Pharmaceutical compositions for the treatment of diseases mediated by trpv1 activity

Abstract

The present invention relates to a new therapeutic pharmaceutical composition, and more particularly, to a pharmaceutical composition for treating a disease mediated by TRPV1 activity. Since the composition for treating pain using the TRPV1 activity inhibitory effect of the present invention shows an excellent inhibitory effect on neuropathic pain caused by spinal nerve injury, the composition can be used as a new pain treatment for various pain conditions and diseases related to the TRPV1 channel such as arthritis and diabetic peripheral neuropathy. Furthermore, since the composition effectively inhibits TRPV1 activity, the composition can be used as a therapeutic drug for treating various diseases mediated by TRPV1 activity.

Description

Technical Field

[0001] This invention relates to a novel therapeutic pharmaceutical composition, and more specifically, to a pharmaceutical composition for treating TRPV1-mediated diseases and a method for treating TRPV1-mediated diseases. Background Technology

[0002] Pain is "an unpleasant sensory and emotional experience associated with actual or potential tissue damage," and generally, pain is relieved as the damaged tissue heals. However, when pain becomes chronic, it can continue to occur without any stimulation, even when the damaged area has seemingly healed completely, and the pain may be caused by harmless stimuli that would not normally cause pain.

[0003] Meanwhile, transient receptor potential vanilloid type 1 (TRPV1), a member of the transient receptor potential (TRP) family, is one of the important nociceptors in the cellular mechanisms of pain induced by noxious stimuli. Furthermore, TRPV1 is a non-selective cation channel that is activated by various stimuli such as noxious heat (42°C or higher), capsaicin, resiniferatoxin (RTX), and protons. It is a key molecule primarily expressed at the terminals of nociceptive nerve fibers and involved in the development of peripheral and central sensitization leading to chronic pain. TRPV1 is considered a major target for the treatment of chronic pain; however, pharmacological inhibition of TRPV1 can lead to unexplained severe high fever, and it is known to be difficult to develop TRPV1 inhibitors into therapeutic drugs.

[0004] GDF11 is a protein belonging to the TGF-β family, and it is known to increase its expression in young animals to promote neurogenesis and angiogenesis. While the role of TGF-β, belonging to the same family, in controlling chronic pain has been extensively studied, the role of GDF11 in alleviating chronic pain remains unclear.

[0005] Korean Patent Publication No. 10-2017-0093286 relates to compositions including GDF11 and their uses, and provides information on the fibroblast proliferation effect of human adult stem cell culture containing GDF11. However, there are no studies or publications regarding the chronic pain relief effect of GDF11. Summary of the Invention

[0006] [Technical Issues]

[0007] Because the inventors have made efforts to provide a substance that can solve the side effects of TRPV1 inhibition, such as severe high fever, while treating chronic pain through TRPV1 inhibition, the inventors completed this invention by verifying that GDF11 has the effect of relieving pain caused by various reasons.

[0008] Therefore, one object of the present invention is to provide a pharmaceutical composition for treating TRPV1-mediated diseases and pain-related diseases, including chronic pain, without side effects. However, these problems are exemplary, and the scope of the invention is not limited thereto.

[0009] [Technical Solution]

[0010] According to one aspect of the present invention, a pharmaceutical composition for treating TRPV1-mediated diseases is provided, comprising a growth differentiation factor 11 (GDF11) peptide containing the amino acid sequence of SEQ ID NO:1, a polynucleotide encoding the peptide, or an expression vector containing the polynucleotide as an active ingredient.

[0011] According to another aspect of the present invention, a method for treating TRPV1-mediated diseases is provided, comprising administering to a patient suffering from TRPV1-mediated diseases a composition comprising a growth differentiation factor 11 (GDF11) peptide containing the amino acid sequence of SEQ ID NO:1, a polynucleotide encoding the peptide, or an expression vector comprising the polynucleotide.

[0012] Terminology definition:

[0013] As used in this article, the term “growth differentiation factor 11 (GDF11),” also known as bone morphogenetic protein 11 (BMP11), refers to the protein expressed by the GDF11 gene located on human chromosome 12.

[0014] As used in this article, the term "transient receptor potential channel vanillin subfamily member 1 (TRPV1)" belongs to a large family of TRP channels composed of non-voltage-gated cation channels, including thermal, visual, gustatory, olfactory, and tactile regions. TRPV1 triggers nociceptive signals through synergistic activation of interacting heat, protons, and endogenous substances.

[0015] As used in this article, the term "vector" refers to a tool used for gene delivery. It can be viewed as a concept corresponding to drug delivery vehicles. Therefore, vectors in a broad sense not only include DNA vectors composed of the same nucleic acid molecules as the transgene to be introduced, similar to general plasmid vectors, but also nanoparticles made of silica or gold, liposomes, extracellular vesicles and phospholipid membrane structures such as exosomes, chitosan, cationic polymers such as polyethyleneimine and polylysine, and reagents for DNA transfection such as calcium phosphate. Detailed Implementation

[0016] According to one aspect of the present invention, a pharmaceutical composition for treating TRPV1-mediated diseases is provided, comprising: a growth differentiation factor 11 (GDF11) peptide containing the amino acid sequence of SEQ ID NO:1, a polynucleotide encoding the peptide, or an expression vector containing the polynucleotide as an active ingredient.

[0017] In this pharmaceutical composition, the expression vector can be a viral vector or a non-viral vector, and the viral vector can be an adeno-associated virus (AAV) vector, adenovirus vector, alphavirus vector, herpes simplex virus vector, vaccinia virus vector, Sendai virus vector, flavivirus vector, rhabdovirus vector, retroviral vector, or lentiviral vector. Furthermore, the serotype of the adeno-associated virus (AAV) vector can be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, or AAV16.

[0018] In this pharmaceutical composition, the non-viral vector may be a DNA vector, nanoparticles, cationic polymers, exosomes, extracellular vesicles, or liposomes, while the DNA vector may be a plasmid vector, a granular vector, a phage vector, or an artificial human chromosome.

[0019] In this pharmaceutical composition, the TRPV1-mediated diseases can be selected from the group consisting of pain, hypertension, stroke, myocardial ischemia, urinary incontinence, bladder hypersensitivity, irritable bowel syndrome, urgency, gastroduodenal ulcer, gastroesophageal reflux disease (GERD), Crohn's disease, hemorrhoids, asthma, chronic obstructive pulmonary disease, pruritus, psoriasis, hearing loss, tinnitus, cough, hirsutism, and alopecia.

[0020] In this pharmaceutical composition, the pain can be nociceptive pain, psychogenic pain, inflammatory pain, or pathological pain, and the pathological pain can be neuropathic pain, cancer pain, chemotherapy-induced pain, postoperative pain, trigeminal neuralgia, idiopathic pain, diabetic neuropathy, or migraine.

[0021] According to another aspect of the invention, a method for relieving or treating pain is provided, comprising administering the pharmaceutical composition.

[0022] The pain described according to the present invention includes nociceptive pain, psychogenic pain, inflammatory pain related to tissue damage and immune cell invasion, and pathological pain (dysfunctional pain such as fibromyalgia, irritable bowel syndrome, and tension headache), which are disease states caused by damage to the nervous system or its abnormal function. Furthermore, pain can include anatomically different types of back pain. Pain can include pain such as neuropathic pain, migraines, as well as neck pain, mid-back pain, lower back pain, or coccyx pain. Neuropathic pain is a chronic neurological disorder caused by damage to the nervous system due to various reasons such as trauma, inflammation, ischemic injury, and metabolites, and it may be caused by damage or disease affecting the somatosensory system. Generally, it is a non-malignant chronic pain caused by abnormalities in the nerves, spinal cord, and brain, and it is estimated that more than 1% of the population suffers from this type of pain.

[0023] Neuropathic pain can be associated with atypical sensations called hypoesthesia, atypical pain where pain is felt even with harmless stimuli that do not cause pain, and hyperalgesia where pain is felt more intensely and for longer duration in response to harmful stimuli such as fever. Furthermore, neuropathic pain can be persistent and / or intermittent (epileptic seizures). The latter is likened to an electric shock. Common characteristics include burning, cold, tingling, numbness, and itching. Depending on whether the peripheral or central nervous system is affected, it can be classified as peripheral neuropathic pain or central neuropathic pain. In contrast, nociceptive pain typically manifests as pain. Additionally, migraine is a chronic disorder associated with many symptoms of the autonomic nervous system and causes headaches ranging from normal to severe. The exact mechanisms of these migraines have not yet been determined. The basic theory involves increased excitability of the cerebral cortex and abnormal regulation of pain neurons in the trigeminal nucleus of the brainstem. For example, pain can be selected from one or more groups including neuropathic pain, cancer pain, postoperative pain, trigeminal neuralgia, idiopathic pain, diabetic neuropathy, migraine, etc.

[0024] In the pharmaceutical composition of the present invention, the effective amount of the compound can vary depending on the type of affected area, application site, number of treatments, treatment duration, dosage form, patient condition, type of adjuvant, etc. There are no particular limitations on the amount used, but it can range from 0.01 μg / kg / day to 10 mg / kg / day. The daily dose can be administered once daily, divided into 2-3 doses at appropriate intervals, or administered intermittently at several-day intervals.

[0025] In the pharmaceutical compositions of the present invention, based on the total weight of the composition, an amount of 0.01 wt% to 100 wt% of the compound may be included. The pharmaceutical compositions of the present invention may also include suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. Furthermore, solid or liquid additives for formulation may be used in the preparation of the pharmaceutical composition. The additives for formulation may be organic or inorganic. Examples of excipients include lactose, sucrose, white sugar, glucose, corn starch, starch, talc, sorbitol, crystalline cellulose, dextrin, kaolin, calcium carbonate, silica, etc. Examples of binders include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth gum, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, dextrin, pectin, etc. Examples of lubricants include magnesium stearate, talc, polyethylene glycol, silica, hydrogenated vegetable oil, etc. Any coloring agents permitted to be added to conventional pharmaceuticals may be used. Depending on other needs, these tablets and granules may be suitably coated with sugar coating and gelatin coating. In addition, preservatives, antioxidants, etc. can be added as needed.

[0026] The pharmaceutical compositions of the present invention can be prepared in any dosage form conventionally prepared in this field, and there are no particular limitations on the form of the formulation.

[0027] The pharmaceutical composition of the present invention can be administered orally or enterally, and preferably enterally, it can be administered via intravenous injection, subcutaneous injection, intraventricular injection, intracerebrospinal fluid injection, intrathecal injection, transforaminal injection, intramuscular injection, intraperitoneal injection, etc.

[0028] This invention provides a method for relieving and / or treating pain, comprising administering a therapeutically effective amount of a pharmaceutical composition to a patient requiring pain relief and / or treatment. The method may further include a step of identifying the patient as requiring pain relief and / or treatment prior to the administration step. The term "therapeutically effective amount" may depend on the amount of an activity gradient capable of achieving the desired effect, pain relief, and / or therapeutic effect.

[0029] The pharmaceutical compositions of the present invention are suitable for the prevention and treatment of diseases associated with TRPV1 activity, including pain such as acute pain, chronic pain, neuropathic pain, postoperative pain, rheumatoid arthritis pain, arthritis pain, posterior herpes neuralgia, neuralgia, headache, toothache, pelvic pain, migraine, bone cancer pain, chemotherapy-induced pain, breast pain, and visceral pain; neurological diseases such as neuropathy, HIV-related neuropathy, nerve damage, neurodegeneration, and stroke; diabetic peripheral neuropathy; constipation; irritable bowel syndrome; inflammatory bowel disease; and gastrointestinal diseases such as gastroesophageal reflux. GERD, peptic ulcers, and Crohn's disease; respiratory disorders such as asthma, chronic obstructive pulmonary disease, and cough; urinary incontinence; bladder hypersensitivity; neurogenic / allergic / inflammatory skin diseases such as psoriasis, pruritus, pruritus, and dermatitis; hyperacusis; hearing loss; tinnitus; vestibular hypersensitivity; heart diseases such as myocardial ischemia; hemorrhagic shock; hair growth-related disorders such as hirsutism, effluvium, and alopecia; rhinitis; pancreatitis; cystitis; vulvar pain; mental disorders such as anxiety or fear; obesity; type 1 and type 2 diabetes, but not limited to these.

[0030] The GDF11 of this invention is a protein encoded by the growth differentiation factor gene 12. GDF11 acts as a cytokine and has the same molecular structure in humans and mice. This bone morphogenetic proteome is characterized by multibasic protein hydrolysis sites, which are cleaved to produce a protein containing seven conserved cysteine ​​residues. Systemic GDF11 treatment improves vascular structure in the hippocampus and cortex of aged mice, thereby improving neurogenesis. Systemic GDF11 supplementation also improves β-cell survival and morphology in both non-genetic and genetic type 2 diabetic mouse models and improves glucose metabolism.

[0031] GDF11 induces phenotypes such as calorie restriction without affecting appetite or blood GDF15 levels. It restores the insulin / IGF-1 signaling pathway, acts directly on adipocytes to stimulate adiponectin secretion from white adipose tissue, and restores neurogenesis in the brains of older adults. Furthermore, it is a regulator of skin biology, playing a crucial role in the production of procollagen I and hyaluronic acid, activating the Smad2 / 3 phosphorylation pathway in skin endothelial cells, and improving the skin's vascular system. Increased GDF11 levels in aged mice also improve muscle structure and function, and increase strength and endurance.

[0032] Furthermore, GDF11 has been found to reduce oxidative stress, decrease levels of AGEs, protein oxidation, and lipid peroxidation, slow the accumulation of age-related histological markers, and significantly inhibit the decline in CAT, GPX, and SOD activities. Members of the GDF11 family are regulators of cell growth and differentiation in embryonic and adult tissues, and studies in mice and African clawed frogs (Xenopus) have shown that this protein is involved in mesodermal formation and neurogenesis during embryonic development.

[0033] Human GDF11 represented by SEQ ID NO:2 and GDF11 derived from other mammals having at least 96% or more homology with it may also be used. GDF11 from other mammals may be mice (Musmusculus, SEQ ID NO:3), chimpanzees (Pan troglodytes, SEQ ID NO:4), gorillas (Gorilla beringeigraueri, SEQ ID NO:5), canaries-proboscis monkeys (Rhinopithecus roxellana, SEQ ID NO:6), northern white-cheeked gibbons (Nomascus leucogenys, SEQ ID NO:7), Bengal monkeys (Macaca mulatta, SEQ ID NO:8), bonobos (Pan paniscus, SEQ ID NO:9), or pigs (Sus scrofa domesticus, SEQ ID NO:10).

[0034] The human-derived GDF11 protein is a protein with very high homology to GDF11 proteins derived from other primates, and can utilize all or part of the full-length peptide or mature peptide derived from other animals. Specifically, the mature peptide corresponds to amino acid positions 299-407 (SEQ ID NO:1), and amino acid positions 1-298 are cleaved portions of the signal peptide and precursor protein. Because the mature peptide has almost 100% homology with other mammals, genes or proteins from other animals can therefore be used. Furthermore, the polynucleotide encoding the mature peptide can be represented by SEQ ID NO:11.

[0035] This invention can be implemented through gene therapy using genes instead of proteins or peptides. For such gene therapy, recombinant vectors are used to facilitate gene amplification and manipulation, in which a gene construct encoding a polynucleotide of GDF11, operatively linked to a regulatory sequence, is inserted into various expression vectors. These expression vectors are classified as viral vectors and non-viral vectors. For viral vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, alphavirus vectors, herpes simplex virus vectors, vaccinia virus vectors, Sendai virus vectors, flavivirus vectors, rhabdovirus vectors, retrovirus vectors, lentivirus vectors, etc., have been used. Viral vectors used for gene therapy are described in detail in the literature. The aforementioned literature is incorporated herein by reference. However, in the case of retroviruses or lentiviruses in these viral vectors, the transgene is inserted into the host's genome, but because it is a random insertion, there are concerns about unintended side effects such as cancer. Therefore, adeno-associated viruses that insert only into specific locations in the host cell genome have attracted attention.

[0036] Adeno-associated virus (AAV) is a single-stranded DNA virus and an helper-dependent human parvovirus. Its genome is approximately 4.7 kbp in size, with the N-terminus encoding the `rep` gene, which is involved in viral replication and gene expression, and the C-terminus encoding the `cap` gene, which encodes the viral capsid protein. Furthermore, it consists of inverted terminal repeats (ITRs) with approximately 145 bases inserted at both ends. Four proteins, classified by molecular weight as `rep78`, `rep68`, `rep52`, and `rep40`, are translated from the `rep` region and play important roles in AAV DNA replication. Three proteins, VP1, VP2, and VP3, are translated from the `cap` region and are structural proteins required for AAV viral assembly.

[0037] Based on various viral infection methods known in the art, the AAV vector of the present invention can be used to transfer exogenous gene sequences into cells, and the method is not particularly limited.

[0038] Meanwhile, according to exemplary embodiments of the present invention, the expression vector for gene therapy can be inserted into a DNA vector, such as a non-viral expression vector, which includes a gene construct in which a polynucleotide encoding GDF11 is operatively linked to a regulatory sequence, particularly a plasmid vector, etc.

[0039] As used in this article, the term “operably ligated” refers to the ligation of a target nucleic acid sequence to a regulatory sequence in a manner that allows for expression in a living organism into which the transgene is to be introduced.

[0040] The term "regulatory sequence" refers to a sequence that includes promoters, enhancers, and other regulatory elements (e.g., polyadenylation signals). This regulatory sequence includes indicators that the target nucleic acid can always be expressed in many host cells, indicators that the target nucleic acid can only be expressed in cells of a specific tissue (e.g., tissue-specific regulatory sequences), and indicators that the expression is induced by a specific signal (e.g., inducible regulatory sequences). Those skilled in the art will understand that the design of expression vectors used in gene therapy can vary depending on a variety of factors, such as the choice of host to which the transgene is to be introduced, the desired protein expression level, etc. The expression vector of the present invention can be introduced into a host to express the GDF11 protein. Therefore, the expression vector according to an exemplary embodiment of the present invention must have eukaryotic regulatory sequences capable of expressing a foreign gene in eukaryotic cells, particularly mammals. Regulatory sequences that enable the expression of a foreign gene in such eukaryotic cells are well known to those skilled in the art. As mentioned above, they typically include regulatory sequences responsible for transcription initiation and optionally include poly-A signals responsible for terminating and stabilizing transcript transcription. In addition to transcription regulators, additional regulatory sequences may include translation enhancers and / or naturally occurring or heterologous promoter regions. For example, regulatory sequences that allow expression in mammalian cells include the CMV-HSV thymidine kinase promoter, SV40, the RSV promoter (Rous sarcoma virus), the human kidney element 1α-promoter, the glucocorticoid-inducible MMTV-promoter (Moloni mouse tumor virus), metallothionein-inducible or tetracycline-inducible promoters, or amplifiers such as CMV amplifiers or SV40 amplifiers. For expression in neurons, neurofilament promoters, PGDF-promoters, NSE-promoters, PrP-promoters, or thy-1 promoters are considered suitable. Such promoters are known in the art and described in the literature. The regulatory sequence may include a transcription termination signal, such as an SV40-poly-A site or a TK-poly-A site downstream of a polynucleotide according to an exemplary embodiment of the present invention. In this invention, suitable expression vectors are known in the art, and examples include the Okayama-Berg cDNA expression vector pcDV1 (Parmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pSPORT1 (GIBCO BRL), pGX-27, pX, yeast two-hybrid vectors, and, for example, pEG202, dpJG4-5, etc. In addition to the nucleic acid molecules of this invention, the vector may also include polynucleotides encoding secretion signals. These secretion signals are well known to those skilled in the art.Furthermore, depending on the expression system used, the leader sequence capable of guiding GDF11 to a cell compartment can be combined with a coding sequence of a polynucleotide according to an exemplary embodiment of the present invention, and preferably, it is a leader sequence capable of directly secreting the translated protein or its protein into the periplasm or extracellular matrix.

[0041] Furthermore, the expression vector of the present invention can be prepared, for example, by standard recombinant DNA techniques, including, for example, ligation of blunt and sticky ends, treatment with restriction enzymes to provide suitable ends, removal of phosphate groups by alkaline phosphatase treatment to prevent improper binding, enzymatic ligation by T4 DNA ligase, etc. The vector of the present invention can be prepared by recombining DNA encoding a signal peptide obtained through chemical synthesis or gene recombination technology and DNA encoding the bispecific fusion protein of the present invention into a vector containing a suitable regulatory sequence. Vectors containing regulatory sequences are commercially available or can be prepared.

[0042] The expression vector may also include a polynucleotide encoding a secretion signal sequence that induces the secretion of an intracellularly expressed recombinant protein outside the cell. This secretion signal sequence may be a tissue plasminogen activator (tPA) signal sequence, a herpes simplex virus glycoprotein Ds (HSV gDs) signal sequence, or a growth hormone signal sequence.

[0043] According to an exemplary embodiment of the present invention, the non-viral expression vector used for gene therapy can be a DNA vector capable of expressing GDF11 in host cells, and the DNA vector can be in any form, such as a plasmid vector, a granular vector, a phage vector, an artificial human chromosome, etc.

[0044] Furthermore, gene delivery methods used in gene therapy employing the aforementioned DNA expression vectors include electroporation, gene guns, ultrasonic microbubble therapy, and magnetic transfection. Transfection methods using vectors such as gold or silica nanoparticles, cationic polymers, liposomes, nanoparticles, and exosomes are also available. These gene transfer methods are well-known in the industry. Recently, a method has been developed to directly inject DNA vaccine compositions into human muscle using irreversible electroporation with a small electroporation device.

[0045] TRPV1 is one of the families of permeable, non-selective cation channels whose activity induces Ca2+. 2+It is uptaken and inhibited by certain antagonists (such as capsazepine). Furthermore, it is directly activated by capsaicin, heat, low pH, bradykinin, PGE2, ATP, etc., and this activation condition indicates that TRPV1 is a major biosensor for thermochemical stimulation and tissue damage. TRPV1 has been reported to be present in various tissues, such as brain, kidney, bronchial epithelial cells, and epidermal keratinocytes, and capsaicin stimulation increases calcium levels in the cytoplasm of keratinocytes. 2+ The concentration of TRPV1 is high, while capsaicin can inhibit this concentration. Although TRPV1 antagonists have been reported as therapeutic agents for chronic pain, the pharmacological inhibition of TRPV1 causes high fever, thus necessitating the development of analgesics without the effect of high fever. Therefore, the inventors investigated pain treatment compositions that can effectively inhibit TRPV1 and confirmed that GDF11 can be a new pain treatment antagonist. Consequently, the inventors developed a pain treatment composition using an inhibitory effect on the activity of TRPV1. The inventors used intracellular calcium (Ca) 2+ Imaging and whole-cell patch-clamp experiments revealed that recombinant GDF11 inhibited TRPV1 channel function in small primary mouse dorsal root ganglia (DRG) in a concentration-dependent manner. Furthermore, similar results were confirmed in TRPV1-transfected HEK293 cell lines, demonstrating that the GDF11 of this invention can be used as a novel pain treatment agent antagonizing TRPV1 channels in chronic pain, and that it can be used as a therapeutic agent to cure various mediator diseases associated with TRPV1 activity.

[0046] According to another aspect of the present invention, a method for treating TRPV1-mediated disease is provided, comprising administering to a patient suffering from TRPV1-mediated disease a composition comprising a growth differentiation factor 11 (GDF11) peptide including the amino acid sequence of SEQ ID NO:1 and a polynucleotide encoding the peptide or an expression vector comprising the polynucleotide.

[0047] Since the expression vector or TRPV1-mediated disease is the same concept as that used in pharmaceutical compositions, its description is replaced with the above description.

[0048] [Beneficial Effects]

[0049] Because the composition for treating pain using the inhibitory effect of TRPV1 activity of the present invention exhibits excellent effects in inhibiting neuropathic pain caused by spinal nerve injury, 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. Furthermore, because it effectively inhibits TRPV1 activity, it can be used as a therapeutic agent for various TRPV1-mediated diseases. Of course, the scope of the invention is not limited by these effects. Attached Figure Description

[0050] Figure 1 This is a schematic diagram illustrating a method for evaluating the inhibitory effect of GDF11 treatment according to the present invention on (a) thermal hyperalgesia and (b) the inhibitory effect on TRPV1 ion channel activity by preparing a mouse model of chronic pain.

[0051] Figure 2a This is a graph showing the delay time of avoidance response induced by radiative heat stimulation in a mouse model of spinal nerve transection in which GDF11 of the present invention was injected.

[0052] Figure 2b This is a graph showing the delay time of avoidance response caused by radiative heat stimulation in a mouse model of chemotherapy-induced peripheral neuropathy, based on the analysis of GDF11 (0.1 mg / kg) of the present invention injected into the mouse model.

[0053] Figure 2c This is a graph showing the delay time of avoidance response caused by radiative heat stimulation in a mouse model of chemotherapy-induced peripheral neuropathy, based on the analysis of GDF11 (0.2 mg / kg) of the present invention injected into the mouse model.

[0054] Figure 3a This is a graph showing the calcium inflow curve in mouse sensory neurons treated with the GDF11 and capsaicin of this invention.

[0055] Figure 3b This is a graph comparing the calcium influx concentration in mouse sensory neurons treated with the GDF11 and capsaicin of this invention to the control group.

[0056] Figure 4a This is a graph showing the calcium influx curves of human embryonic kidney 293 (HEK293) cells transformed with the pcDNA-TRPV1 vector and treated with the GDF11 and capsaicin of the present invention.

[0057] Figure 4b This is a graph analyzing the ratio of calcium influx concentration in human embryonic kidney 293 (HEK293) cells transformed with the pcDNA-TRPV1 vector to that of the control group after treatment with GDF11 and capsaicin of the present invention.

[0058] Figure 5a It is a graph showing the inward current curves in mouse sensory neurons treated with the GDF11 and capsaicin of the present invention.

[0059] Figure 5b It is a graph analyzing the ratio of the magnitude of the inward current (by a concentration meter) in mouse sensory neurons treated with the GDF11 and capsaicin of the present invention to the control group.

[0060] Figure 6a This is a graph showing the inward current curve of TRPV1 treated with GDF11 and capsaicin of the present invention in HEK293 cells transformed with the pcDNA-TRPV1 vector.

[0061] Figure 6b This is a graph showing the ratio of the magnitude of the inward current (measured by a concentration meter) in HEK293 cells transformed with the pcDNA-TRPV1 vector and treated with the GDF11 and capsaicin of this invention to the control group.

[0062] Figure 7 The study showed changes in body temperature in a mouse model of chemotherapy-induced peripheral neuropathy after administration of the GDF11 of the present invention. Even when GDF11 was administered, it was confirmed that there were no sudden changes in the mice's body temperature.

[0063] Figure 8 The adeno-associated virus vector AAV-GDF11 of the present invention, which expresses GDF11, is shown to stably express GDF11 in mouse sensory neurons (L4 DRG or L5 DRG).

[0064] Figure 9 This is a graph showing the delay time of avoidance response induced by radiative heat stimulation when the adeno-associated virus vector AAV-GDF11 expressing GDF11 of the present invention was administered to a mouse model of neuropathic pain.

[0065] [Optimal Mode of the Invention]

[0066] General methods

[0067] Preparation of a mouse model of spinal cord transection for neurogenic pain

[0068] The inventors have developed an animal model of spinal nerve transection (SNT). Specifically, 6-week-old male C57BL / 6 mice were acclimatized for one week in a laboratory setting. Then, their right hind paws were subjected to radiant heat stimulation using an infrared thermal stimulation device, and the time until an avoidance response was measured to establish a reference value. Subsequently, experimental groups (n = 5-6 mice / group) were randomly separated, and the spinal nerve transection surgery was performed. First, sodium pentobarbital (60 mg / kg) was injected intraperitoneally. Under anesthesia, the skin of the mice was incised to sever the L5 spinal nerve, and the incised muscle and skin were sutured with surgical sutures.

[0069] Construction of animal models of peripheral neuropathy

[0070] The inventors have prepared an animal model of chemotherapy-induced peripheral neuropathy (CIPN).

[0071] Specifically, 6-week-old male C57BL / 6 mice were acclimatized for one week in a laboratory setting. 10 mg of paclitaxel (Sigma Aldrich, USA) was dissolved in 1.66 mL of a solution containing Cremophor EL and anhydrous ethanol at a 1:1 ratio to prepare a 6 mg / mL stock solution. This solution was then aliquoted into 100 μL portions and stored at -20°C. Shortly before administration, the paclitaxel stock solution was dissolved, diluted 1 / 30 with saline, and administered intraperitoneally at a dose of 2 mg / kg (cumulative dose 10 mg / kg) once daily for 5 days to induce chemotherapy-induced pain. Thermal injury perception was assessed 7 days after the last administration; subjects with an avoidance response time to radiant heat stimuli of less than 6 seconds were considered to have chemotherapy-induced pain.

[0072] Measurement of thermal hyperalgesia

[0073] An acrylic chamber with an open bottom was placed on a glass plate of a thermal pain measurement device (plantar analgesia tester), and mice were placed in this chamber for 2 hours to 3 days to complete the stabilization process. Basic pain response values ​​to radiative thermal stimulation were measured before establishing the spinal cord transection mouse model. Infrared intensity was set to 25 or 30 to obtain a basic pain avoidance response time of 8–10 seconds. The right hind paw of the mouse was subjected to radiative thermal stimulation three times at intervals of at least 1 minute per subject, and the avoidance response time was measured; the average value was used as the thermal pain response value. The duration of thermal stimulation was limited to 20 seconds when evaluating the thermal pain response to prevent thermal damage to the plantar tissues. After measuring the basic pain response value, the rate of change of each subject's response value was calculated based on the average value of all subjects, and subjects within ±50% of the overall average were selected. A neuropathic pain model was constructed using only the selected subjects, and thermal hyperalgesia was measured on day 5 after nerve injury under the above conditions. When measuring thermal pain response, experimental animals were placed in an acrylic chamber for 2 hours each time. After stabilization, they were subjected to radiant heat stimulation, and the thermal pain response was measured between 4 PM and 6 PM. Subsequently, the neuropathic pain animal model was randomly divided into two groups, and either recombinant human GDF11 or saline was administered intrathecally. The analgesic effect of the thermal pain response was then evaluated. Figure 2a For the experimental group, a researcher in a dark-blind state measured the thermal pain response, and after completing the planned thermal pain response evaluation test, the results were analyzed using information from the drug-treated group for statistical processing.

[0074] Intraspinal administration of drugs

[0075] Under isoflurane inhalation anesthesia, the drug is injected directly into the spinal cord using a syringe. A 30-gauge needle is attached to a Hamilton syringe, filled with GDF11 or saline, and injected into the space between the 5th and 6th vertebrae, administering GDF11 at a concentration of 10 ng / 5 μL.

[0076] Culture of mouse sensory neurons

[0077] The inventors first cultured sensory neurons from the dorsal root ganglion (DRG) of mice. Specifically, 6-9 week old C57BL / 6 mice were anesthetized with isoflurane for 5 minutes, disinfected with 70% ethanol, and their backs were cut open. Subsequently, all sites of the spinal cord's dorsal root ganglia were dissected and placed in a solution mixed with 10X HBSS and 10 mM HEPES (Wood, et al., J Neurosi. 8, pp3208-3220, 1988). Then, the dorsal root ganglia were cultured in a solution mixed with 0.2 mg / mL collagenase A and 3 mg / mL dispersin II, washed once with DMEM medium containing 10% FBS, and crushed using a Pasteur pipette. Subsequently, an average of 100-300 cells were dispersed on coverslips coated with poly-D-lysine and cultured for 1 hour. Then, 2 mL of neural basal medium containing 10% FBS, 1% penicillin / streptomycin and 1X B27 supplement was added for further culture.

[0078] Intracellular Ca 2+ Inflow Experiment

[0079] For calcium influx measurement experiments, primary cultured sensory neurons were exposed to DMEM medium containing 2 μM Fura-2AM for up to 40 min, and the extent of calcium ion influx into cells was subsequently measured by the F340 / F380 ratio. The inhibition rate of calcium influx by GDF11 pretreatment on the F340 / F380 ratio was measured for an increase of 200 nM capsaicin. Specifically, intracellular calcium ion concentration was measured using a microscope equipped with a camera and a connected fluorescence spectrometer. The fluorescence wavelength ratio (F340 / F380) observed during fluorescence measurement reflected the calcium ion concentration in the cells. A drug-containing perfusion solution was infused at a rate of 2 mL / min–3 mL / min. This perfusion solution consisted of 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES, and the pH was adjusted to 7.4 by adding NaOH. Figure 1 (b)

[0080] Single-cell patch-clamp experiment

[0081] The inhibition rate of capsaicin-induced inward current by GDF11 was measured using a patch-clamp experiment. The solution in the patch pipette consisted of 126 mM K-gluconate, 10 mM NaCl, 1 mM MgCl2, 0.1 mM Na2GTP, 2 mM Na2ATP, 10 mM HEPES, and 10 mM EGTA, with the pH adjusted to 7.4 by adding KOH. The extracellular perfusion solution consisted of 140 mM NaCl, 2 mM EGTA, 1 mM MgCl2, 10 mM glucose, 5 mM KCl, and 10 mM HEPES, with the pH adjusted to 7.4 by adding NaOH. The extracellular perfusion solution containing the drug was infused by gravity at a rate of 2-3 mL / min, and capsaicin and GDF11 were perfused into nociceptive neurons using this solution to measure the inhibition rate of capsaicin-induced inward current by GDF11.

[0082] pcDNA TRPV1 expression vector transformed into HEK293 cells

[0083] The inventors simultaneously transformed the TRPV1 gene and the green fluorescent protein (GFP) gene into HEK293 cells, and subsequently used intracellular Ca2+ to transform the cells. 2+ Calcium influx and inward current were determined using influx measurement experiments and single-cell patch-clamp experiments. Specifically, this was achieved by using Lipofectamine. TM In 2000 (Invitrogen, USA), the pcDNATRPV1 expression vector was transformed using the following method. First, the culture medium for HEK293 cells cultured in 35 mm cell culture dishes was removed, and only DMEM medium was added. The cells were then wet-cultured at 37°C and 5% CO2, with pcDNA-TRPV1 (1 μg) and Lipofectamine (10 μL) dissolved in each DMEM medium. After waiting 5 minutes, the two media were mixed, and the mixture was allowed to stand for another 20 minutes. Next, the DMEM medium attached to the HEK293 cells was removed, and the medium containing the pre-prepared pcDNA was dispersed. The cells were then wet-cultured at 37°C and 5% CO2 for 4 hours. The medium was then replaced with DMEM containing 10% FBS and 1% penicillin / streptomycin. After culturing for 12 hours, calcium imaging or patch-clamp experiments were performed within 24 hours.

[0084] Preparation of viral vectors

[0085] The inventors attempted to prepare a viral vector containing the GDF11 gene.

[0086] Specifically, the AAV5 serotype and CMV promoter were selected to induce sustained expression of GDF11 in infected or neuronal-infected cells. Furthermore, the internal ribosome entry site (IRES) and enhanced green fluorescent protein (eGFP) nucleotide sequences were added after the GDF11 nucleotide sequence, enabling independent expression of GDF11 and GFP proteins in the infected cells. The distribution of infected cells and GDF11 expression were evaluated in experimental animal tissues. After preparing a viral vector by loading the GDF11 gene into an AAV5 vector, the insertion of the normal GDF11 gene was verified by nucleotide sequencing, and the titer of the tested GDF11-expressing virus was 1.22 × 10⁻⁶. 13 GC / mL.

[0087] Immunostaining

[0088] The inventors performed tissue immunostaining to verify the expression of GDF11 in sensory neurons after intrathecal injection of a viral vector containing the gene.

[0089] Specifically, sodium pentobarbital (60 mg / kg) was injected intraperitoneally into the experimental animals to anesthetize them. A 30 mL syringe was filled with 30 mL of 0.9% NaCl solution, and the right atrium of the heart was incised. The syringe was then inserted into the left ventricle to draw blood. Subsequently, 30 mL of 10% formalin solution was administered to the left ventricle for fixation, and samples of L4 and L5 DRG tissue were collected. The removed DRG tissue was further fixed by soaking in 10% formalin solution for 16 hours, and then transferred to 30% sucrose solution for dehydration for two days. Afterward, the DRG tissue was frozen at -20°C and then prepared into 12 μm thick sections. Microtubule antibody (Santa Cruz, USA) was diluted 1 / 100 in 0.1 M PBS and treated on the DRG tissue sections for 24 hours. After washing with 0.1 M PBS, fluorescence images were captured using confocal microscopy.

[0090] Experimental statistics

[0091] Statistical analysis was performed using Prism (Graphpad, version 501) software. Specifically, after confirming the normality of the distribution through a normality test, parametric or nonparametric tests were used. For parametric and nonparametric tests, t-tests were performed on two or more groups, and for three or more groups, one-way or two-way ANOVA tests were used for analysis. Error bars were visualized using the standard error of measurement (SEM), and if the experimental result was significant, it was indicated by * or # [1 indicates P < 0.05, 2 indicates P < 0.005, 3 indicates P < 0.001].

[0092] [Example 1]

[0093] Evaluation of thermal hyperalgesia

[0094] <1-1> Chronic pain mouse model of spinal cord transection

[0095] The inventors established a mouse model of chronic pain through spinal nerve transection (SNT). After intraspinal administration of GDF11 (10 ng), thermal pain response was evaluated using a Hargreaves device. The results showed that, compared to the control group, administration of GDF11 to the chronic pain mouse model increased the delay time of the hindfoot avoidance response by inhibiting thermal hyperalgesia. Figure 2a ).

[0096] <1-2> Mouse model of peripheral neuropathy

[0097] The inventors prepared a mouse model of chemotherapy-induced peripheral neuropathy (CIPN) and evaluated the thermal pain response using a Hargreaves device after administering 0.1 mg / kg or 0.2 mg / kg of GDF11 intraperitoneally.

[0098] The results confirmed that, Figure 2b and Figure 2c As shown, administration of GDF11 increased the delay in avoidance response by inhibiting thermal hyperalgesia. Specifically, it was found that GDF11 inhibited thermal hyperalgesia for a longer period of time (at a dose 650 times less than gabapentin (Sigma Aldrich, USA), one of the representative pain management agents). Figure 2b ).

[0099] [Example 2]

[0100] Analysis of calcium influx into mouse sensory neurons and HEK293 cells

[0101] The calcium influx in primary cultured mouse sensory neurons and HEK293 cells transformed with pcDNA-TRPV1 vector was compared and analyzed due to treatment with GDF11 (10 nM) and capsaicin (200 nM) of the present invention, and the calcium influx was also compared based on the concentration of GDF11 (0.1 nM, 1 nM or 10 nM).

[0102] Compared with the control group not treated with GDF11, the results showed that the GDF11 treatment of the present invention reduced TRPV1-mediated Ca in a concentration-dependent manner. 2+ Inflow (Ca) 2+ Transient phenomena Figure 3a , Figure 3b , Figure 4a and Figure 4b The extent of calcium inflow was normalized and displayed based on the magnitude of the first calcium response to capsaicin.

[0103] [Example 3]

[0104] Analysis of the magnitude of inward currents in mouse sensory neurons and HEK293 cells

[0105] The magnitudes of inward currents in primary cultured mouse sensory neurons and HEK293 cells transformed with the pcDNA-TRPV1 vector were compared and analyzed under treatment with GDF11 (10 nM) and capsaicin (200 nM) according to the present invention, and under treatment with different GDF11 concentrations (0.1 nM, 1 nM, or 10 nM). The results showed that, compared with the control group not treated with GDF11, the GDF11 of the present invention reduced the concentration-dependent TRPV1-mediated inward currents induced by capsaicin in mouse sensory neurons. Figure 5a , Figure 5b , Figure 6a and Figure 6b The measurement of the inward current was standardized and displayed based on the magnitude of the first response of capsaicin.

[0106] The results of Examples 1-3 above show that GDF11 treatment effectively inhibits TRPV1 ion channel activity at low concentrations, and can be used as an effective antagonist for TRPV1.

[0107] [Example 4]

[0108] Verification of GDF11 side effects

[0109] Existing TRPV1 antagonists are known to have side effects such as abnormal fever or hypothermia when administered under conditions that demonstrate analgesic effects, and therefore the applicant sought to verify whether such side effects occurred with the administration of GDF11.

[0110] Specifically, BCTC (30 mg / kg) or GDF11 (0.1 mg / kg or 0.2 mg / kg) was administered intraperitoneally to a concentration similar to that used by capsaicin to inhibit TRPV1 activity in chemotherapy-induced pain model mice whose skin was exposed by shaving part of the hair on the back of their bodies. The temperature of the exposed skin was then measured at time intervals using a thermal imaging camera.

[0111] The results are as follows Figure 7 As shown, even after administration of GDF11, the mice's body temperature did not change significantly, confirming that administration of GDF11 does not cause abnormal fever or hypothermia as side effects. On the other hand, abnormal fever was observed as a side effect when BCTC, a representative TRPV1 antagonist, was administered intraperitoneally at a dose of 30 mg / kg.

[0112] [Example 5]

[0113] Validation of expression of viral vector GDF11

[0114] The inventors sought to determine whether administering the prepared viral vector would induce GDF11 expression in vivo.

[0115] Specifically, 5 μL of GDF11 viral vector was administered at 1.22 × 10⁻⁶ ppm. 11 GC / mL was injected into the spinal cord of a neuropathic pain model induced by peripheral spinal nerve injury. Five weeks later, mice were sacrificed, and L4 and L5 DRG tissues were sampled and lyophilized. The tissues were immunostained with a tubulin antibody, and the GFP fluorescence signal was used as the expression level of GDF11 for imaging under a confocal microscope.

[0116] The results confirmed that GDF11 is expressed in L4 or L5 DRG neurons in mice, such as Figure 8 As shown.

[0117] [Example 6]

[0118] Verification of the pain-relieving effect of viral vector

[0119] The inventors sought to determine whether the prepared viral vector AAV5-GDF11 exhibited analgesic effects in an animal model of neuropathic pain.

[0120] Specifically, after determining the basic pain response value before establishing the pain model, a neuropathic pain model was created by severing peripheral spinal nerves. Three days after the pain model surgery, the thermal pain response was evaluated, and subjects with an avoidance response threshold of less than 6 seconds were considered as pain models. In this pain model, 5 μL of a 1.22 × 10⁻⁶ ppm solution was used. 11GDF11 viral vector at GC / mL was injected into the spinal cord, and the thermal pain response was evaluated weekly using a Hargreaves device.

[0121] The results confirmed that, Figure 9 As shown, administration of AAV5-GDF11 increased the delay time of avoidance response by inhibiting thermal hyperalgesia.

[0122] In summary, since the GDF11 of the present invention (which is a composition for treating pain by utilizing the inhibitory effect of TRPV1 activity) has been shown to rapidly and effectively inhibit the TRPV1 channel, it can be used as a new therapeutic agent for treating various pain conditions and diseases related to the TRPV1 channel, such as arthritis and diabetic peripheral neuropathy.

[0123] The present invention has been described with reference to the exemplary embodiments described above, but these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent other exemplary embodiments are possible therein. Therefore, the true scope of protection of the present invention should be determined by the spirit of the appended claims.

[0124] [Industrial Applicability]

[0125] Because the composition for treating pain utilizing the TRPV1 activity inhibition of the present invention exhibits excellent efficacy in inhibiting neuropathic pain caused by spinal cord nerve injury, 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. Furthermore, due to its effective inhibition of TRPV1 activity, it can be used as a therapeutic agent for various TRPV1-mediated diseases, thus possessing high industrial applicability.

[0126] [Free text for sequence lists]

[0127] SEQ ID NO:1 shows the amino acid sequence of the mature peptide of growth differentiation factor 11 (GDF11).

[0128] SEQ ID NO:2 shows the full-length amino acid sequence of human GDF11 peptide.

[0129] SEQ ID NO:3 shows the amino acid sequence of mouse GDF11.

[0130] SEQ ID NO:4 shows the amino acid sequence of chimpanzee GDF11.

[0131] SEQ ID NO:5 shows the amino acid sequence of gorilla GDF11.

[0132] SEQ ID NO:6 shows the amino acid sequence of GDF11 golden snub-nosed monkey.

[0133] SEQ ID NO:7 shows the amino acid sequence of the northern white-cheeked gibbon GDF11.

[0134] SEQ ID NO:8 shows the amino acid sequence of GDF11 from the Bengal macaque.

[0135] SEQ ID NO:9 shows the amino acid sequence of bonobo GDF11.

[0136] SEQ ID NO:10 shows the amino acid sequence of porcine GDF11.

[0137] SEQ ID NO:11 shows the polynucleotide sequence encoding the GDF11 peptide (SEQ ID NO:1). sequence list <110> Rudacourt Company <120> Pharmaceutical compositions for treating TRPV1-mediated diseases <130> PCT5040671 <150> KR 10-2020-0007433 <151> 2020-01-20 <160> 11 <170> PatentIn version 3.2 <210> 1 <211> 109 <212> PRT <213> artificial <220> <223> GDF-11 mature peptide <400> 1 Asn Leu Gly Leu Asp Cys Asp Glu His Ser Ser Glu Ser Arg Cys Cys 1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu 35 40 45 Tyr Met Phe Met Gln Lys Tyr Pro His Thr His Leu Val Gln Gln Ala 50 55 60 Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser 65 70 75 80 Pro Ile Asn Met Leu Tyr Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly 85 90 95 Lys Ile Pro Gly Met Val Val Asp Arg Cys Gly Cys Ser 100 105 <210> 2 <211> 407 <212> PRT <213> Artificial <220> <223> Full sequence of GDF-11 CDS <400> 2 Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu 1 5 10 15 Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu Arg Ser 35 40 45 Ser Arg Pro Ala Pro Ser Val Ala Pro Glu Pro Asp Gly Cys Pro Val 50 55 60 Cys Val Trp Arg Gln His Ser Arg Glu Leu Arg Leu Glu Ser Ile Lys 65 70 75 80 Ser Gln Ile Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro Asn Ile Ser 85 90 95 Arg Glu Val Val Lys Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln Gln 100 105 110 Ile Leu Asp Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp 115 120 125 Phe Leu Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser 130 135 140 Met Ala Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro Leu 145 150 155 160 Cys Cys His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu 165 170 175 Lys Ala Gln Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro Ala Thr 180 185 190 Val Tyr Leu Gln Ile Leu Arg Leu Lys Pro Leu Thr Gly Glu Gly Thr 195 200 205 Ala Gly Gly Gly Gly Gly Gly Arg Arg His Ile Arg Ile Arg Ser Leu 210 215 220 Lys Ile Glu Leu His Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe 225 230 235 240 Lys Gln Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly 245 250 255 Ile Glu Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr 260 265 270 Ser Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg 275 280 285 Val Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys 290 295 300 Asp Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 305 310 315 320 Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 325 330 335 Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys 340 345 350 Tyr Pro His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala 355 360 365 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 370 375 380 Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val 385 390 395 400 Val Asp Arg Cys Gly Cys Ser 405 <210> 3 <211> 405 <212> PRT <213> Artificial <220> <223> Mus musculus <400> 3 Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu 1 5 10 15 Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu Arg Ser Ser Arg 35 40 45 Pro Ala Pro Ser Ala Pro Pro Glu Pro Asp Gly Cys Pro Val Cys Val 50 55 60 Trp Arg Gln His Ser Arg Glu Leu Arg Leu Glu Ser Ile Lys Ser Gln 65 70 75 80 Ile Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro Asn Ile Ser Arg Glu 85 90 95 Val Val Lys Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln Gln Ile Leu 100 105 110 Asp Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp Phe Leu 115 120 125 Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser Met Ala 130 135 140 Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro Leu Cys Cys 145 150 155 160 His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu Lys Ala 165 170 175 Gln Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro Ala Thr Val Tyr 180 185 190 Leu Gln Ile Leu Arg Leu Lys Pro Leu Thr Gly Glu Gly Thr Ala Gly 195 200 205 Gly Gly Gly Gly Gly Arg Arg His Ile Arg Ile Arg Ser Leu Lys Ile 210 215 220 Glu Leu His Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe Lys Gln 225 230 235 240 Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly Ile Glu 245 250 255 Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr Ser Leu 260 265 270 Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg Val Leu 275 280 285 Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys Asp Glu 290 295 300 His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp Phe 305 310 315 320 Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr Lys Ala 325 330 335 Asn Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys Tyr Pro 340 345 350 His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala Gly Pro 355 360 365 Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr Phe Asn 370 375 380 Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val Val Asp 385 390 395 400 Arg Cys Gly Cys Ser 405 <210> 4 <211> 405 <212> PRT <213> Artificial <220> <223> Chimpanzee (Pan troglodytes) <400> 4 Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu 1 5 10 15 Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu Arg Ser Ser Arg 35 40 45 Pro Ala Pro Ser Val Ala Pro Glu Pro Asp Gly Cys Pro Val Cys Val 50 55 60 Trp Arg Gln His Ser Arg Glu Leu Arg Leu Glu Ser Ile Lys Ser Gln 65 70 75 80 Ile Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro Asn Ile Ser Arg Glu 85 90 95 Val Val Lys Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln Gln Ile Leu 100 105 110 Asp Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp Phe Leu 115 120 125 Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser Met Ala 130 135 140 Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro Leu Cys Cys 145 150 155 160 His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu Lys Ala 165 170 175 Gln Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro Ala Thr Val Tyr 180 185 190 Leu Gln Ile Leu Arg Leu Lys Pro Leu Thr Gly Glu Gly Thr Ala Gly 195 200 205 Gly Gly Gly Gly Gly Arg Arg His Ile Arg Ile Arg Ser Leu Lys Ile 210 215 220 Glu Leu His Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe Lys Gln 225 230 235 240 Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly Ile Glu 245 250 255 Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr Ser Leu 260 265 270 Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg Val Leu 275 280 285 Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys Asp Glu 290 295 300 His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp Phe 305 310 315 320 Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr Lys Ala 325 330 335 Asn Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys Tyr Pro 340 345 350 His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala Gly Pro 355 360 365 Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr Phe Asn 370 375 380 Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val Val Asp 385 390 395 400 Arg Cys Gly Cys Ser 405 <210> 5 <211> 407 <212> PRT <213> Artificial <220> <223> Lowland Gorilla (Gorilla gorilla) <400> 5 Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu 1 5 10 15 Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu Arg Ser 35 40 45 Ser Arg Pro Ala Pro Ser Val Ala Pro Glu Pro Asp Gly Cys Pro Val 50 55 60 Cys Val Trp Arg Gln His Ser Arg Glu Leu Arg Leu Glu Ser Ile Lys 65 70 75 80 Ser Gln Ile Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro Asn Ile Ser 85 90 95 Arg Glu Val Val Lys Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln Gln 100 105 110 Ile Leu Asp Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp 115 120 125 Phe Leu Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser 130 135 140 Met Ala Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro Leu 145 150 155 160 Cys Cys His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu 165 170 175 Lys Ala Gln Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro Ala Thr 180 185 190 Val Tyr Leu Gln Ile Leu Arg Leu Lys Pro Leu Thr Gly Glu Gly Thr 195 200 205 Ala Gly Gly Gly Gly Gly Gly Arg Arg His Ile Arg Ile Arg Ser Leu 210 215 220 Lys Ile Glu Leu His Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe 225 230 235 240 Lys Gln Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly 245 250 255 Ile Glu Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr 260 265 270 Ser Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg 275 280 285 Val Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys 290 295 300 Asp Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 305 310 315 320 Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 325 330 335 Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys 340 345 350 Tyr Pro His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala 355 360 365 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 370 375 380 Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val 385 390 395 400 Val Asp Arg Cys Gly Cys Ser 405 <210> 6 <211> 406 <212> PRT <213> Artificial <220> <223> Golden Snub-Nosed Monkey (Rhinopithecus roxellana) <400> 6 Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu 1 5 10 15 Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu Arg Ser Ser 35 40 45 Arg Pro Ala Pro Ser Val Ala Pro Glu Pro Asp Gly Cys Pro Val Cys 50 55 60 Val Trp Arg Gln His Ser Arg Glu Leu Arg Leu Glu Ser Ile Lys Ser 65 70 75 80 Gln Ile Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro Asn Ile Ser Arg 85 90 95 Glu Val Val Lys Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln Gln Ile 100 105 110 Leu Asp Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp Phe 115 120 125 Leu Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser Met 130 135 140 Ala Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro Leu Cys 145 150 155 160 Cys His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu Lys 165 170 175 Ala Gln Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro Ala Thr Val 180 185 190 Tyr Leu Gln Ile Leu Arg Leu Lys Pro Leu Thr Gly Glu Gly Thr Ala 195 200 205 Gly Gly Gly Gly Gly Gly Arg Arg His Ile Arg Ile Arg Ser Leu Lys 210 215 220 Ile Glu Leu His Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe Lys 225 230 235 240 Gln Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly Ile 245 250 255 Glu Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr Ser 260 265 270 Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg Val 275 280 285 Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys Asp 290 295 300 Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp 305 310 315 320 Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr Lys 325 330 335 Ala Asn Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys Tyr 340 345 350 Pro His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala Gly 355 360 365 Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr Phe 370 375 380 Asn Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val Val 385 390 395 400 Asp Arg Cys Gly Cys Ser 405 <210> 7 <211> 409 <212> PRT <213> Artificial <220> <223> Gibbon (Nomascus leucogenys) <400> 7 Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu 1 5 10 15 Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu 35 40 45 Arg Ser Ser Arg Pro Ala Pro Ser Val Ala Pro Glu Pro Asp Gly Cys 50 55 60 Pro Val Cys Val Trp Arg Gln His Ser Arg Glu Leu Arg Leu Glu Ser 65 70 75 80 Ile Lys Ser Gln Ile Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro Asn 85 90 95 Ile Ser Arg Glu Val Val Lys Gln Leu Leu Pro Lys Ala Pro Pro Leu 100 105 110 Gln Gln Ile Leu Asp Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro 115 120 125 Glu Asp Phe Leu Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val 130 135 140 Ile Ser Met Ala Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser 145 150 155 160 Pro Leu Cys Cys His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys 165 170 175 Val Leu Lys Ala Gln Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro 180 185 190 Ala Thr Val Tyr Leu Gln Ile Leu Arg Leu Lys Pro Leu Thr Gly Glu 195 200 205 Gly Thr Ala Gly Ala Gly Gly Gly Gly Arg Arg His Ile Arg Ile Arg 210 215 220 Ser Leu Lys Ile Glu Leu His Ser Arg Ser Gly His Trp Gln Ser Ile 225 230 235 240 Asp Phe Lys Gln Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn 245 250 255 Trp Gly Ile Glu Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala 260 265 270 Val Thr Ser Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu 275 280 285 Leu Arg Val Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu 290 295 300 Asp Cys Asp Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu 305 310 315 320 Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys 325 330 335 Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met 340 345 350 Gln Lys Tyr Pro His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly 355 360 365 Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met 370 375 380 Leu Tyr Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly 385 390 395 400 Met Val Val Asp Arg Cys Gly Cys Ser 405 <210> 8 <211> 408 <212> PRT <213> Artificial <220> <223> Macaca mulatta <400> 8 Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu 1 5 10 15 Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu Arg 35 40 45 Ser Ser Arg Pro Ala Pro Ser Val Ala Pro Glu Pro Asp Gly Cys Pro 50 55 60 Val Cys Val Trp Arg Gln His Ser Arg Glu Leu Arg Leu Glu Ser Ile 65 70 75 80 Lys Ser Gln Ile Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro Asn Ile 85 90 95 Ser Arg Glu Val Val Lys Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln 100 105 110 Gln Ile Leu Asp Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu 115 120 125 Asp Phe Leu Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile 130 135 140 Ser Met Ala Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro 145 150 155 160 Leu Cys Cys His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val 165 170 175 Leu Lys Ala Gln Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro Ala 180 185 190 Thr Val Tyr Leu Gln Ile Leu Arg Leu Lys Pro Leu Thr Gly Glu Gly 195 200 205 Thr Ala Gly Gly Gly Gly Gly Gly Arg Arg His Ile Arg Ile Arg Ser 210 215 220 Leu Lys Ile Glu Leu His Ser Arg Ser Gly His Trp Gln Ser Ile Asp 225 230 235 240 Phe Lys Gln Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp 245 250 255 Gly Ile Glu Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val 260 265 270 Thr Ser Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu 275 280 285 Arg Val Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp 290 295 300 Cys Asp Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr 305 310 315 320 Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg 325 330 335 Tyr Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln 340 345 350 Lys Tyr Pro His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser 355 360 365 Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu 370 375 380 Tyr Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met 385 390 395 400 Val Val Asp Arg Cys Gly Cys Ser 405 <210> 9 <211> 263 <212> PRT <213> Artificial <220> <223> Bonobo (Pan paniscus) <400> 9 Met Ala Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro Leu 1 5 10 15 Cys Cys His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu 20 25 30 Lys Ala Gln Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro Ala Thr 35 40 45 Val Tyr Leu Gln Ile Leu Arg Leu Lys Pro Leu Thr Gly Glu Gly Thr 50 55 60 Ala Gly Gly Gly Gly Gly Gly Arg Arg His Ile Arg Ile Arg Ser Leu 65 70 75 80 Lys Ile Glu Leu His Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe 85 90 95 Lys Gln Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly 100 105 110 Ile Glu Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr 115 120 125 Ser Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg 130 135 140 Val Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys 145 150 155 160 Asp Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 165 170 175 Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 180 185 190 Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys 195 200 205 Tyr Pro His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala 210 215 220 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 225 230 235 240 Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val 245 250 255 Val Asp Arg Cys Gly Cys Ser 260 <210> 10 <211> 404 <212> PRT <213> Artificial <220> <223> Domestic pig (Sus scrofa domesticus) <400> 10 Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu 1 5 10 15 Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala Ala Ala Gly Ala Gly Gly Glu Arg Ser Ser Arg Pro 35 40 45 Ala Ala Ser Val Ala Pro Glu Pro Asp Gly Cys Pro Val Cys Leu Trp 50 55 60 Arg Gln His Ser Arg Glu Leu Arg Leu Glu Ser Val Lys Ser Gln Ile 65 70 75 80 Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro Asn Ile Ser Arg Glu Val 85 90 95 Val Lys Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln Gln Ile Leu Asp 100 105 110 Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp Phe Leu Glu 115 120 125 Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser Met Ala Gln 130 135 140 Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro Leu Cys Cys His 145 150 155 160 Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu Lys Ala Gln 165 170 175 Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro Ala Thr Val Tyr Leu 180 185 190 Gln Ile Leu Arg Leu Lys Pro Leu Thr Gly Glu Gly Thr Ala Gly Gly 195 200 205 Gly Gly Gly Gly Arg Arg His Ile Arg Ile Arg Ser Leu Lys Ile Asp 210 215 220 Leu His Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe Lys Gln Val 225 230 235 240 Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly Ile Glu Ile 245 250 255 Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr Ser Leu Gly 260 265 270 Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg Val Leu Glu 275 280 285 Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys Asp Glu His 290 295 300 Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp Phe Glu 305 310 315 320 Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr Lys Ala Asn 325 330 335 Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys Tyr Pro His 340 345 350 Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys 355 360 365 Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr Phe Asn Asp 370 375 380 Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val Val Asp Arg 385 390 395 400 Cys Gly Cys Ser <210> 11 <211> 330 <212> DNA <213> Artificial <220> <223> GDF-11 polynucleotide <400> 11 aacctgggtc tggactgcga cgagcactca agcgagtccc gctgctgccg atatcccctc 60 acagtggact ttgaggcttt cggctgggac tggatcatcg cacctaagcg ctacaaggcc 120 aactactgct ccggccagtg cgagtacatg ttcatgcaaa aatatccgca tacccatttg 180 gtgcagcagg ccaatccaag aggctctgct gggccctgtt gtacccccac caagatgtcc 240 ccaatcaaca tgctctactt caatgacaag cagcagatta tctacggcaa gatccctggc 300 atggtggtgg atcgctgtgg ctgctcttaa 330

Claims

1. Use of a pharmaceutical composition for the manufacture of a medicament for the treatment of a TRPVl mediated disease, wherein, The pharmaceutical composition comprises: The growth differentiation factor 11 (GDF11) peptide with the amino acid sequence shown in SEQ ID NO: 1, the polynucleotide encoding the peptide, or the expression vector containing the polynucleotide as an active ingredient. The TRPV1-mediated diseases are selected from the group consisting of cancer pain, chemotherapy-induced pain, postoperative pain, diabetic neuropathic pain, migraine, and joint pain.

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

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

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

5. The application according to claim 2, wherein the non-viral vector is a DNA vector, nanoparticles, cationic polymers, exosomes, extracellular vesicles, or liposomes.

6. The application according to claim 5, wherein the DNA vector is a plasmid vector, a granular vector, a phage vector, or an artificial human chromosome.

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