portion encodes the cap gene encoding the viral capsid protein, and it consists of an inter-translational repeat region (ITR) with approximately 145 bases inserted at both ends. Four proteins are translated from the rep region, which are classified as rep78, rep68, rep52, and rep40 according to their molecular weights, and perform important functions in AAV DNA replication. Three proteins, VP1, VP2, and VP3, are translated from the cap region and are structural proteins required for AAV particle formation (virus assembly) (Muzyczka N. Curr. Top Microbiol. Immunol. 158: 97-129, 1992).

[0081] The AAV vector of the present invention can be used to deliver a foreign gene sequence into a cell according to various viral infection methods known in the art, and the method is not particularly limited.

[0082] The activity of TRPV1, a member of the non-selective cation channel family, is Ca 2+TRPV1 induces an influx of stimuli and is inhibited by specific antagonists such as capsazepine. It is also directly activated by capsaicin, heat, low pH, bradykinin, PGE2, or ATP, which indicate that TRPV1 is a primary biological sensor for thermochemical stimuli and tissue damage. TRPV1 is reported to be present in various tissues such as brain, kidney, bronchial epithelial cells, and epidermal keratinocytes, and capsaicin stimulation induces an induction of cytoplasmic Ca in keratinocytes. 2+ The concentration of TRPV1 increases and this is inhibited by capsazepine. The above TRPV1 antagonist has been reported as a treatment for chronic pain, but since pharmacological inhibition of TRPV1 causes hyperthermia, the development of an analgesic without hyperthermia effect was required. Accordingly, the present inventors conducted a study on a pain treatment composition that can effectively inhibit TRPV1 and confirmed that the two peptides, exendin 9-39 and exendin 20-29 peptides, can be novel antagonist molecules for pain treatment, and developed the pain treatment composition using TRPV1 activity inhibition of the present invention. The present inventors found that the recombinant exendin 9-39 and exendin 20-29 peptides, and the two peptides, can increase intracellular calcium (Ca 2+ ) We observed inhibition of the function of TRPV1 channels in mouse primary small sensory neurons (DRG) using imaging and whole-cell patch clamping. In addition, similar results as in DRG neurons were confirmed in rat TRPV1-transfected HEK293 cell line and human TRPV1-overexpressing CHO K1 cell line. Therefore, the results suggest that the exendin 9-39 and exendin 20-29 peptides of the present invention, the two peptides, can be utilized as novel pain therapeutic agents that antagonize TRPV1 channels in chronic pain and can be utilized as therapeutic agents for treating various diseases mediated by TRPV1 activity.

[0083]

[0084] Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the examples disclosed below, but can be implemented in various different forms. The following examples are provided to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention.

[0085] General method

[0086] Culture of mouse sensory neurons

[0087] We performed a primary culture of sensory neurons from the dorsal root ganglion (DRG) of the mouse spinal cord. Specifically, 6-9 week-old C57BL / 6 mice were anesthetized with isoflurane for 5 minutes, disinfected with 70% ethanol, and the dorsal region was incised. Spinal DRGs from all regions were dissected and placed in a solution containing 10X HBSS and 10 mM HEPES (Wood et al., J. Neurosi. 8: pp. 3208-3220, 1988). Afterwards, the dorsal root ganglia were cultured in a solution containing 200 U / ml collagenase A and 0.25 mg / ml dispase II, 10% fetal bovine serum was added, washed at least once with glucose DMEM medium, and pulverized using a fire-polish pasteur pipette. Then, an average of 100-300 cells were seeded on poly-D-lyrine-coated coverslips, and after 1 hour of culture, they were cultured for up to 2-3 days under the same conditions as the cell culture medium, Neurobasal, supplemented with 10% bovine calf serum, 1% penicillin streptomycin, and 1X B27 supplement.

[0088] Single-cell patch-clamp experiments

[0089] The inhibition of capsaicin (100 nM)-induced inward current by exendin 9-39 and exendin 20-29 peptides was measured using patch-clamp experiments. The solution in the patch pipette contained 126 mM K-gluconate, 10 mM NaCl, 1 mM MgCl2, 0.1 mM Na2GTP, 2 mM Na2ATP, 10 mM HEPES, and 10 mM EGTA, and the pH was adjusted to 7.4 with KOH. The extracellular perfusate contained 140 mM NaCl, 2 mM EGTA, 1 MgCl2, 10 mM glucose, 5 mM KCl, and 10 mM HEPES, and the pH was adjusted to 7.4 with NaOH. Extracellular perfusate containing drugs was perfused by gravity at a rate of 1 to 2 ml / min, and the inhibition rate of capsaicin-induced inward current by capsaicin, exendin 9-39 and exendin 20-29 peptides, and the two peptides was measured in mouse sensory neurons, HEK293T cells transfected with pcDNA-rat TRPV1 vector, and CHO K1 cells overexpressing human TRPV1.

[0090] Intracellular Ca2+ influx experiment

[0091] For calcium influx experiments, mouse sensory neurons, HEK293T cells transfected with pcDNA-rat TRPV1 vector, and CHO K1 cells overexpressing human TRPV1 were exposed to 2 μM Fura-2AM for 40 minutes, and the degree of calcium ion influx into the cells was measured through the F340 / F380 ratio. The effects of pretreatment with exendin 9-39 and exendin 20-29 peptides and the two peptides on the increased F340 / F380 induced by 100 nM capsaicin were studied. Specifically, intracellular calcium ion concentrations were measured using a microscope (olympus; BX51WI) equipped with an imaging camera (Q-imaing; optiMOS, sCMOS camera) and a fluorescence measurement device (sutter instrument; High speed wavelength switch, LAMBDA DG-4) connected thereto. When the fluorescence intensity change that appears during fluorescence measurement is alternately excited with light of wavelengths of 340 nm and 380 nm, the ratio of the fluorescence intensity emitted at 510 nm (F340 / F380) reflects the intracellular calcium ion concentration. The measurement and analysis of fluorescence signals were performed using Slidebook6 64X software, and fura2AM (Invitrogen; F1221), which reflects the calcium ion concentration, was used as a fluorescent dye to measure the intracellular calcium response. Fura-2AM was treated at a concentration of 2 μM to the primary cultured neurons and loaded into the cells through wet culture at 37℃, 5% CO2 for 40 minutes in a light-blocked condition. After washing three times with DMEM medium (10% FBS, 1% P / S), the coverslip with attached sensory neurons was placed on an introductory microscope, and the perfusate was perfused at a rate of 1 to 2 ml / min by gravity.The composition (mM) of the above perfusion solution was composed of 140 NaCl, 1 CaCl2ㅇH2O, 5 MgCl2ㅇH2O, 10 glucose, 1 KCl, and 10 HEPES, and the pH was adjusted to 7.4 by adding 5N NaOH.

[0092] Transfection of HEK293T cells with pcDNA rat TRPV1 expression vector

[0093] The present inventors simultaneously transfected HEK293T cells with the rat TRPV1 gene and the GFP (green fluorescence protein) gene, and then measured calcium influx and inward current after 24 hours by transferring them to poly-D-lysine-coated coverslips. Specifically, the pcDNA TRPV1 expression vector was transfected using Lipofectamine TM 2000 transfection reagent (Invitrogen; 11668-019) according to the following method. First, the medium of HEK293T cells cultured in a 35π cell culture dish was removed, and only DMEM solution was added, followed by wet culture at 5% CO2 and 37°C. rTRPV1-cDNA (1 μg / μl) and lipofectamine (10 μl), kept cool in a cool-rack, were dissolved in each DMEM solution, waited for 5 minutes, and then the two solutions were mixed and waited for another 20 minutes. Afterwards, the DMEM solution from the dish with HEK293T cells attached was removed, and the prepared solution was dispensed and cultured in a wet atmosphere at 37°C, 5% CO2 for 4 hours. The medium was replaced with DMEM medium containing 10% FBS and 1% P / S, and calcium imaging experiments were performed after at least 12 hours.

[0094] Pull-down assay

[0095] To confirm the direct interaction between exendin 9-39, one of four GLP-1 analogues, and TRPV1, we performed a pull-down assay. Specifically, using the Pull-down PolyHis Protein:Protein Interaction Kit (Thermo Scientific), we treated the cell lysate of a CHO K1 cell line overexpressing human TRPV1 with a peptide tagged with the N-terminus of exendin 9-39 and the cell lysate of a naive CHO K1 cell line for comparison. Subsequently, Western blot was performed using an anti-human TRPV1 antibody (ACC-030, Alomone labs) to visualize the binding of the peptide tagged with the N-terminus of exendin 9-39 to the protein, i.e., TRPV1.

[0096] Cytoimmunological staining

[0097] To confirm the direct interaction between exendin 9-39, one of the four GLP-1 analogues, and TRPV1, we performed immunocytochemical staining. Specifically, CHO K1 cell lines overexpressing human TRPV1 and naive-CHO K1 cell lines for comparison were cultured in confocal dishes, and after 24 hours, the next day, anti-human TRPV1 antibody (ACC-030, Alomone labs) was treated in a 0.1 M PBS solution, and after washing with a 0.1 M PBS solution, the cells were treated once again with a peptide tagged with FITC fluorescence at the N-terminus of exendin 9-39, washed with a 0.1 M PBS solution, and then fluorescence images were captured using a confocal microscope.

[0098] Creation of a mouse pain model

[0099] To determine whether exendin 9-39 and exendin 20-29 peptides exert analgesic effects, we developed a mouse model of acute pain hypersensitivity induced by capsaicin. Specifically, 6-week-old C57BL / 6 male mice were acclimated to a laboratory environment for one week. Acute pain was induced by administering a 10 μL dose of capsaicin (1.6 μg) to the paw pad of the mice. Spontaneous pain, thermal-induced hypersensitivity, and mechanical-induced hypersensitivity were measured.

[0100] Spontaneous pain measurement

[0101] Mice were placed on a mesh bottom with a hole in it and covered with an acrylic chamber of appropriate size to allow minimal movement. The mice were allowed to stabilize for 2 hours each day for 3 days. Five mice per group were injected into the footpad with 10 μL of vehicle (saline), 5 μg exendin 9-39, 10 μg exendin 9-39, and 0.5 μg BCTC for comparison. After 30 minutes, capsaicin (1.6 μg) was administered in a 10 μL volume, and the mice's behavior was recorded for 5 minutes, after which spontaneous pain behavior (licking) was analyzed. A researcher blinded to the experimental groups measured spontaneous pain responses, and after the planned thermal pain assessment test was completed, the results were analyzed and statistically processed using information from the drug administration group.

[0102] Measurement of thermal hyperalgesia (Hargreavest test)

[0103] An acrylic chamber with a hole in the bottom was placed on the glass plate of a thermal pain measurement device (Plantar test analgesia meter), and mice were placed in the chamber for 2 hours each day for 3 days to stabilize. The radiant heat intensity value (Active intensity) was set to 25 or 30 to obtain a basic pain avoidance reaction time of 8-10 seconds. Radiant heat stimulation was applied to the right hind paw of each mouse three times at intervals of at least 1 minute, and the avoidance reaction time was measured, and the average value was presented as the thermal pain response value. To prevent thermal damage to the paw tissue due to the radiant heat stimulation during the thermal pain response evaluation, the duration of the thermal stimulation was limited to 20 seconds. When measuring the thermal pain response, the experimental animals were placed in the acrylic chamber for 2 hours each time, and after stabilization, the radiant heat stimulation was applied, and the thermal pain response was measured between 4:00 and 6:00 PM. Five mice per group were injected into the footpad with 10 μL of vehicle (saline), 5 μg exendin 9-39, 10 μg exendin 9-39, and 0.5 μg BCTC for comparison. After 30 minutes, capsaicin (1.6 μg) was administered in a 10 μL volume, and the analgesic effect of heat nociception was evaluated at 30-minute intervals from 0 to 120 minutes (0, 30, 60, 90, and 120 minutes). Pain responses were measured by a researcher who was blinded to the experimental groups, and after the planned heat nociception evaluation test was completed, the results were analyzed and statistically processed using information from the drug administration group.

[0104] Measurement of mechanical hyperalgesia (Von Frey test)

[0105] The von Frey test is an experiment in which a pain-inducing substance is injected into only one paw of a white rat to induce pain. When even a very small mechanical stimulus is applied to the painful leg, the weight difference between the painful paw and the normal paw is measured by the thickness of the hair that stimulates it. Weinstein von Frey aesthesiometer (Semmes, http: / www.2biol.com / 2biol_analgesia.htm; Pitcher GM et al., Journal of Neuroscience Methods, 87(2): 185-193, 1999). The mouse was placed on a mesh with an opening in the bottom and an acrylic chamber of an appropriate size was covered over the mouse to allow only minimal movement. The mouse was placed in the chamber for 2 hours each time for 3 days to stabilize. Five mice per group were injected into the footpad with 10 μL of vehicle (saline), 5 μg exendin 9-39, 10 μg exendin 9-39, and 0.5 μg BCTC for comparison. After 30 minutes, capsaicin (1.6 μg) was administered in a 10 μL volume, and the analgesic effect of mechanical nociceptive responses was evaluated at 30-minute intervals from 0 to 120 minutes (0, 30, 60, 90, and 120 minutes). Pain responses were measured by a researcher who was blinded to the experimental groups, and after the planned mechanical nociceptive response evaluation test was completed, the results were analyzed and statistically processed using information from the drug administration group.

[0106] Evaluation of thermal and mechanical hyperalgesia

[0107] CFA (20 μl) was injected into the plantar surface to induce inflammatory pain, and one day later, vehicle (saline), 5 μg exendin 9-39, and 10 μg exendin 9-39 were injected into the plantar surface. Thermal nociception was assessed using the Hargreaves apparatus, and mechanical nociception was assessed using the Von Frey apparatus on days 1, 2, 4, 6, and 7. As a result, it was found that administration of exendin 9-39 peptide inhibited thermal hyperalgesia, increasing the latency of withdrawal response, and inhibited mechanical hyperalgesia, increasing the threshold.

[0108] Confirmed reduction in edema

[0109] CFA (20 μl) was injected into the plantar surface to induce inflammatory pain. One day later, vehicle (saline), 5 μg exendin 9-39, and 10 μg exendin 9-39 were injected into the plantar surface. After CFA injection, the inflammatory response-induced edema of the plantar surface was measured using a digital caliper on days 1, 2, 4, 6, and 7. The results showed that the edema decreased with exendin 9-39 peptide administration, indicating that the inflammatory response was attenuated.

[0110] Measuring mouse body temperature

[0111] To determine whether exendin 9-39 exhibited the same febrile side effects as conventional TRPV1 antagonists, body temperature measurements were performed. Specifically, 6-week-old C57BL / 6 male mice were acclimated to an animal laboratory environment for a week. Body temperature was measured by inserting a digital thermometer (Therma-1, ETI. Ltd., West Sussex, UK) soaked in lubricant into the rectum of the mice. Six mice per group were intraperitoneally injected with 200 μL of vehicle (saline), 50 μg / kg exendin 9-39, and 5 mg / kg BCTC for comparison. Body temperature changes were measured at 15-minute intervals from 0 to 180 minutes (0, 30, 60, 90, 120, and 180 minutes).

[0112] Experimental statistics

[0113] Statistical analysis was performed using Prism (graphpad, version 5.01) software. Specifically, after confirming normal distribution through normality test, parametric or nonparametric test method was used. The parametric and nonparametric test method was performed with t-test for two or more groups, and one-way ANOVA test or two-way ANOVA test for three or more groups. In addition, error bars were visualized as SEM (standard error of measurement), and if the experimental result was significant, it was indicated with *, #, or † [one for P < 0.05, two for P < 0.01, three for P < 0.001, and four for P < 0.0001].

[0114]

[0115] Example 1: Analysis of inward current size and calcium influx in mouse sensory neurons

[0116] The magnitude of the inward current and the amount of calcium influx induced by the exendin 9-39 peptide (100 nM) of the present invention and capsaicin (100 nM) treatment in primary cultured mouse sensory neurons were compared and analyzed using patch clamp and calcium imaging. As a result, compared to the control group treated with capsaicin alone, the exendin 9-39 peptide of the present invention was found to reduce capsaicin-induced TRPV1-mediated inward current (Fig. 1) and calcium influx (Fig. 2) in mouse sensory neurons. The comparison of the inward current and calcium influx was normalized to the magnitude of the first response induced by capsaicin and expressed.

[0117] Example 2: Analysis of calcium influx in transfected HEK293T cells and human TRPV1-CHO K1 cells.

[0118] Calcium influx was compared and analyzed by treatment with exendin 9-39 (100 nM) of the present invention and capsaicin (100 nM) in HEK293T cells transfected with pcDNA-rat TRPV1 vector. In addition, calcium influx according to treatment with different concentrations of the peptide was compared and analyzed in CHO K1 cell lines overexpressing human TRPV1, and IC50 values ​​were derived.

[0119] As a result, compared to the control group treated with capsaicin alone, treatment with the exendin 9-39 peptide of the present invention significantly increased rat TRPV1 and human TRPV1-mediated calcium influx (Ca 2+ The shape of the transient influx was reduced (Fig. 3) and was concentration-dependent (Fig. 4). The extent of calcium influx was normalized to the size of the first calcium response induced by capsaicin.

[0120] The results of Example 1 above suggest that treatment with a GLP-1 receptor antagonist directly inhibits TRPV1 ion channel activity at low concentrations, and thus can be utilized as an effective TRPV1 antagonist. The results of Examples 1 and 2 above suggest that treatment with a GLP-1 receptor antagonist directly inhibits TRPV1 ion channel activity at low concentrations, and thus can be utilized as an effective TRPV1 antagonist.

[0121] Example 3: Confirmation of interaction between exendin 9-39 peptide and TRPV1 among four GLP-1 analogues.

[0122] Human TRPV1-overexpressing CHO K1 cell lines were treated with FITC-tagged exendin 9-39 or His-tagged exendin 9-39, and pull-down assays and immunological staining were performed, respectively, to compare the reactions with anti-human TRPV1 antibodies.

[0123] As a result, it was shown that the exendin 9-39 peptide of the present invention directly interacted with TRPV1 compared to the naive-CHO K1 cell line control group without TRPV1 expression (Figs. 5 and 6).

[0124] Example 4: Confirmation of proton-induced changes in TRPV1 response by exendin 9-39 peptide in vitro

[0125] Existing TRPV1 antagonists, when administered under conditions that exhibit analgesic efficacy, have been accompanied by side effects such as hyperthermia or hypothermia. This has been revealed to be due to the modulation of proton-activated TRPV1 among the activation modes of TRPV1. Therefore, the inventors of the present invention compared and analyzed the inward current and calcium influx induced by treatment with exendin 9-39 (1 μM), one of the four peptides of the present invention, and proton (pH 5.5) in a CHO K1 cell line overexpressing human TRPV1.

[0126] As a result, it was confirmed that even when 1 μM, a concentration approximately 30 times greater than the IC50 value of exendin 9-39 for capsaicin, was used, there was no effect on TRPV1 ion channel activity due to protons (Figs. 7 to 10).

[0127] Example 5: Evaluation of capsaicin-induced acute pain hypersensitivity due to Exendin 9-39 administration

[0128] 1-1: Spontaneous Pain Assessment

[0129] The present inventors injected 10 μL of vehicle (saline), 5 μg exendin 9-39, 10 μg exendin 9-39, and 0.5 μg BCTC for comparison into the paw pads of five mice per group. After 30 minutes, capsaicin (1.6 μg) was injected in a 10 μL dose, and the mice's pain behavior (licking the paw pad) was evaluated. As a result, it was confirmed that the spontaneous pain response was suppressed by exendin 9-39 peptide administration, thereby reducing the duration of licking (Fig. 11).

[0130] 1-2: Evaluation of thermal hyperalgesia

[0131] The present inventors injected 10 μL of vehicle (saline), 5 μg exendin 9-39, 10 μg exendin 9-39, and 0.5 μg BCTC for comparison into the footpad of five mice per group. After 30 minutes, capsaicin (1.6 μg) was injected in 10 μL and the thermal pain response was evaluated using a Hargreaves apparatus. As a result, it was found that the thermal hyperalgesia phenomenon was suppressed in response to exendin 9-39 peptide administration, and the withdrawal response latency was increased (Fig. 12, left).

[0132] 1-3: Mechanical hyperalgesia assessment

[0133] The present inventors injected 10 μL of vehicle (saline), 5 μg exendin 9-39, 10 μg exendin 9-39, and 0.5 μg BCTC for comparison into the footpads of five mice per group. After 30 minutes, capsaicin (1.6 μg) was injected in 10 μL and mechanical nociceptive responses were evaluated using a Von Frey apparatus. As a result, it was found that exendin 9-39 peptide administration suppressed mechanical nociceptive hypersensitivity and increased the threshold (Fig. 12, right).

[0134] Example 8: Confirmation of the febrile side effect of exendin 9-39 peptide

[0135] Existing TRPV1 antagonists are known to cause side effects such as fever or hypothermia under administration conditions that exhibit analgesic efficacy. Therefore, the inventors of the present invention sought to determine whether administration of exendin 9-39, one of the four peptides, caused the above side effects. Specifically, after intraperitoneal administration of BCTC (5 mg / kg) at a concentration that causes febrile side effects or exendin 9-39 (50 mg / kg), which is ten times that concentration, the rectal temperature of mice was measured at different time points.

[0136] As a result, it was found that even when exendin 9-39 was administered, there was no significant change in the body temperature of the mice, confirming that exendin 9-39 administration did not induce adverse effects such as abnormal fever or hypothermia. On the other hand, when BCTC, a representative TRPV1 antagonist, was administered intraperitoneally at a dose of 5 mg / kg, an adverse effect of abnormal fever was observed (Fig. 13).

[0137] Example 9: Analysis of calcium influx and inward current magnitude in human TRPV1-CHO K1 cells treated with Exendin 20-29.

[0138] The amount of calcium influx and the size of the inward current by treatment with exendin 20-29 (100 nM) and capsaicin (100 nM) of the present invention were compared and analyzed through calcium imaging and patch clamp. As a result, compared to the control group treated with capsaicin alone, treatment with the exendin 20-29 peptide of the present invention significantly increased human TRPV1-mediated calcium influx (Ca 2+ The shape of the transient influx was reduced (Fig. 14) and the inward current was also reduced (Fig. 15). The magnitude of calcium influx and inward current was normalized to the magnitude of the first calcium response and inward current induced by capsaicin.

[0139] Example 10: Evaluation of capsaicin-induced acute pain hypersensitivity due to administration of Exendin 20-29

[0140] 1-1: Spontaneous Pain Assessment

[0141] The inventors injected 10 μL of vehicle (saline), 20 μg exendin 9-39, and 0.5 μg BCTC for comparison into the paw pads of five mice per group. After 30 minutes, capsaicin (1.6 μg) was injected 10 μL, and the mice's pain behavior (licking the paw pad) was evaluated. As a result, it was confirmed that exendin 20-29 peptide administration suppressed spontaneous pain responses and reduced licking duration (Fig. 16).

[0142] 1-2: Evaluation of thermal hyperalgesia

[0143] The inventors injected 10 μL of vehicle (saline), 20 μg exendin 9-39, and 0.5 μg BCTC for comparison into the footpads of five mice per group. After 30 minutes, capsaicin (1.6 μg) was injected in 10 μL, and the thermal pain response was evaluated using a Hargreaves apparatus. As a result, it was found that administration of the exendin 20-29 peptide suppressed thermal hyperalgesia and increased the latency of the withdrawal response (Fig. 17, left).

[0144] 1-3: Mechanical hyperalgesia assessment

[0145] The inventors injected 10 μL of vehicle (saline), 20 μg exendin 9-39, and 0.5 μg BCTC for comparison into the footpads of five mice per group. After 30 minutes, capsaicin (1.6 μg) was injected in 10 μL, and mechanical nociceptive responses were assessed using a Von Frey apparatus. As a result, it was found that exendin 20-29 peptide administration suppressed mechanical nociceptive hypersensitivity and increased the threshold (Fig. 17, right).

[0146] Example 11: Confirmation of proton-induced changes in TRPV1 response by exendin 20-29 peptide in vitro

[0147] Existing TRPV1 antagonists have been shown to have side effects such as hyperthermia or hypothermia under administration conditions that exhibit analgesic efficacy. This has been revealed to be due to the modulation of proton-activated TRPV1 among the activation modes of TRPV1. Therefore, the inventors of the present invention compared and analyzed the inward current (Figs. 18 and 19) and calcium influx (Fig. 20) induced by exendin 20-29 (1 μM) of the present invention and proton (pH 5.5) treatment in a CHO K1 cell line overexpressing human TRPV1.

[0148] As a result, we were able to confirm that exendin 20-29 in response to capsaicin did not affect proton-induced TRPV1 ion channel activation.

[0149] Example 12: Evaluation of the effect of exendin 20-29 peptide administration on GLP-1 receptor function

[0150] The present inventors injected 200 μL of vehicle (saline), 10 μg / kg exendin 20-29, and, for comparison, 10 μg / kg exendin-4, a GLP-1 receptor agonist, intraperitoneally. Fifteen minutes later, 2 g / kg glucose was injected intraperitoneally using the same method, and changes in blood glucose were measured at -15, 0, 30, 60, 90, and 120 minutes using an Accu-Choda Perfoma glucometer. As a result, it was confirmed that exendin 20-29 did not affect blood glucose control compared to exendin-4, which lowered blood glucose (Fig. 21).

[0151] Example 13: Evaluation of chronic pain hypersensitivity in the CFA inflammatory pain model.

[0152] 1-1: Evaluation of thermal and mechanical hyperalgesia

[0153] CFA (20 μl) was injected into the plantar surface to induce inflammatory pain, and one day later, vehicle (saline), 5 μg exendin 9-39, and 10 μg exendin 9-39 were injected into the plantar surface (Fig. 22). Thermal nociceptive responses were evaluated using the Hargreaves apparatus, and mechanical nociceptive responses were evaluated using the Von Frey apparatus on days 1, 2, 4, 6, and 7. As a result, it was found that administration of exendin 9-39 and exendin 20-29 peptides inhibited thermal hyperalgesia, increasing the latency time of withdrawal response (Figs. 23 and 26), and inhibited mechanical hyperalgesia, increasing the threshold (Figs. 24 and 27).

[0154] 1-2: Confirm reduction in edema

[0155] CFA (20 μl) was injected into the sole of the foot to induce inflammatory pain, and one day later, vehicle (saline), 5 μg exendin 9-39, and 10 μg exendin 9-39 were injected into the sole. After CFA injection, the inflammatory response-induced edema of the sole was measured using a digital caliper on days 1, 2, 4, 6, and 7. As a result, it was found that the edema was reduced (Figs. 25 and 28) in response to exendin 9-39 peptide administration, indicating a reduction in the inflammatory response.

[0156] Example 14: Evaluation of chronic pain hypersensitivity in the SNI neuropathic pain model.

[0157] 1-1: Evaluation of thermal and mechanical hyperalgesia

[0158] Under continuous isoflurane anesthesia, mice underwent surgical manipulation to expose the left sciatic nerve by separating muscle tissue. After the sciatic nerve was identified, the peroneal and tibial nerves were ligated with silk thread and transected below the ligation site, leaving the sciatic nerve intact. The surgical site was then sutured and disinfected with iodine. After a 14-day recovery period, the mice were administered 20 μL or 200 μL of vehicle solution, Exendin 9-39, or Exendin 20-29 intraplantarly or intraperitoneally, respectively (Fig. 29). As a result, it was found that administration of exendin 9-39 (Figs. 30 and 31) and exendin 20-29 (Fig. 32) peptides suppressed thermal hyperalgesia and increased the latency of the withdrawal response.

[0159]

[0160] The present invention relates to a novel therapeutic pharmaceutical composition that acts as an antagonist targeting TRPV1, and more specifically, provides a pharmaceutical composition for treating TRPV1 activity-mediated diseases, containing exendin 9-39 and exendin 20-29 as active ingredients that alleviate or treat various pain and TRPV1-related diseases without side effects.

[0161]

[0162] Sequence number 1: Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser

[0163] Sequence number 2: Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly

Claims

1. A pharmaceutical composition comprising GLP-1 analogue peptides Exendin 9-39 and / or Exendin 20-29, a polynucleotide encoding the peptides, or the polynucleotide as an active ingredient, and for treating a disease mediated by TRPV1 activity by inhibiting TRPV1 activity.

2. A pharmaceutical composition according to claim 1, wherein the GLP-1 analogue peptide is at least one selected from the group consisting of Exendin 9-39 peptide and Exendin 20-29.

3. A pharmaceutical composition according to claim 1 or 2, wherein the TRPV1 activity-mediated disease is selected from the group consisting of pain, hypertension, stroke, myocardial ischemia, urinary incontinence, urinary bladder hypersensitivity, irritable bowel syndrome, fecal urgency, stomach-duodenal ulcer, gastroesophageal reflux disease (GERD), Crohn's disease, hemorrhoids, asthma, chronic obstructive pulmonary disease, pruritus, psoriasis, tinnitus, cough, hypertrichosis, and alopecia.

4. A pharmaceutical composition according to claim 3, wherein the pain is selected from the group consisting of neuropathic pain, cancer pain, chemotherapy-induced peripheral neuropathy, postoperative pain, trigeminal neuralgia pain, diabetic neuropathic pain, migraine, arthralgia, and rheumatoid arthritis pain.

5. A pharmaceutical composition according to claim 2, wherein the Exendin 9-39 peptide is a peptide represented by SEQ ID NO: 1, and Exendin 20-29 is a peptide represented by SEQ ID NO:

2.

6. A pharmaceutical composition comprising an exendin 9-39 peptide represented by the amino acid sequence of SEQ ID NO: 1; a polynucleotide encoding the peptide; or an expression vector comprising the polynucleotide, as an active ingredient, for treating a disease mediated by TRPV1 activity by inhibiting pain or TRPV1 activity.

7. A pharmaceutical composition comprising an exendin 20-29 peptide represented by the amino acid sequence of SEQ ID NO: 2; a polynucleotide encoding the peptide; or an expression vector comprising the polynucleotide, as an active ingredient, for treating a disease mediated by TRPV1 activity by inhibiting pain or TRPV1 activity.

8. A pharmaceutical composition according to claim 6 or 7, wherein the TRPV1 activity-mediated disease is selected from the group consisting of pain, hypertension, stroke, myocardial ischemia, urinary incontinence, urinary bladder hypersensitivity, irritable bowel syndrome, fecal urgency, stomach-duodenal ulcer, gastroesophageal reflux disease (GERD), Crohn's disease, hemorrhoids, asthma, chronic obstructive pulmonary disease, pruritus, psoriasis, tinnitus, cough, hypertrichosis, and alopecia.

9. A pharmaceutical composition according to claim 8, wherein the pain is selected from the group consisting of neuropathic pain, cancer pain, chemotherapy-induced peripheral neuropathy, postoperative pain, trigeminal neuralgia pain, diabetic neuropathic pain, migraine, arthralgia, and rheumatoid arthritis pain.

10. Exendin 20-29 peptide represented by the amino acid sequence of sequence number 2.

11. A polynucleotide encoding an exendin 20-29 peptide represented by the amino acid sequence of SEQ ID NO: 2; or an expression vector comprising the polynucleotide.

Images