Composition for mimicking acupuncture treatment

Abstract

The present invention relates to a composition for mimicking acupuncture treatment, comprising a Gq-DREADD expression vector administered to an acupoint as an active ingredient. The present invention provides a technique for mimicking acupuncture treatment by applying chemical genetics to an acupoint, thereby non-invasively enabling the expression of Gq-DREADD in neurons of an individual and the activation of the Gq-DREADD so as to enhance the cognitive and / or motor skills of the individual, and enabling the reduction of neuropathic pain and the alleviation of atopic dermatitis symptoms, and thus the present invention is expected to be used as a means for replacing acupuncture treatment in which the same result cannot be guaranteed depending on the operator.

Description

Composition for simulating acupuncture treatment

[0001] The present invention relates to a composition for simulating acupuncture treatment, including a vector for expressing Gq-DREADD administered to an acupoint as an active ingredient.

[0002] Chemical genetics, also known as chemical biology or chemical genomics, is an applied technology that helps understand physiological functions by regulating cellular activity by applying proteins that respond to unknown small-molecule synthetic substances. Proteins such as kinases, non-kinase enzymes, G protein-coupled receptors (GPCRs), and ligand-gated ion channels have been created through chemical genetics. DREADDs (designer receptors exclusively activated by designer drugs), which are the most widely used tool in chemical genetics, are proteins in which some mutations have been made to reduce the reactivity to acetylcholine (Ach) from the wild-type muscarinic receptor and increase the reactivity to new synthetic substances (specifically, clozapine-N-oxide (CNO), perlapine, and compound 21).

[0003] Gq-DREADD, a type of DREADD, also known as hM3Dq, is a receptor that promotes neuronal activity and can respond to stimulation by CNO, perlapine, and compound 21, and can interact with G proteins. Gq-DREADDs are known to be activated at low concentrations (nM) of CNO, increasing intracellular calcium secretion and promoting neuronal activity. Research on DREADDs for the treatment of neurological diseases is actively underway, and it is known that Gq-DREADD and Gi-DREADD can improve the symptoms of neurodegenerative diseases such as Parkinson's disease with muscular dystrophy (Van Den Herrewegen et al. Mol Brain (2021) 14:144). However, studies on the treatment of neurological diseases related to DREADDs known to date have been conducted by directly stimulating the central nervous system. However, direct injection of DREADD expression vectors into the brain requires surgery and has a high risk of side effects such as inflammation and infection.

[0004] In traditional medicine, acupuncture points are points where needles or moxibustion are inserted, and they refer to points along the pathways (also called 'meridians') through which the reactions of the five viscera and six bowels appear on the body's surface. The World Health Organization (WHO) Western Pacific Regional Office (WPRO) has been working to standardize the locations of acupuncture points in traditional medicine to standardize traditional medicine, and through agreements between Korea, China, and Japan, established 361 standard acupuncture points and published the WHO / WPRO Standard Acupuncture Point Locations in 2008.

[0005] The present inventors conducted extensive research to develop a technique for simulating acupuncture treatment that does not show differences in results depending on the practitioner, and as a result, confirmed that by applying chemical genetics to acupoints, various neurological diseases including Parkinson's disease and neuropathic pain can be treated, and further, the effects of simulating acupuncture treatment, such as improvement of atopic symptoms, can be obtained, thereby completing the present invention.

[0006] The technical problem to be achieved by the present invention is to provide a technique for simulating acupuncture treatment by applying chemical genetics to acupoints, and to provide a composition and kit for simulating acupuncture treatment for implementing the technique and its use.

[0007] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0008] To solve the above problem, the present invention provides a composition for simulating acupuncture treatment, which comprises a vector expressing Gq-DREADD (hM3Dq) as an active ingredient.

[0009] The present invention also provides the use of a vector expressing Gq-DREADD (hM3Dq) for the preparation of a composition for simulating acupuncture treatment.

[0010] The acupuncture treatment simulating composition of the present invention is characterized in that it is administered to an acupoint.

[0011] As one embodiment of the present invention, the pharmaceutical composition may be administered in combination with a ligand of Gq-DREADD, and may be administered prior to administration of the ligand.

[0012] In another embodiment of the present invention, the vector may be a viral vector or a non-viral vector, but is preferably a viral vector.

[0013] As another embodiment of the present invention, the viral vector may be a retroviral vector, an adenoviral vector, or an adeno-associated viral vector, but is preferably a retroviral vector.

[0014] As another embodiment of the present invention, the acupuncture point may be a point located at one or more meridians selected from a group consisting of 361 acupuncture points presented in the WHO / WPRO standard acupuncture point locations published in 2008.

[0015] In addition, the present invention provides an acupuncture treatment-mimetic kit (in other words, a chemogenetic acupuncture kit) comprising the acupuncture treatment-mimetic composition and a ligand of Gq-DREADD.

[0016] Meanwhile, the acupuncture treatment-simulating composition of the present invention can be provided as a pharmaceutical composition for preventing or treating nervous system diseases, neuropathic pain, and / or atopic dermatitis.

[0017] Specifically, the present invention provides a pharmaceutical composition for treating a nervous system disease, which comprises the acupuncture treatment-simulating composition as an active ingredient, and the pharmaceutical composition for treating a nervous system disease may be administered to one or more acupoints selected from the group consisting of Gihae, Naegwan, Daechu, Baekhoe, Bulyu, Samyingyo, Susamri, Sinmun, Sinjeong, Yanglingcheon, Yangbo, Woosang, Wijung, Joksamri, Chungyang, Pungryung, Taegye, Taechung, and Hegok.

[0018] In one embodiment of the present invention, the neurological disease may be at least one selected from the group consisting of Stiff-person syndrome, dystrophy not caused by nutritional deficiency, myotubular myopathy, Guillain-Barre syndrome, nemaline myopathy, multiple sclerosis, multiminicore disease, Huntington's disease, Hallervorden-Spatz Syndrome, Parkinson's disease, Alzheimer's disease, dementia, and Lou Gehrig's disease.

[0019] In addition, the present invention provides a pharmaceutical composition for treating neuropathic pain, which comprises the acupuncture treatment-simulating composition as an active ingredient, and the pharmaceutical composition for treating neuropathic pain may be administered to one or more acupoints selected from the group consisting of Gansu, Gyeoksu, Gyeonwoo, Gokji, Kunlun, Gongsun, Gwanwon, Gihae, Naegwan, Naejeong, Daechu, Bokto, Bigwan, Bisoo, Biyang, Samyingyo, Susamri, Shindo, Shinmaek, Shinsu, Shinju, Yanggu, Yanglingcheon, Yeolgyeol, Oesang, Johae, Joksamri, Jokineup, Joktonggok, Jiyang, Cheonchu, Pungryung, Taegye, Taebaek, Taechung, Pyesu, Hamgok, Heogok, Hwando, Hyeolhae, and Hugye.

[0020] In addition, the present invention provides a pharmaceutical composition for treating atopic dermatitis, which comprises the acupuncture treatment-simulating composition as an active ingredient, and the pharmaceutical composition for treating atopic dermatitis may be administered to one or more acupoints selected from the group consisting of Gansu, Gyeoksu, Gokji, Myeongmun, Bisu, Simsu, Samyingyo, Yeogu, Okye, Eumryungcheon, Joksamri, Jiyang, Jiyin, Cheonchu, Feisu, Pungsi, Hegok, and Hyeolhae.

[0021] In addition, the present invention provides a pharmaceutical composition for treating one or more diseases selected from the group consisting of stroke, Parkinson's disease, facial paralysis, arthritis, pain (including neuropathic pain), cholecystitis, cholelithiasis, fatty liver, cerebral palsy, multiple system atrophy, hypertension, and muscular dystrophy, wherein the acupuncture treatment-simulating composition is characterized by being administered to Yangneungcheon.

[0022] In addition, the present invention provides a pharmaceutical composition for treating one or more diseases selected from the group consisting of stroke, Parkinson's disease, Alzheimer's disease and other dementias, facial paralysis, arthritis, pain (including neuropathic pain), gastritis, gastric ulcers, diarrhea and other digestive system diseases, hypertension, sepsis (systemic inflammatory diseases), chorea, dyskinesia, Huntington's disease, diabetes, intellectual disability, various emotional disorders (depression, schizophrenia, etc.), ovarian-related diseases, adrenal-related diseases, various infections, hepatitis, pancreatitis, obesity, and various tumors (breast cancer, etc.), characterized in that the acupuncture treatment-simulating composition is administered to the Zusanli point.

[0023] In addition, the present invention provides a pharmaceutical composition for treating one or more diseases selected from the group consisting of pain (including neuropathic pain), herniated disc, stroke, sickle cell anemia, muscular dystrophy, and neuroma, wherein the composition is characterized by being administered to the patient.

[0024] In addition, the present invention provides a pharmaceutical composition for treating one or more diseases selected from the group consisting of various skin diseases (eczema, rubella, psoriasis, measles, atopic dermatitis), stroke, hypertension, tinnitus, pain, Lou Gehrig's disease (amyotrophic lateral sclerosis), sepsis (systemic inflammatory disease), and constipation, wherein the composition for simulating acupuncture treatment is characterized by being administered to the stomach.

[0025] The present invention also provides a chemical genetic acupuncture treatment method comprising the following steps:

[0026] (1) a step of administering a vector for Gq-DREADD expression to an acupoint of an individual; and

[0027] (2) A step of administering a ligand of Gq-DREADD to the acupoint of the above object.

[0028] As one embodiment of the present invention, the subject may be a mammal, including a human.

[0029] The present invention provides a technique for simulating acupuncture treatment by applying chemical genetics to acupoints, thereby expressing and activating Gq-DREADDs in sensory neurons connected to acupoints of an individual in a minimally invasive manner, thereby improving cognitive and / or motor abilities of an individual, reducing neuropathic pain, and alleviating symptoms of atopic dermatitis. It is expected to be used as a more reproducible treatment method to replace acupuncture treatment.

[0030] Figure 1 shows the results of applying chemical genetic stimulation to acupoints (specifically, Yanglingcheon) and confirming the activation of brain neurons. (A) is a schematic diagram of the experiment, (B) is a confocal fluorescence micrograph of mouse brain tissue, and (C) is a graph comparing the number of c-Fos+ cells in mouse brain tissue treated with chemical genetic stimulation and the control group.

[0031] Figure 2 shows the results of performing motor behavior and cognitive behavior tests on experimental animals after applying chemical and genetic stimulation to acupoints (specifically, Yanglingchuan) in an animal model of Parkinson's disease. (A) is a schematic diagram of the experiment, (B) is the result of measuring the time it took for a mouse to fall from the rod in the rotarod treadmill test, (C) is the result of measuring the number of times a mouse stood with its forepaws against the wall in the cylinder test, (D) is the result of measuring the change rate in the Y-maze test, and (E) is the result of measuring the object exploration time of a mouse in the NOR test.

[0032] Figure 3 shows the results of confirming that the number of cells and the density of nerve fibers of dopamine neurons expressing tyrosine hydroxylase increased in the striatum and substantia nigra of Parkinson's disease-induced animals that received chemical and genetic stimulation at acupoints (specifically, Yanglingcheon).

[0033] Figure 4 shows the results of the Von Frey test (A) and the Open field test (B) confirming the reduction of pain and anxiety symptoms in unilateral sciatic nerve injury animals after chemical-genetic stimulation of acupoints (specifically, bilateral Zusanli, Yanglingquan, and Hwando).

[0034] Figure 5 shows the improvement of dermatitis in animals induced with atopic dermatitis by chemical genetic stimulation of acupoints (specifically, bilateral Gokji). Figure 5a is a photograph of the dermatitis-induced area of ​​each experimental animal, and Figure 5b is a graph showing the degree of erythema, dryness, and skin picking of each experimental animal and the severity of dermatitis as a score.

[0035] Figure 6 shows the results confirming that motor ability and anxiety symptoms were improved by applying chemical and genetic stimulation to acupoints of animals induced with atopic dermatitis.

[0036] The present inventors have confirmed that the effect of acupuncture treatment simulation can be obtained when chemical genetics is applied to acupoints, and completed the present invention.

[0037] In this specification, “acupoint” means acupuncture point located on a meridian, and is indicated according to the WHO / WPRO standard acupoint location published in 2008.

[0038] In the present invention, the term "acupuncture treatment mimicking technique" refers to the application of chemical genetics to acupuncture points, specifically, inducing the expression of Gq-DREADD in peripheral nerves located at the acupuncture point and administering a compound capable of activating the Gq-DREADD to the acupuncture point. In this specification, the term "acupuncture treatment mimicking technique" is used interchangeably with the terms "chemogenetic stimulation" and "chemogenetic acupuncture."

[0039] Accordingly, in the present invention, the “acupuncture treatment simulating composition” means a composition administered to an acupoint for the chemical genetic acupuncture stimulation.

[0040] Traditional acupuncture involves inserting needles into acupoints to physically stimulate the skin and muscles. Regardless of the location of the acupoint, the goal is to induce specific sensations (tingling, tingling, heaviness, pulling, etc.). In other words, the therapeutic effect of acupuncture is expected to be induced by stimulation of sensory nerves. The chemogenetic stimulation of the present invention directly stimulates peripheral afferent fibers (sensory nerves) at the acupoint site, achieving the same goal as conventional acupoint stimulation using needles. Therefore, it is expected to be effective regardless of the location of the acupoint.

[0041] Specifically, the present inventors locally injected a vector expressing Gq-DREADD, a type of DREADD, into the Yanglingchen to induce the expression of Gq-DREADD in the sensory nerve terminals of the acupoint site, and confirmed that when CNO (clozapine-N-oxide) was administered by injecting it into the acupoint, the neurons in the hypothalamus in the brain were activated. Subsequently, the present inventors confirmed that the symptoms of the disease were significantly improved by administering a Gq-DREADD expression vector and CNO into the Yanglingchen using an MPTP-induced Parkinson's disease animal model.

[0042] In traditional acupuncture, "Yanglingcheon" is known as an acupuncture point for the treatment of stroke, Parkinson's disease, facial paralysis, arthritis, pain (including neuropathic pain), cholecystitis, cholelithiasis, fatty liver, cerebral palsy, multiple system atrophy, hypertension, and muscular atrophy.

[0043] Therefore, the present inventors sought to determine whether chemogenetic acupuncture could be effective in treating diseases other than Parkinson's disease, for which traditional acupuncture has shown therapeutic effects. Among these diseases, neuropathic pain was selected and, using a PSNL-induced neuropathic pain animal model, chemogenetic acupuncture applied to the Yangling Hot Spring was used to determine whether pain could be controlled. As a result, chemogenetic acupuncture applied to the Yangling Hot Spring resulted in a reduction in pain.

[0044] From the above, it can be seen that diseases for which specific acupoints have a therapeutic effect in traditional acupuncture treatment can also have the same therapeutic effect when chemical genetic acupuncture treatment is performed on the acupoints.

[0045] Next, the inventors of the present invention sought to determine whether chemical genetic acupuncture could achieve the same therapeutic effects when performed on acupuncture points used for the treatment of specific diseases in traditional acupuncture. In addition to Yanglingchuan, acupuncture points used for Parkinson's disease treatment include Gihae, Naegwan, Daechu, Baihui, Bulyu, Sanyinjiao, Susamli, Shenmin, Sinjeong, Yangbo, Yuesang, Wijung, Joksamli, Chongyang, Fenglong, Taegye, Taechung, and Hegu (PMID: 34221005). In addition to Yanglingcheon, acupoints used in acupuncture for the treatment of neuropathic pain include Gansu, Gyeoksu, Gyeonwoo, Gokji, Kunlun, Gongsun, Gwanwon, Gihae, Naegwan, Naejeong, Daechu, Bokto, Bigwan, Bisoo, Biyang, Sanyinjiao, Susamri, Shindo, Shinmaek, Shinsu, Shinju, Yanggu, Yeolgyeol, Oesang, Johae, Joksanri, Jokineup, Joktonggok, Jiyang, Cheonchu, Pungryung, Taegye, Taebaek, Taechung, Feisu, Hamgok, Heogok, Hwando, Hyeolhae, and Hugye (PMID: 29197180).

[0046] Accordingly, the present inventors conducted an identical experiment using a PSNL-induced neuropathic pain animal model by selecting Hwandu and Joksamli, in addition to Yanglingchuan, among the acupoints used to treat neuropathic pain in order to determine whether all acupoints used for the treatment of the above-mentioned diseases in traditional acupuncture could be utilized in chemogenetic acupuncture. As a result, it was confirmed that when chemogenetic acupuncture was performed at Hwandu and Joksamli, the pain of the experimental animals was reduced in the same manner as when chemogenetic acupuncture was performed at Yanglingchuan.

[0047] From the above, it was found that the chemical genetic acupuncture of the present invention has the same effect as traditional acupuncture treatment, and that acupuncture points that are stimulated by needles in traditional acupuncture treatment can be freely used to obtain a therapeutic effect for a specific disease, and that diseases for which acupuncture points have a therapeutic effect in traditional acupuncture treatment can have the same effect when chemical genetic acupuncture treatment is applied to the acupuncture points.

[0048] To verify this once again, we applied chemogenetic acupuncture for the treatment of atopic dermatitis. The acupoints used in acupuncture for the treatment of atopic dermatitis include Gansu, Gyeoksu, Gokji, Myeongmun, Bisu, Simsu, Sanyinjiao, Yeogu, Okye, Yinlingcheon, Joksamli, Jiyang, Jieum, Cheonchu, Feisu, Fengsi, Hegu, and Hyeolhae (PMID: 31495184). Therefore, the present inventors selected Gokji among the acupoints for the treatment of atopic dermatitis and performed chemogenetic acupuncture on both Gokji points of an atopic dermatitis-induced animal model. As a result of observing the degree of dermatitis, we were able to confirm that dermatitis was improved through chemogenetic stimulation.

[0049] Meanwhile, "Joksamri" is known as an acupuncture point for treating stroke (apoplexy), Parkinson's disease, Alzheimer's disease and other dementias, facial paralysis, arthritis, pain (including neuropathic pain), gastritis, gastric ulcers, diarrhea and other digestive system diseases, hypertension, sepsis (systemic inflammatory disease), chorea, dyskinesia, Huntington's disease, diabetes, intellectual disability, various emotional disorders (depression, schizophrenia, etc.), ovarian diseases, adrenal diseases, various infections, hepatitis, pancreatitis, obesity, various tumors (breast cancer, etc.) (PMID: 35095910; PMID: 28810910), and "Hwando" is known as an acupuncture point for treating pain (including neuropathic pain), herniated disc, stroke (apoplexy), sickle cell anemia, muscular dystrophy, neuromas, etc. Additionally, "Gokji" is known as an acupuncture point for treating various skin diseases (eczema, rubella, psoriasis, measles, atopic dermatitis), stroke (apocrine disruption), high blood pressure, tinnitus, pain, Lou Gehrig's disease (amyotrophic lateral sclerosis), sepsis (systemic inflammatory disease), constipation, etc.

[0050] From the above, the inventors of the present invention have confirmed that the same effect as traditional acupuncture treatment can be obtained by inducing and activating Gq-DREADD expression in peripheral nerves located at acupoints rather than in the central nervous system, and they intend to provide a composition for application to the technique of simulating traditional acupuncture treatment.

[0051] In this specification, DREADD (designer receptors exclusively activated by designer drugs) refers to a receptor in which a mutation has occurred in a part of a wild-type muscarinic receptor, and in the present invention, chemical genetics refers to a technology using Gq-DREADD (hM3Dq) among DREADDs that promotes the activity of nerve cells.

[0052] In the present invention, the Gq-DREADD used for realizing chemical genetics can be provided in the form of a genome that can be expressed as a protein in a cell, and the genome means an oligonucleotide composed of DNA and / or RNA encoding Gq-DREADD or a vector containing the oligonucleotide, and the genome can be provided loaded onto a gene delivery system.

[0053] In the present invention, a "vector" refers to a genetic construct that is an expression vector capable of expressing a target gene in a suitable host cell and includes a regulatory element operably linked to express a gene insert contained in the vector.

[0054] In the present invention, "operably linked" refers to a functional linkage between a nucleic acid expression regulatory sequence performing a general function and a nucleic acid sequence encoding a target gene. In the vector of the present invention, each component is considered to be operably linked to one another unless explicitly stated to be directly linked.

[0055] For example, when a Gq-DREADD coding sequence is operably linked to a promoter, the expression of the Gq-DREADD coding sequence is under the influence or control of the promoter. Two nucleic acid sequences (the Gq-DREADD coding sequence and the promoter region sequence at the 5' end of the sequence) are operably linked when the promoter action is induced to transcribe the Gq-DREADD coding sequence, and the linkage characteristics between the two sequences do not induce a frameshift mutation, and the expression control sequence does not inhibit the expression of the Gq-DREADD. The operably linked sequence with the recombinant vector can be prepared using a genetic recombination technique well known in the art, and the site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art.

[0056] The term "promoter" as used herein refers to a portion of DNA involved in the binding of RNA polymerase to initiate transcription. It is typically located adjacent to and upstream of a target gene, and is a site where RNA polymerase or a transcription factor, a protein that induces RNA polymerase, binds, thereby directing the enzyme or protein to the correct transcription start site. Specifically, it is located at the 5' end of the gene to be transcribed on the sense strand, and induces RNA polymerase to bind to that site directly or through a transcription factor, thereby initiating mRNA synthesis for the target gene. It has a specific genetic sequence.

[0057] The vector according to the present invention comprises, in addition to expression control elements such as a promoter, operator, initiation codon, termination codon, polyadenylation signal, and enhancer, a signal sequence for membrane targeting, and can be manufactured in various ways depending on the purpose. In addition, the vector of the present invention may further comprise a regulatory element capable of increasing the expression level of Gq-DREADD in a cell. The promoter of the vector may be constitutive or inducible. In addition, the expression vector may comprise a selectable marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, may comprise an origin of replication. The vector may be self-replicating or integrated into the host DNA.

[0058] The vector according to the present invention may be a plasmid vector, a cosmid vector, a viral vector, etc., and preferably may be a viral vector. The viral vector may be a vector derived from a retrovirus, for example, human immunodeficiency virus (HIV), murine leukemia virus (MLV), avian sarcoma / leucosis virus (ASLV), spleen necrosis virus (SNV), Rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), but is not limited thereto. The expression vector according to the present invention may most preferably be a retroviral vector.

[0059] In the present invention, the term "gene delivery system" refers to a system capable of increasing expression efficiency by increasing the delivery efficiency of a desired gene and / or nucleic acid sequence into a cell, and can be classified into a viral-mediated system and a non-viral system.

[0060] Viral vector systems utilize viral vectors, such as retroviral vectors and adenoviral vectors, and are known to have relatively higher intracellular gene transfer efficiency than non-viral systems because they utilize the virus's unique intracellular penetration mechanism that causes infection in human cells. Furthermore, non-viral vectors have the problem of genes being degraded in endolysosomes after fusion of endosomes with lysosomes after entering the cell. However, viral vectors have the advantage of high gene transfer efficiency due to low gene loss due to a mechanism that delivers genes into the nucleus without passing through lysosomes.

[0061] The viral vector that can be used in the present invention may be a vector derived from a retrovirus, adenovirus, adeno-associated virus, etc., as described above. Such a viral vector can be assembled into a viral particle and then introduced into a cell through a transduction method such as infection.

[0062] In the present invention, “prevention” means any act of suppressing or delaying the onset of a target disease by administering a pharmaceutical composition according to the present invention or applying a kit containing the composition.

[0063] In the present invention, “treatment” means any act of improving a target disease or beneficially changing its symptoms by administering a pharmaceutical composition according to the present invention or applying a kit containing the composition.

[0064] In the present invention, “target disease” means a nervous system disease, neuropathic pain, and / or atopic dermatitis.

[0065] In the present invention, "neurological disease" includes central nervous system disease and peripheral nervous system disease, and means a disease that has as a symptom a decrease in motor ability and / or cognitive ability of an individual. Non-limiting examples of the neurological disease include Stiff-person syndrome, dystrophy not caused by nutritional deficiency, myotubular myopathy, Guillain-Barre syndrome, nemaline myopathy, multiple sclerosis, multiminicore disease, Huntington's disease, Hallervorden-Spatz Syndrome, Parkinson's disease, Alzheimer's disease, dementia, Lou Gehrig's disease, etc.

[0066] The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent. Examples of pharmaceutically acceptable carriers, excipients and diluents that can be used in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, calcium carbonate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil and the like.

[0067] The pharmaceutical composition of the present invention should be administered parenterally according to the intended method, and may be administered as an injection into an acupoint.

[0068] When the pharmaceutical composition of the present invention is administered as an injection into an acupoint, there is no limitation on the depth of injection. However, in order to obtain the same results regardless of the operator, to alleviate pain in the operator, and to reduce the possibility of side effects in the operator, it is sufficient if the pharmaceutical composition can reach the peripheral nerves of the acupoint area.

[0069] The injection according to the present invention may be dispersed in a sterile medium so that it can be administered directly to a patient, or it may be dispersed in an appropriate concentration by adding distilled water for injection and then administered. Furthermore, when manufactured as an injection, it may be mixed with buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., and may be manufactured in a unit dosage ampoule or multiple dosage form.

[0070] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition and weight, the severity of the disease, the drug form, the route of administration, and the time of administration, but can be appropriately selected by those skilled in the art. Meanwhile, the pharmaceutical composition of the present invention is not used alone, but is administered in combination with an agent capable of activating Gq-DREADD. In this case, it is preferable that the pharmaceutical composition of the present invention be administered prior to the administration of the agent capable of activating Gq-DREADD.

[0071] In this specification, the agent capable of activating Gq-DREADD refers to a ligand of Gq-DREADD, and is not limited to any agent that binds to Gq-DREADD and activates signal transduction, but may be at least one selected from the group consisting of CNO (clozapine-N-oxide), perlapine, and compound 21.

[0072] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0073]

[0074] [Example]

[0075] Example 1. Production of retro-adeno-associated virus for hM3Dq expression

[0076] To chemically and genetically activate the neural circuit connecting acupoints to the brain, we constructed a retrovirus that infects nerve terminals, axons, and neuronal cell bodies. This retrovirus was constructed by inserting the nucleotide sequence of the hM3Dq gene, a receptor activated by CNO binding, into a retrograde adeno-associated virus (AAVretro) vector.

[0077]

[0078] Example 2. Confirmation of neuronal activation in the brain following chemical and genetic stimulation of acupoints.

[0079] We aimed to determine whether melanocyte-concentrating hormone-secreting neurons in the lateral hypothalamus of the central nervous system would be activated when a virus expressing hM3Dq was injected into a peripheral acupoint instead of directly into the brain and CNO was injected into the same acupoint to chemically and genetically stimulate peripheral neurons connected to the acupoint. To this end, Ai14 transgenic mice expressing the tdTomato fluorescent protein in a Cre-dependent manner were anesthetized with 2% isoflurane with their heads fixed in a stereotaxic apparatus. 1.5 μL of an adeno-associated virus (AAV-pMCH-Cre, 5.7x1013 GC / mL) designed to express Cre protein under the pMCH promoter in the lateral hypothalamus was injected. Two weeks later, using the same method as described above, a retro-adeno-associated virus (AAVretro-hSynapsin-hM3Dq-mCherry, 3.53 x 1013 GC / mL, 1.5 μL) expressing hM3Dq under the human synapsin promoter, a pan-neuronal targeting promoter, was injected into the subcutaneous fat layer (1 mm), superficial muscle layer (1.5 mm), and muscular layer (3 mm) of the Yanglingcheon using a stereotaxic apparatus. Two weeks after the virus injection, chemogenetic stimulation was performed by intravenous injection of CNO (1 mg / kg, 50 μL) into the same acupoint at 1.5 mm from the surface. One hour and 30 minutes after the CNO injection, the animals were fixed by cardiac perfusion with a 4% solution of saline and paraformaldehyde, and the brain tissues were obtained and cryosectioned at 30 μm. The brain sections were stained with c-Fos primary antibody (sc-253, Santa Cruz Biotechnology, 1:500) and a fluorescent secondary antibody conjugated to it. The intensity of c-Fos fluorescence was quantitatively measured in melanocyte-secreting neurons expressing tdTomato fluorescence using a confocal fluorescence microscope.As a result, it was confirmed that chemical genetic stimulation applied to acupoints increased the activity of lateral hypothalamic neurons in the brain compared to the control group (Fig. 1).

[0080]

[0081] Example 3. Animal experiment to induce Parkinson's disease

[0082] This study aimed to determine whether chemical and genetic stimulation of acupoints could be used to treat Parkinson's disease. To this end, C57BL / 6 mice were injected with a virus expressing hM3Dq at an acupoint (GB34) 14 days before inducing Parkinson's disease, and then MPTP (1-methyl4-phenyl-1,2,3,6-tetrahydro-pyridine, 30 mg / kg) was administered intraperitoneally once daily for 5 days to induce Parkinson's disease and left in place for 7 days. The experimental animals were divided into a CNO administration group, which received 1 mg / kg of CNO once daily at the Yanglingcheon (GB34) point from the first day of Parkinson's disease induction until the end of the experiment, and a saline administration group, which received an equal amount of saline locally injected at the same site. From the 10th day of induction of Parkinson's disease, motor and cognitive behavioral tests such as Rotarod test, Cylinder test, Y-maze test, and NOR test were performed for 3 days (A in Figure 2). After the experiment, the mice were sacrificed to obtain substantia nigra pars compacta (SNpc) tissue and striatum tissue, and immunohistochemical staining was performed to identify dopamine neurons.

[0083]

[0084] 3-1. Motor behavior test

[0085] (1) Rotarod test

[0086] After acclimation at a fixed speed of 12 rpm using a rotarod treadmill, the speed was accelerated from 4 rpm to 40 rpm for 5 minutes, and the time it took for the mice to fall from the rod was measured.

[0087] As a result, compared to normal mice (Na_ve), Parkinson's disease-induced mice fell from the rod significantly faster, but it was confirmed that Parkinson's disease-induced mice subjected to chemical-genetic stimulation at acupoints had an increased time spent on the rod similar to that of normal mice (B in Fig. 2).

[0088] (2) Cylinder test

[0089] The mice were placed in a cylinder, raised, and the number of times they stood with their front paws against the wall for 5 minutes was measured.

[0090] As a result, compared to normal mice, the number of movements performed by Parkinson's disease-induced mice was significantly reduced, but it was confirmed that the Parkinson's disease-induced mice that received chemical genetic stimulation to acupoints showed an increase in standing behavior (C in Figure 2).

[0091]

[0092] 3-2. Cognitive Behavioral Test

[0093] (1) Y-maze test

[0094] Mice were placed in the center of a Y-shaped enclosed maze with three branches arranged at a constant 120° angle and allowed to move freely. The mice were observed through a camera installed on the maze ceiling, and a computer program measured the number of times and the order in which they entered each branch to assess the spontaneous alteration rate (%). The three branches of the maze were designated as areas A, B, and C, respectively, and an alteration was awarded 1 point if the mouse entered three different branches sequentially (in the order ABC, BCA, CAB); otherwise, no point was awarded.

[0095] As a result, compared to normal mice, the mice induced with Parkinson's disease had a lower change rate, but the mice induced with Parkinson's disease to which chemical genetic stimulation was applied to acupoints showed a change rate that increased close to normal, indicating that stimulation applied to acupoints improved the short-term memory of animals induced with Parkinson's disease (D in Figure 2).

[0096] (2) NOR test (Novel Object Recognition Test)

[0097] The experiment was conducted in a box measuring 40Х40Х30 cm. On the first day, the mice were allowed to move freely in the box for 5 minutes, and on the second day, two identical objects were placed 5 cm apart from the walls of the box, and the mice were placed in the center of the box and allowed to explore the objects. On the third day, one of the objects was replaced with a novel object, and the mice were placed in the center of the box again. The time for which the mice exhibited exploratory behavior toward the existing object and the novel object was measured, and the ratio of the exploration time for the novel object to the exploration time for the existing object was calculated.

[0098] As a result, the time for exploring a novel object and an existing object was almost the same in Parkinson's disease-induced mice, but the time for exploring a novel object was longer in Parkinson's disease-induced mice that were chemically and genetically stimulated at acupoints, indicating that stimulation applied to acupoints improved the learning and memory ability of Parkinson's disease-induced animals (Fig. 2E).

[0099]

[0100] 3-3. Immunohistochemical analysis

[0101] After the experiment, mice were sacrificed, and the striatum and substantia nigra brain tissues were selected, washed with PBS, reacted with Tyrosine Hydroxylase (TH) antibody, and then developed using DAB. TH-positive cells were counted in the substantia nigra tissues, and optical density (OD) was measured in the striatum tissues.

[0102] As a result, it was observed that the number of dopamine neurons and the expression of nerve fibers in the substantia nigra and striatum, respectively, were significantly reduced in Parkinson's disease-induced mice, but that chemical genetic acupuncture stimulation at acupoints restored this (Fig. 3).

[0103]

[0104] Example 4. Animal experiment on partial sciatic nerve ligation (PSNL)

[0105] This study aimed to determine whether chemical-genetic stimulation of acupoints could reduce neuropathic pain. To this end, a unilateral sciatic nerve ligation (PSNL) model was created, in which the sciatic nerve was exposed at the upper thigh and ligated with 8-0 silk sutures one-third or one-half of the nerve distal to the point where the posterior semitendinosus nerve branches off from the sciatic nerve.

[0106] The experimental animals were divided into two groups: a CNO group, which received local injections of 1 mg / kg of CNO once a day from the 7th day after surgery to the end of the experiment, at the points of Zusanli (ST36), Yanglingchuan (GB34), and Hwando (GB30) on both sides; and a saline group, which received local injections of the same amount of saline at the same sites. The Von Frey test was performed to evaluate pain before surgery (Base), and on days 0, 4, 9, 14, 21, and 28 after CNO administration. The Open Field test was performed on the 27th day of CNO administration to evaluate anxiety symptoms.

[0107] As a result, as shown in Fig. 4, the pain in the neuropathic pain-induced mice increased compared to the control group, but the pain in the neuropathic pain-induced mice that received chemical-genetic stimulation to the acupoints was confirmed to decrease (Fig. 4A).

[0108] In addition, when the Open field test was performed to evaluate anxiety symptoms, it was found that the anxiety symptoms increased in the neuropathic pain-induced mice, but the anxiety symptoms decreased in the neuropathic pain-induced mice that were chemically and genetically stimulated at the acupoints (B in Figure 4).

[0109]

[0110] Example 5. Atopic dermatitis-induced animal experiment

[0111] We sought to determine whether chemical and genetic stimulation of acupoints could treat atopic dermatitis. To this end, atopic dermatitis was induced by applying MC903 to the cheeks of mice after removing hair.

[0112] The experimental animals were divided into two groups: a CNO group, which received local injections of 1 mg / kg of CNO once daily into the calf (LI11) on both sides from day 0 of atopic dermatitis induction until the end of the experiment, and a saline group, which received local injections of the same amount of saline into the same location. The severity of erythema, xerosis, and excoriation in the atopic dermatitis-induced areas of the mice was measured to determine the dermatitis severity score.

[0113] As a result, as shown in Figures 5a and 5b, compared to normal mice, atopic dermatitis-induced mice showed an increased severity score, but atopic dermatitis-induced mice subjected to chemical-genetic stimulation at acupoints showed a decreased dermatitis severity score (Figure 5).

[0114] Next, a behavioral assessment (Open Field Test) was performed on atopic dermatitis-induced mice to determine their motor abilities and anxiety recovery. As shown in Figure 6, compared to normal mice, atopic dermatitis-induced mice exhibited decreased motor abilities and increased anxiety symptoms. However, atopic dermatitis-induced mice subjected to chemical and genetic stimulation at acupoints exhibited improved motor abilities and anxiety symptoms (Figure 6).

[0115]

[0116] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0117] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims described below.

[0118] The present invention provides a highly reproducible acupuncture treatment simulation technology, which can be used to treat various neurological diseases and neuropathic pain and to improve atopic symptoms.

Claims

1. A composition for simulating acupuncture treatment, comprising a vector expressing Gq-DREADD (hM3Dq) as an active ingredient. A composition characterized in that the composition is administered to an acupoint.

2. In paragraph 1, A composition wherein the above vector is a viral vector.

3. In paragraph 2, A composition wherein the above viral vector is a retroviral vector.

4. In paragraph 1, The composition is administered in combination with a ligand of Gq-DREADD (hM3Dq).

5. In paragraph 4, A composition wherein the composition is administered in advance before administration of the ligand.

6. In paragraph 4, A composition wherein the ligand is at least one selected from the group consisting of CNO (clozapine-N-oxide), perlapine, and compound 21.

7. A kit for simulating acupuncture treatment, comprising the composition of paragraph 1 and a ligand of Gq-DREADD (hM3Dq) as an active ingredient.

8. In paragraph 7, A kit wherein the above ligand is at least one selected from the group consisting of CNO (clozapine-N-oxide), perlapine, and compound 21.

9. In paragraph 7, A kit wherein the above ligand is administered to an acupoint.

10. A pharmaceutical composition for treating a nervous system disease, comprising the composition of paragraph 1, The above composition is characterized in that it is administered to acupoints, A pharmaceutical composition wherein the acupuncture points are at least one selected from the group consisting of Gihae, Naegwan, Daechu, Baekhoe, Bulyu, Samyingyo, Susamri, Sinmun, Sinjeong, Yanglingcheon, Yangbo, Woosang, Wijung, Joksamri, Chungyang, Pungryung, Taegye, Taechung, and Hegok.

11. A pharmaceutical composition for treating neuropathic pain comprising the composition of paragraph 1, The above composition is characterized in that it is administered to acupoints, A pharmaceutical composition, wherein the acupuncture points are at least one selected from the group consisting of Gansu, Gyeoksu, Gyeonwoo, Gokji, Kunlun, Gongsun, Gwanwon, Gihae, Naegwan, Naejeong, Daechu, Bokto, Bigwan, Bisu, Biyang, Samyingyo, Susamri, Shindo, Shinmaek, Shinsu, Shinju, Yanggu, Yanglingcheon, Yeolgyeol, Oesang, Johae, Joksamri, Jokineup, Joktonggok, Jiyang, Cheonchu, Pungryung, Taegye, Taebaek, Taechung, Pyesu, Hamgok, Heopgok, Hwando, Hyeolhae, and Hugye.

12. A pharmaceutical composition for treating atopic dermatitis comprising the composition of paragraph 1, The above composition is administered to acupoints, A pharmaceutical composition, wherein the above acupoints are at least one selected from the group consisting of Gansu, Gyeoksu, Gokji, Myeongmun, Bisu, Simsu, Samyingyo, Yeogu, Okye, Eumryeongcheon, Joksamri, Jiyang, Jieum, Cheonchu, Feisu, Pungsi, Hegok, and Hyeolhae.

13. A chemical genetic acupuncture treatment method comprising the steps: (1) a step of administering a vector for Gq-DREADD expression to an acupoint of an individual; and (2) A step of administering a ligand of Gq-DREADD to the acupoint of the above object.

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