Novel peptides and their applications

Abstract

Novel peptides are disclosed.

Description

Technical Field

[0001] The present invention relates to a novel peptide and a novel pharmaceutical composition containing the same. More specifically, the present invention relates to a novel peptide and a pharmaceutical composition for treating TRPV1-mediated diseases (such as pain, etc.) containing the same, or a method for treating TRPV1-mediated diseases using the peptide.

Background Art

[0002] TRPV1 (transient receptor potential vanilloid type 1) is a non-selective Ca 2+ permeable cation channel and a core protein involved in the development of peripheral and central sensitization, which can be activated by physical and chemical stimuli such as exogenous factors (such as capsaicin, resiniferatoxin, etc.), endogenous factors (such as arachidonic acid metabolites, anandamide, etc.), heat or pH.

[0003] TRPV1 has been presented as a main target for the treatment of chronic pain, and research on TRPV1 agonists and antagonists has been actively carried out.

[0004] TRPV1 is mainly present in sensory nerves, but also exists in other tissues such as the brain, bronchus, kidney, bladder, epithelial cells, and epidermal keratinocytes.

[0005] Recently, TRPV1-mediated diseases have been actively studied.

[0006] Although it is known that the GDF11 protein is involved in the TRPV1 channel and can be used as a therapeutic use for TRPV1-mediated diseases, since the GDF11 protein consists of 109 amino acid sequences, there are still difficulties in industrially producing the GDF11 protein and using it as a pharmaceutical.

[0007] Therefore, there is a need to develop peptides that are relatively short in length so that they can be used as a pharmaceutical alternative to GDF11 and that are capable of treating TRPV1-mediated diseases. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The inventors conducted research to develop a peptide with a relatively short length that can be used as a pharmaceutical substitute for GDF11 and that can treat TRPV1-mediated diseases. As a result, they confirmed that even short-length peptides have a therapeutic effect on TRPV1-mediated diseases.

[0009] In particular, by treating and alleviating pain, a typical TRPV1-mediated disease, it can be useful in treating persistent pain even after the stimulus has disappeared and the injured area has clearly healed, or pain that occurs in the absence of any stimulus, injury, or disease. [Means for solving the problem]

[0010] The present invention aims to solve the above problems and relates to a peptide containing any one of the sequences from SEQ ID NOs. 1 to 49, and preferably consists of 25 or fewer amino acids.

[0011] Furthermore, the present invention relates to a peptide comprising the sequence represented by the following formula 1, which preferably consists of 25 or fewer amino acids:

[0012] [Formula 1] X1-X2-X3-X4-YM-X7-X8-Q-X10-W

[0013] In the above formula 1, X1 is A, S, or Y. X2 is G, K, S, or L. X3 is Q, M, R, I, T, L, or S. X4 is A, C, W, M, P, or G. X7 is F, W, or Y. X8 is M, F, Y, S, G, H, or P. X10 is any amino acid.

[0014] In the above formula 1, it is preferable that X1 is Y, X2 is G, X3 is Q, X4 is C, X7 is W or Y, and X8 is a peptide that is a hydrophobic amino acid. Here, the hydrophobic amino acid may be M, F, or Y.

[0015] Furthermore, in formula 1, X10 may be any amino acid, but is preferably K, Y, W, G, E, H, S, C, A, or M.

[0016] Furthermore, the present invention relates to oligopeptides containing the above-mentioned peptides.

[0017] The peptide according to the present invention has a therapeutic effect on TRPV1-mediated diseases. Therefore, the present invention relates to a pharmaceutical composition for the treatment of TRPV1-mediated diseases comprising the above-mentioned peptide.

[0018] TRPV1 activity-mediated diseases may include 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.

[0019] Furthermore, 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.

[0020] Therefore, the present invention relates to a pharmaceutical composition for treating the above-mentioned TRPV1 activity-mediated diseases. The pharmaceutical composition according to the present invention can be administered into the body in an oral or parenteral form (e.g., in an injectable form).

[0021] In addition, the present invention can be a polynucleotide encoding the peptide or an expression vector containing the polynucleotide.

[0022] Therefore, the present invention relates to a pharmaceutical composition for treating TRPV1 activity-mediated diseases, which contains, as an active ingredient, the peptide, or a polynucleotide encoding the same, or an expression vector containing the polynucleotide.

[0023] In the present specification, the single-letter alphabet used to represent amino acids is the one used to represent specific amino acids as shown in Table 1 below:

[0024]

Table 1

[0025] In the present specification, "GDF11 (growth differentiation factor 11)" can be represented by SEQ ID NO: 50 and is also called BMP-11 (bone morphogenetic protein 11). It is a protein expressed by the GDF11 gene present on human chromosome 12.

Advantages of the Invention

[0026] The peptide according to the present invention has a relatively short length so that it can be used for pharmaceutical purposes instead of GDF11, and can effectively suppress TRPV1 activity. Therefore, it can be usefully used as a pharmaceutical composition for treating various TRPV1 activity-mediated diseases.

Brief Description of the Drawings

[0027] [Figure 1] This is the result of an experiment on the inhibitory effect of the peptides of SEQ ID NO: 5 and SEQ ID NO: 6 according to the present invention on calcium influx into the TRPV1 channel. [Figure 2A]This is the result of an experiment investigating the effect of the concentrations of the peptides of SEQ ID NO: 5 and SEQ ID NO: 6 according to the present invention on suppressing calcium inflow into the TRPV1 channel. [Figure 2B] This is the result of an experiment investigating the effect of the concentrations of the peptides of SEQ ID NO: 5 and SEQ ID NO: 6 according to the present invention on suppressing calcium inflow into the TRPV1 channel. [Figure 3] This is the result of experiments on the effect of the peptides of SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 12 according to the present invention on suppressing calcium inflow into the TRPV1 channel. [Figure 4] This is the result of testing the pain-relieving efficacy of formalin on the peptide according to the present invention. [Figure 5] This is the result of testing the pain-relieving efficacy of capsaicin on the peptide according to the present invention. [Figure 6] This is the result of testing the pain-suppressing efficacy of the peptide according to the present invention in a CCI pain model. [Figure 7] This is the result of testing the pain-inhibiting efficacy of the peptide according to the present invention in an SNL pain model. [Figure 8] This is the result of testing the pain-suppressing efficacy of the peptide according to the present invention in a CIPN pain model. [Modes for carrying out the invention]

[0028] The present invention will be described in detail below based on examples. However, the following examples are merely illustrative of the present invention, and the concept and scope of the present invention are not limited thereto.

[0029] Example A. Peptide Design 1. Design of 3 types of peptides TRPV1 plays a crucial role in acute and / or chronic pain disorders. Therefore, finding substances that antagonize TRPV1 could be a strategy for pain control.

[0030] The GDF11 protein, sequence number 50, is known as a protein that acts as an antagonist to TRPV1.

[0031] If a peptide sequence can be found that maintains the pain-suppressing effect while shortening the length of the amino acid sequence of the GDF11 protein, it could be usefully used as a pharmaceutical. This is because shorter peptide sequences are generally easier to synthesize and mass-produce, are more cost-effective, and are expected to have high in vivo stability and low toxicity.

[0032] Protein-protein docking simulation analysis of TRPV1 and GDF11 proteins predicted that the Gln(Q) residue at position 91 of the GDF11 protein would bind to the capsaicin-binding sites of TRPV1: Tyr at position 513, Thr at position 552, and Asn at position 553.

[0033] Therefore, the inventors designed three peptides, shown in Table 2 below, which are 11mer, 15mer, and 19mer peptides centered around the 91st Gln residue of GDF11:

[0034] [Table 2]

[0035] 2. Modification of three types of peptides The TRPV1 activity inhibitory effect was tested on peptides modified by substituting, deleting, inserting, or adding one or more amino acids in the amino acid sequence of each of the three peptides mentioned above. The results showed that the two peptides listed in Table 3 below exhibited the best TRPV1 inhibitory rate (the testing methods for the effects will be described later):

[0036] [Table 3]

[0037] Furthermore, by modifying each of the peptides of Sequence ID No. 5 and No. 6 by substituting one or more amino acids in the amino acid sequence, we designed 11 more peptides as shown in Table 4 below:

[0038] [Table 4]

[0039] 4. Additional peptide design Further selection of the aforementioned peptides and testing for TRPV1 inhibitory activity as shown in the following examples revealed that 49 peptides listed in Tables 5 and 6 below possessed TRPV1 activity:

[0040] [Table 5]

[0041] [Table 6]

[0042] The amino acid sequence according to the present invention can be configured to be protected from protease enzymes by capping the N-terminus with lipoic acid and / or the C-terminus with p-nitroanilide. For example, it can be synthesized by capping the N-terminus of the amino acid sequence of Sequence ID No. 2 with lipoic acid and the C-terminus with p-nitroanilide.

[0043] Furthermore, the amino acid sequence according to the present invention can be constructed cyclically by linking the N-terminus and C-terminus with peptide bonds. For example, the amino acid sequence of Sequence ID No. 8 can be synthesized cyclically by linking the N-terminus and C-terminus with peptide bonds.

[0044] Example B. Evaluation of the TRPV1 inhibitory efficacy of the peptide. The TRPV1 inhibitory efficacy of various short-length peptides, including the 49 peptides listed in Tables 5 and 6, was evaluated using the following method.

[0045] 1. Cell line culture All reagents used in cell culture are sterile products. The cell line culture media are as shown in Table 7 below, and the culture conditions were 50% CO2 and 37°C in a cell incubator. When using thawed frozen cells (stock), they were subcultured 2-3 times before use in the experiment.

[0046] [Table 7]

[0047] After removing the cell culture medium by suction, the cells were washed with 10 ml of DPBS, and the DPBS was removed by suction. 2 ml of 0.05% trypsin-EDTA was added, and the cells were cultured in a 37°C cell incubator for 3-5 minutes. Microscopic examination confirmed that cells attached to the surface of the culture dish had detached, and 8 ml of cell culture medium containing 10% FBS was added to inactivate the trypsin-EDTA. The cells in the culture dish were collected, pipetted, and then transferred to a 15 ml tube.

[0048] After centrifuging at 800 rpm for 5 minutes and removing all of the supernatant liquid except for the cell pellet, add the culture medium and carefully break up the pellet.

[0049] After measuring the cell count with a hemocytometer, 1 × 10¹⁶ cells were placed in the center of a 12 mm poly-D-lysine coated coverslip. 3 Dispensed 10 μl of cells per coverslip.

[0050] A 35π petri dish containing a coverslip was placed in a cell incubator. After 1-2 hours, when the cells had attached to the coverslip, 2 ml of cell culture medium was added and the dish was placed back in the cell incubator.

[0051] 2. Preparation of reagents and experimental materials a. Preparation of extracellular solution The samples were added to 200 ml of distilled water (DW) and mixed according to the composition shown in Table 8 below.

[0052] [Table 8]

[0053] After adjusting the pH to within the range of 7.3-7.4 using 5N NaOH, distilled water (DW) was added to bring the final volume to 250 ml.

[0054] b. Production of capsaicin A 10 mM stock solution was prepared by dissolving capsaicin in 100% ethanol. Before use in the experiment, the TRPV1 active substance, capsaicin, was diluted in an extracellular solution to a concentration of 0.1–1 mM.

[0055] c. Preparation of test substances The test substance (peptide according to the present invention) was dissolved in a solvent and then diluted to an appropriate concentration in an extracellular solution before use in the experiment.

[0056] d. Manufacturing of Fura-2AM 50 mg of Fura 2AM powder, used as a membrane-permeable fluorescent marker, was mixed with 24.95 ml of DMSO to prepare a 2 mM Fura-2AM solution. After the experiment, any remaining Fura-2AM was stored at -20°C for reuse and covered with silver foil to prevent exposure to light.

[0057] 3. Loading the Fura-2AM A 2 mM Fura-2AM solution was diluted to 1 / 1000 in a cell culture medium that did not contain FBS or antibiotics to prepare 1 ml (based on a 35π Petri dish) of the final 2 mM solution. After removing the cell culture medium from the 35π Petri dish, it was washed once with 2 ml of DPBS. The 1 ml of the prepared Fura-2AM solution was transferred to the 35π Petri dish and cultured in a cell incubator for 40 minutes.

[0058] 4. Analysis of calcium images To power the calcium imaging equipment, the IRIS-9 camera was first turned on, followed by the computer. To power the perfusion system, the suction device was turned on, and 20-30 ml of distilled water was placed in each perfusion syringe and suctioned into the chamber (syringe wash).

[0059] Syringes washed with distilled water were filled with pre-prepared extracellular solutions and test substances, respectively, and it was confirmed that the solutions flowed smoothly from each syringe into the chamber. At this time, all syringe lines except for the one being tested were sealed with clips. In other words, it was confirmed that the solution flowed with the clip removed from only one syringe line (if the solution flows through two or more lines, the solutions are mixed).

[0060] Once it was confirmed that the solution was flowing well in each syringe, the test was prepared by flowing only the extracellular solution until the coverslip with the cells attached was placed in the chamber.

[0061] The cells to be used in the experiment were positioned in the center of the microscope lens and observed with a 40x magnification objective lens to set the cell region for calcium imaging using filter 1 (bright filter). Then, filter 4 (Fura-2AM wavelength filter) was switched on, the fluorescence light source (CoolLED) was powered on, and the Fura-2AM loaded cells were observed. Filter 4 was maintained for the experiment to proceed.

[0062] The calcium imaging-driven software (MetaFluor) was run to measure the F340nm / F380nm ratio at 1-second intervals, and the test material was processed according to the time schedule shown in the following figure to observe the change in the ratio of Ca 2+ The inflow level was measured.

[0063] JPEG0007904628000009.jpg63170

[0064] 5. Experimental Results a. Peptide screening using three modified peptides (SEQ ID NOs: 51, 52, and 53) The experimental results showed that injecting peptides of sequence number 5 or 6 significantly reduced calcium inflow (see Figure 1: in Figure 1, the y-axis of each graph is ΔF / F and the x-axis is time (minutes)).

[0065] b. Confirmation of hTRPV1 inhibitory IC50 of peptides SEQ ID NOs. 5 and 6. The inhibition of hTRPV1 activity was confirmed by treating cells with peptides of sequence numbers 5 and 6, which showed high efficacy in inhibiting hTRPV1, at different concentrations. After pretreatment of cultured cells with the peptides, the ratio values ​​(ΔF / F) induced by capsaicin and the ratio values ​​(ΔF / F) induced during the third capsaicin treatment were normalized to the ratio value (ΔF / F) induced by the first capsaicin treatment. The experimental results are shown in Figure 2.

[0066] The peptide of SEQ ID NO: 5 was inhibited by approximately 20% when treated with 1 nM compared to the control group, and its activity was inhibited by 53% at 5 nM, 75% at 10 nM, 79% at 50 nM, 77% at 100 nM, and 95% at 500 nM. The IC50 was confirmed to be 3.056 nM. The experimental results are shown in Figure 3.

[0067] The peptide of Sequence ID No. 6 showed approximately 9% inhibition of activity when treated with 0.5 nM compared to the control group, 16% inhibition at 1 nM, 51% inhibition at 5 nM, and 70% inhibition at 100 nM. The IC50 was confirmed to be 6.399 nM.

[0068] Statistical significance was analyzed using two-way ANOVA. Compared to the control group, *; P<005, **; p<001, ***; p<0001.

[0069] c. Peptide screening modified by substituting some sequences from peptides of SEQ ID NOs. 5 and 6. Experiments with the amino acids of SEQ ID NO: 5 or 6, and peptides modified by substituting one or more amino acids in each amino acid sequence, showed that peptides such as SEQ ID NO: 1 to 49 possessed TRPV1 inhibitory activity. In particular, peptides SEQ ID NO: 5, 6, 9, 11, and 12 showed high TRPV1 inhibition rates.

[0070] Example C. Confirmation of pain-reducing effect by formalin test In vivo studies were conducted on peptides of sequence numbers 11 and 12.

[0071] 1. Examination System Information

[0072] [Table 9]

[0073] 2. Preparation and administration of the control substance a. Preparation of negative control: Finished saline solution (0.9% NaCl) was used. b. Method of administration of the negative control substance: The negative control substance was administered intraperitoneally. c. Number of doses of the negative control substance: It was administered as a single dose, similar to the test substance. d. Preparation of positive control substance: Pregabalin was uniformly dissolved in saline solution at a concentration of 3 mg / mL and used. e. Method of administration of the positive control substance: The positive control substance was administered intraperitoneally. f. Number of doses of the positive control substance: It was administered as a single dose, similar to the test substance.

[0074] 3. Preparation and administration of the test substance a. Preparation of test substances: The peptide of SEQ ID NO: 11 was uniformly dissolved in saline solution at a concentration of 0.8 mg / mL, and the peptide of SEQ ID NO: 12 was prepared in saline solution at a concentration of 0.4 mg / mL. b. Setting of the test substance dosage: The dosage was set considering that the typical intraperitoneal dose for mice is 200 μL. c. Route of administration of the test substance: Both general systemic administration and intraperitoneal administration, which facilitates drug absorption, were performed. d. Method of administering the test substance: The test substance was administered intraperitoneally using a 1 mL syringe. e. Number of doses of the test substance: A single dose was administered.

[0075] 4. Composition of the test group The composition of the test group is as follows:

[0076] [Table 10]

[0077] 5. Test Method a. Administer the saline solution, pregabalin, peptide of sequence number 11, and peptide of sequence number 12 corresponding to the administered substance for each group approximately one hour before formalin administration. b. One hour after administration of the test substance to each group, 20 μL of 2% formalin will be subcutaneously injected into the sole of the right foot. c. After administering 2% formalin to each individual, measure the time it takes for pain behavioral responses (licking, biting, etc.) to appear at intervals of 0-5 minutes and 20-40 minutes. d. Grooming behavior, such as grooming hair in areas other than the formalin-administered site, is not counted. e. Statistics were analyzed using the T-test and expressed as Mean±SEM (*=p<0.01, **=p<0.01).

[0078] 6. Test Results In animal experiments, injecting formalin subcutaneously induces sustained excitation in olfactory nerve cells of the spinal cord, resulting in hypersensitive behavioral responses to external stimuli. This phenomenon is called central facilitation, and the resulting symptoms are called hyperalgesia.

[0079] The formalin test is a method for evaluating the degree of pain in rats by observing their behavior after subcutaneous injection of formalin into the top of their hind paw. The degree of pain is evaluated by the number and duration of flinching reactions.

[0080] The pain response typically exhibits an abnormal pattern, with a temporary, severe pain reaction appearing for several minutes immediately after formalin subcutaneous injection, gradually decreasing, and then resuming with a persistent, strong pain reaction after 10 minutes. The first phase response is attributed to the direct activation of thin nociceptive afferent nerve fibers (A and C fibers) distributed in the injured tissue, while the second phase response is an inflammatory response, resulting in pain caused by peripheral and central sensitization resulting from repeated stimulation of C fibers due to inflammation.

[0081] The experimental results showed that the groups administered 8 mg / kg of peptide SEQ ID NO: 11 and 4 mg / kg of peptide SEQ ID NO: 12 showed a statistically significant reduction in second-phase pain behavioral responses compared to the saline administration group. (See Figure 4: Mean ± SEM, *=p<0.01, **=p<0.01).

[0082] The results above show that administering the peptides of SEQ ID NO: 11 or SEQ ID NO: 12 suppresses pain caused by peripheral and central sensitization, confirming that this has a similar effect to pregabalin, which is widely used as a chronic pain treatment agent.

[0083] Example D. Confirmation of pain-reducing effect by capsaicin test In vivo studies were conducted on peptides with sequence numbers 5, 6, 9, and 11.

[0084] 1. Examination System Information

[0085] [Table 11]

[0086] 2. Preparation and administration of the control substance a. Preparation of negative control: Finished saline solution (0.9% NaCl) was used. b. Method of administration of the negative control substance: The negative control substance was administered intraperitoneally. c. Number of doses of the negative control substance: It was administered as a single dose, similar to the test substance. d. Preparation of positive control substance: BCTC was uniformly dissolved in saline solution at a concentration of 3 mg / mL and used. e. Method of administration of the positive control substance: The positive control substance was administered intraperitoneally. f. Number of doses of the positive control substance: It was administered as a single dose, similar to the test substance.

[0087] 3. Preparation and administration of the test substance a. Preparation of test substances: Peptides of SEQ ID NOs. 5, 6, 9, and 11 were prepared in saline solution at a concentration of 0.05 mg / mL. b. Setting of the test substance dosage: The dosage was set considering that the typical intraperitoneal dose for mice is 200 μL. c. Route of administration of the test substance: It was administered via the common systemic method and intraperitoneal administration, which facilitates drug absorption. d. Method of administering the test substance: The test substance was administered intraperitoneally using a 1 mL syringe. e. Number of doses of the test substance: A single dose was administered.

[0088] 4. Composition of the test group The composition of the test group is as follows:

[0089] [Table 12]

[0090] 5. Test Method a. Administer the saline solution, BCTC, and peptides corresponding to the administered substance for each group approximately one hour before administering capsaicin. b. One hour after administration of the test substance to each group, 25 μL of 0.01% capsaicin will be subcutaneously injected into the sole of the right foot. c. Measure the time it takes for pain behavioral responses (licking, biting, etc.) to appear in each individual between 0 and 5 minutes after administration of 2% formalin. d. Grooming behavior, such as grooming hair in areas other than the site of capsaicin administration, will not be counted. e. Statistics were analyzed using the T-test and expressed as Mean±SEM (*=p<0.05, **=p<0.01, **=p<0.001).

[0091] 6. Test Results Injecting large amounts of capsaicin into experimental animals immediately after birth results in the loss of pain-sensing nerves that express TRPV1 channels, leading to permanent desensitization to painful stimuli. Therefore, capsaicin administration has long been a useful method for studying the function of pain-sensing nerves.

[0092] Previous studies have confirmed the pain-suppressing effect via the formalin test. To confirm whether this pain-suppressing phenomenon is inhibited via TRPV1, we investigated the pattern of the pain response after subcutaneous injection of capsaicin, one of the TRPV1 active components.

[0093] Experimental results showed that peptides SEQ ID NOs. 5, 6, and 11 statistically significantly reduced pain behavioral responses (see Figure 5: Mean ± SEM, *=p<0.01, **=p<0.01).

[0094] Based on the above results, it was determined that administering peptides SEQ ID NOs. 5, 6, 9, and 11 suppresses pain caused by peripheral and central sensitization, thus confirming that capsaicin-induced pain suppression is related to TRPV1.

[0095] Example: Confirmation of pain reduction effect in the E.CCI pain model 1. Examination System Information

[0096] [Table 13]

[0097] 2. Preparation and administration of the control substance a. Preparation of negative control: Finished saline solution (0.9% NaCl) was used. b. Method of administration of the negative control substance: The negative control substance was administered intraperitoneally. c. Number of doses of the negative control substance: It was administered as a single dose, similar to the test substance. d. Preparation of positive control substance: Finished morphine (1 mg / mL) was used. e. Method of administration of the positive control substance: The positive control substance was administered intraperitoneally. f. Number of doses of the positive control substance: It was administered as a single dose, similar to the test substance.

[0098] 3. Preparation and administration of the test substance a. Preparation of test substances The peptide of sequence number 11 (2 mg) was uniformly dissolved in saline solution (20 mL), and the solution was dispensed into 2 mL aliquots and stored. (Final concentration: 0.1 mg / mL) The peptide of sequence number 12 (2 mg) was uniformly dissolved in saline solution (20 mL), and the solution was dispensed into 2 mL aliquots and stored. (Final concentration: 0.1 mg / mL) b. Setting the dosage of the test substance. This was determined considering that the typical intraperitoneal dose for mice is 200 μL. c. Route of administration of the test substance: It was administered intraperitoneally, which is a common route for systemic administration and facilitates drug absorption. d. Method of administering the test substance: The test substance was administered intraperitoneally using a 1 mL syringe. e. Number of doses of the test substance: A single dose was administered.

[0099] 4. Composition of the test group The test group was structured as follows:

[0100] [Table 14]

[0101] 5. Test Method a. Test animals were adapted to the measurement equipment chamber for at least one hour three times according to the allodynia response behavior experiment. After adaptation to the equipment was complete, the baseline pain response value was measured using the Von Frey test. b. Modeling of CCI-Pain The test animals, whose basic pain response values ​​were measured, were anesthetized, and the hair around the right sciatic nerve was removed to expose the skin. After disinfection, the skin and muscle layer were incised to locate the sciatic nerve. The sciatic nerve was lightly tied 2-3 times with a silk suture. The incision site was sutured again and disinfected. c. Confirmation of pain induction On the 3rd and 7th days post-surgery, the Von Frey test was performed according to the allodynia response behavior experiment to check whether pain was induced. d. Confirmation of drug administration and analgesic efficacy After confirming that pain had been induced, the test drug for each group was administered intraperitoneally. Two hours after administration of the test drug, the Von Frey test was performed according to the allodynia response behavior experiment, and the pain response was measured. The drug was administered as a single dose once daily. Allodynia was measured before and after drug administration to confirm changes in pain caused by the drug. e. Statistical analysis processing The statistics were analyzed using One-Way ANOVA and expressed as Mean±SEM (*=p<0.05, **=p<0.01, ***=p<0.001).

[0102] 6. Test Results Currently, the number of patients with chronic pain is gradually increasing due to various causes, and non-steroidal anti-inflammatory drugs (NSAIDs), GABA-like drugs, and narcotic analgesics are used as treatments for chronic pain. In particular, narcotic analgesics such as morphine are used for extreme pain, including chronic pain and cancer pain.

[0103] This experiment aimed to determine whether administering the peptide of SEQ ID NO: 11 or SEQ ID NO: 12 would show a pain-relieving effect compared to the narcotic analgesic morphine.

[0104] The experimental results showed that the groups administered with either the peptide of SEQ ID NO: 11 or the peptide of SEQ ID NO: 12 exhibited a pain-reducing effect, with an increase in the pain behavioral response threshold after administration compared to before administration, demonstrating statistical significance (see Figure 6: Mean ± SEM, *=p<0.05, **=p<0.01, ***=p<0.001).

[0105] The experimental results showed that when the peptide according to the present invention is administered, it exhibits a similar effect at a lower concentration compared to morphine, a narcotic analgesic. This means that the peptide according to the present invention can be usefully used as a pain-relieving medicine instead of narcotic analgesics.

[0106] Example F. Confirmation of pain reduction effect in the SNL pain model 1. Examination System Information

[0107] [Table 15]

[0108] 2. Preparation and administration of the control substance a. Preparation of negative control: Finished saline solution (0.9% NaCl) was used. b. Method of administration of the negative control substance: The negative control substance was administered intraperitoneally. c. Number of doses of the negative control substance: It was administered as a single dose, similar to the test substance. d. Preparation of positive control substance: Finished morphine (1 mg / mL) was used. e. Method of administration of the positive control substance: The positive control substance was administered intraperitoneally. f. Number of doses of the positive control substance: It was administered as a single dose, similar to the test substance.

[0109] 3. Preparation and administration of the test substance a. Preparation of test substances The peptide of sequence number 11 (2 mg) was uniformly dissolved in saline solution (10 mL) and used. (Final concentration: 0.2 mg / mL) The peptide of sequence number 12 (2 mg) was uniformly dissolved in saline solution (10 mL) and used. (Final concentration: 0.2 mg / mL) b. Setting the dosage of the test substance. This was determined considering that the typical intraperitoneal dose for rats is 2 mL. c. Route of administration of the test substance: Both general systemic administration and intraperitoneal administration, which facilitates drug absorption, were performed. d. Method of administering the test substance: The test substance was administered intraperitoneally using a 3 mL syringe. e. Number of doses of the test substance: A single dose was administered.

[0110] 4. Composition of the test group The test group was structured as follows:

[0111] [Table 16]

[0112] 5. Test Method a. Test animals were adapted to the measurement equipment chamber for at least one hour three times according to the allodynia response behavior experiment. After adaptation to the equipment was complete, the baseline pain response value was measured using the Von Frey test. b. SNL pain modeling The test animals, whose basic pain response values ​​were measured, were anesthetized, and the hair on the left spinal nerve area of ​​the test animals was removed to expose the skin. After disinfection, the skin and muscle layer were incised to locate the left fifth lumbar spinal nerve. The left fifth lumbar spinal nerve was lightly tied once with a silk suture. The incision site was sutured again and disinfected. c. Confirmation of pain induction On days 1, 7, and 14 post-surgery, the Von Frey test was performed according to the allodynia response behavior experiment to check for pain induction. d. Confirmation of drug administration and analgesic efficacy After confirming that pain had been induced, the test drug for each group was administered intraperitoneally. The Von Frey test was performed at 1, 3, and 7 hours after administration of the test drug, following the allodynia response behavior experiment, and the pain response was measured. The drug was administered as a single dose once daily. Allodynia responses were measured before and after drug administration to confirm changes in pain caused by the drug. e. Statistical analysis processing Statistical analysis was performed using the T-test, and the results were expressed as Mean±SEM (*=p<0.05, ***=p<0.001).

[0113] 6. Test Results Chronic pain is known to be very difficult to treat because it is triggered by direct nerve damage or inflammation, and progresses through the simultaneous and complex action of multiple mechanisms. Among chronic pains, neuropathic pain is a representative type of chronic pain syndrome that can arise from various causes such as ischemic injury due to nerve trauma or compression, toxic metabolites, and inflammation, and is accompanied by symptoms such as spontaneous pain, hyperalgesia, and allodynia. Among the neuropathic pain models, the spinal nerve ligation model reliably induces persistent mechanical allodynia and is used in many drug administration experiments because it induces mechanical allodynia more readily than other nerve injury models.

[0114] This experiment aimed to determine whether administering the peptide of SEQ ID NO: 11 or SEQ ID NO: 12 would have a pain-reducing effect on allodynia in an SNL pain animal model, compared to the narcotic analgesic morphine.

[0115] The experimental results showed that the groups administered with either the peptide of SEQ ID NO: 11 or the peptide of SEQ ID NO: 12 exhibited a pain-reducing effect, with an increase in the pain behavioral response threshold after administration compared to before administration, demonstrating statistical significance (see Figure 7: Mean ± SEM, *=p<0.05, ***=p<0.001).

[0116] The results above show that administering the peptide of SEQ ID NO: 11 or SEQ ID NO: 12 suppresses allodynia that appears in the spinal nerve ligation model. This means that the peptides of the present invention can be usefully used as pain-relieving pharmaceuticals instead of narcotic analgesics, which are commonly used as treatments for neuropathic pain.

[0117] Example G. Confirmation of pain reduction effect in the CIPN pain model. 1. Examination System Information

[0118] [Table 17]

[0119] 2. Preparation and administration of the control substance a. Preparation of negative control: Finished saline solution (0.9% NaCl) was used. b. Method of administration of the negative control substance: The negative control substance was administered intraperitoneally. c. Number of doses of the negative control substance: It was administered as a single dose, similar to the test substance. d. Preparation of positive control substance: Pregabalin was uniformly dissolved in saline at a concentration of 30 mg / 10 mL and used. e. Method of administration of the positive control substance: The positive control substance was administered intraperitoneally. f. Number of doses of the positive control substance: It was administered as a single dose, similar to the test substance.

[0120] 3. Preparation and administration of the test substance a. Preparation of test substances The peptide of sequence number 11 (4 mg) was uniformly dissolved in saline solution (10 mL) and used. (Final concentration: 0.4 mg / mL) The peptide of sequence number 12 (2 mg) was uniformly dissolved in saline solution (10 mL) and used. (Final concentration: 0.2 mg / mL) b. Setting the dosage of the test substance. This was determined considering that the typical intraperitoneal dose for rats is 2 mL. c. Route of administration of the test substance: Both general systemic administration and intraperitoneal administration, which facilitates drug absorption, were performed. d. Method of administering the test substance: The test substance was administered intraperitoneally using a 3 mL syringe. e. Number of doses of the test substance: A single dose was administered.

[0121] 4. Composition of the test group The test group was structured as follows:

[0122] [Table 18]

[0123] 5. Test Method a. Test animals were adapted to the measurement equipment chamber for at least one hour three times according to the allodynia response behavior experiment. After adaptation to the equipment was complete, the baseline pain response value was measured using the Von Frey test. b. Modeling of anticancer drug-induced neuropathic pain (CIPN pain) Paclitaxel was dissolved at a rate of 2 mg / 10 ml in a solution of Cremophor EL and absolute ethanol in a 1:1 ratio, and then administered intraperitoneally in 2 ml doses. Paclitaxel was administered once a day for 4 days at 24-hour intervals to induce chemotherapy-induced pain. c. Confirmation of pain induction On days 1, 7, and 14 post-surgery, the Von Frey test was performed according to the allodynia response behavior experiment to check for pain induction. d. Confirmation of drug administration and analgesic efficacy After confirming that pain had been induced, the test drug for each group was administered intraperitoneally. The Von Frey experiment was performed at 1, 3, and 7 hours after administration of the test drug, following the allodynia response behavior experiment, and the pain response was measured. The drug was administered as a single dose once daily. Allodynia responses were measured before and after drug administration to confirm changes in pain caused by the drug. e. Statistical analysis processing Statistical analysis was performed using the T-test, and the results were expressed as Mean±SEM (*=p<0.05, ***=p<0.001).

[0124] 6. Test Results As average life expectancy increases, so does the incidence of cancer, and as cancer treatments advance, the number of cancer survivors is increasing. In this context, improving the quality of life for cancer patients is a very important issue. Chemotherapy-induced peripheral neuropathy (CIPN) is distinct from cancer pain caused by cancer directly invading nerve tissue and compressing nerves or by bone metastases, and is observed in approximately 40% of patients receiving combination chemotherapy.

[0125] This experiment aimed to determine whether administering the peptide of SEQ ID NO: 11 or SEQ ID NO: 12 would have a pain-reducing effect on allodynia in an animal model of CIPN pain, compared to pregabalin.

[0126] The experimental results showed that the groups administered with either the peptide of SEQ ID NO: 11 or the peptide of SEQ ID NO: 12 exhibited a pain-reducing effect, with an increase in the pain behavior response threshold after administration compared to before administration, demonstrating statistical significance (see Figure 8: Mean ± SEM, *=p<0.05, ***=p<0.001).

[0127] The results above show that administration of the peptide of SEQ ID NO: 11 or SEQ ID NO: 12 suppresses allodynia that appears in anti-cancer neuronal models. This means that the peptides of the present invention can be usefully used as pain-relieving pharmaceuticals instead of analgesics such as gabapentin or pregabalin, which are clinically used to treat CIPN. [Industrial applicability]

[0128] This invention can be usefully used as a pain-relieving medicine. [Sequence Listing Free Text]

[0129] Sequence ID 1: MLYFNDKQQI IY Sequence ID 2:PINMLYFNDK QQIIYGKID Sequence ID 3: GINMLYFNDK QQIIYGKIN Sequence ID 4: YCSGQCYMFM QKY Sequence ID 5: YKANYCSGQC Y Sequence ID 6: SGQCYMFMQK Y Sequence ID 7: YKANYCSGQC EY Sequence ID 8: SGQCEYMFMQ KY Sequence ID 9: AGQCYMFMQK Y Sequence ID 10: SGQAYMFMQK Y Sequence ID 11: YGQCYMWMQK W Sequence ID 12: YGQCYMYMQK W Sequence ID 13: YGQCYMWMQY W Sequence ID 14: YGQCYMWMQW W Sequence ID 15: YGQCYMWMQG W Sequence ID 16: YGQCYMWMQK Y Sequence ID 17: YGQCYMWMQE W Sequence ID 18: YGMCYMWMQK W Sequence ID 19: YGRCYMWMQK W Sequence ID 20: YGQCYMWFQK W Sequence ID 21: ICYMWMQK W Sequence ID 22: YGQCYMWMQH W Sequence ID 23: YGQCYMWYQK W Sequence ID 24: YGQCYMWSQK W Sequence ID 25: YGGCYMWMQK W Sequence ID 26: YKQCYMWMQK W Sequence ID 27: YGTCYMWMQK W Sequence ID 28: YGQCYMWGQK W Sequence ID 29: YGQCYMWMQS W Sequence ID 30: YGQCYMWMQC W Sequence ID 31: YGQCYMWMQA W Sequence ID 32: YGQWYMWMQK W Sequence ID 33: YGQMYMWMQK W Sequence ID 34: YGLCYMWMQK W Sequence ID 35: YGQCYMWHQK W Sequence ID 36: YSQCYMWMQK W Sequence ID 37: YGQCYMWMQM W Sequence ID 38: YGQPYMWMQK W Sequence ID 39: YGQCYMWPQK W Sequence ID 40: YGQGYMWMQK W Sequence ID 41: YGSCYMWMQK W Sequence ID 42: YLQCYMWMQK W Sequence ID 43: YKAAYCSGQC Y Sequence ID 44: SGACYMFMQK Y Sequence ID 45: SGQCYAFMQK Y Sequence ID 46: SGQCYMFMQA Y Sequence ID 47: SGQCYMWMQK Y Sequence ID 48: SGQCYMWMQK W Sequence ID 49: SGQCYMYMQK Y Sequence ID 50: NLGLDCDEHS SESRCCRYPL TVDFEAFGWD WIIAPKRYKA NYCSGQCEYM FMQKYPHTHL VQQANPRGSA GPCCTPTKMS PINMLYFNDK QQIIYGKIPG MVVDRCGCS Sequence ID 51:LYFNDKQQII Y Sequence ID 52: NMLYFNDKQQ IIYGK Sequence ID 53:PINMLYFNDK QQIIYGKIP

Claims

1. A peptide containing one of the sequences from SEQ ID NOs: 2-4, SEQ ID NO: 6, and SEQ ID NOs: 9-49.

2. A pharmaceutical composition for the treatment of TRPV1-mediated diseases, comprising the peptide described in claim 1.

3. A pharmaceutical composition for pain treatment comprising the peptide described in claim 1.

4. A polynucleotide encoding the peptide described in claim 1.

5. The peptide according to claim 1, characterized in that the N-terminus and / or C-terminus are capped to protect them from protease enzymes.

6. The peptide according to claim 5, wherein the N-terminus is capped with lipoic acid.

7. The peptide according to claim 5, wherein the C-terminus is capped with p-nitroanilide.

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