Screening method for drugs for treating COVID-19 sequelae and manufacturing method for COVID-19 sequelae model animals

Abstract

To provide a new coronavirus infectious disease aftereffect therapeutic agent.SOLUTION: A new coronavirus infectious disease aftereffect therapeutic agent screening method includes: an administration step for administering a test substance to a new coronavirus infectious disease aftereffect model animal which is a non-human mammal expressing a SARS-CoV-2 S1 protein; and an evaluation step for assessing changes in symptoms related to the new coronavirus in the model animal before and after administration of the test substance.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a therapeutic agent for the sequelae of COVID-19 infection, a method for screening a therapeutic agent for the sequelae of COVID-19 infection, and a method for producing a model animal for the sequelae of COVID-19 infection. [Background technology]

[0002] COVID-19 (also known as novel coronavirus disease), an acute respiratory disease caused by the SARS-CoV-2 virus, is a severe infectious disease that has infected many people worldwide.

[0003] COVID-19 is known to cause severe illness during the acute phase, leading to death in patients, and frequently causes sequelae during the recovery phase (hereinafter referred to as "COVID-19 sequelae"). Among the sequelae of COVID-19, fatigue, depression, and olfactory impairment are said to be particularly common. Furthermore, because COVID-19 sequelae can occur in patients whose acute symptoms are not particularly severe, they are likely to occur even if a vaccine is used to prevent the disease from becoming severe.

[0004] As the name suggests, the aftereffects of COVID-19 are the aftereffects of a viral infection, and therefore the causative virus, SARS-CoV-2, is not replicating when the aftereffects occur. Therefore, even if drugs capable of suppressing viral replication are administered after the onset of COVID-19 aftereffects, they are not expected to be effective against the aftereffects. For this reason, it is thought that therapeutic drugs for the aftereffects of COVID-19 must repair the tissue damage caused by SARS-CoV-2 virus infection.

[0005] There are currently no effective treatments for the aftereffects of COVID-19, which has led to the proliferation of unsubstantiated folk remedies, which are causing health problems. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Do vaccines protect against long COVID? What the data say. Heidi Ledford. Nature. 2021 Nov;599(7886):546-548. [Non-patent document 2] Promotion of non-evidence-based therapeutics within patient-led Long COVID support groups. Vicky van der Togt et.al. Nat Med. 2021 Dec;27(12):2068-2069. [Non-patent document 3] The lesson of ivermectin: meta-analyses based on summary data alone are inherently unreliable. Jack M Lawrence et.al. Nat Med. 2021 Nov;27(11):1853-1854. Summary of the Invention [Problem to be solved by the invention]

[0007] There is a need for treatments for the aftereffects of COVID-19. However, there are currently no effective treatments. In order to conduct research on the aftereffects of COVID-19 for the development of treatments, animal models that exhibit the symptoms of COVID-19 aftereffects are important. However, animal models related to COVID-19 to date have focused on research into infection prevention and acute symptoms, and therefore the development of animal models suitable for research into aftereffects has been insufficient.

[0008] An object of one embodiment of the present invention is to provide a therapeutic drug for the sequelae of novel coronavirus infection. [Means for solving the problem]

[0009] As a result of intensive research to solve the above problems, the inventors have discovered for the first time that the S1 region of the Spike protein of the SARS-CoV-2 virus is the protein responsible for the aftereffects of COVID-19, and have completed an animal model that exhibits symptoms of COVID-19 sequelae. As a result of research on the aftereffects of COVID-19 using an animal model of COVID-19 sequelae, the inventors have discovered for the first time that acetylcholine levels in the brain are reduced in the aftereffects of COVID-19, and that the aftereffects of COVID-19 can be treated or prevented with a receptor agonist.

[0010] That is, one aspect of the present invention is a therapeutic drug for treating sequelae of novel coronavirus infection, which contains an acetylcholine receptor agonist as an active ingredient.

[0011] Another aspect of the present invention is a method for treating COVID-19 sequelae, comprising: administering a test substance to a model animal in which SARS-CoV-2 S1 protein is expressed in a non-human mammal; and an evaluation step of evaluating changes in symptoms associated with the novel coronavirus in the model animal before and after administration of the test substance.

[0012] Another aspect of the present invention is a method for producing a model animal suffering from the sequelae of COVID-19, which includes an expression step of expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector. [Effects of the Invention]

[0013] According to one aspect of the present invention, a therapeutic agent for the aftereffects of COVID-19 can be provided. [Brief explanation of the drawings]

[0014] [Figure 1]Schematic diagram showing the structure of the SARS-CoV-2 Spike protein. [Figure 2] FIG. 1 shows the results of an example, measuring the intracellular calcium concentration in 3T3 cells and A549 cells transformed with an S1 protein expression plasmid or a control plasmid. [Figure 3] FIG. 1 shows the results of an example, measuring the intracellular calcium concentration in 3T3 cells and A549 cells infected with an S1 protein-expressing adenovirus or a control adenovirus. [Figure 4] FIG. 1 shows the results of an example, and is a diagram showing the results of a 10% weighted forced swimming test in control mice and S1 protein-expressing mice (S1 mice). [Figure 5] FIG. 1 shows the results of an example and is a diagram showing the results of a tail suspension test in control mice and S1 mice. [Figure 6] FIG. 1 shows the results of an example, and is a diagram showing the results of analyzing the expression levels of calbindin gene in the olfactory bulb of control mice and S1 mice after administration of LPS. [Figure 7] FIG. 1 shows the results of an example, and is a diagram showing the results of analyzing the expression levels of calbindin gene in the brains of control mice and S1 mice after administration of LPS. [Figure 8] FIG. 1 shows the results of an example and is a diagram showing damage to cholinergic neurons in control mice and S1 mice. [Figure 9] FIG. 1 shows the results of an example and is a diagram showing damage to cholinergic neurons in control mice and S1 mice. [Figure 10] FIG. 1 is a diagram illustrating an administration scheme of donepezil. [Figure 11] FIG. 1 shows the results of an example, and is a diagram showing the results of a 10% weighted forced swimming test after administration of donepezil to control mice and S1 mice. [Figure 12] FIG. 1 shows the results of an example and is a diagram showing the results of a tail suspension test after administration of donepezil to control mice and S1 mice. [Figure 13]FIG. 1 shows the results of an example, and is a diagram showing the results of analyzing the gene expression levels of interleukin 6 (IL-6), tumor necrosis factor (TNFα), and chemokine CC motif ligand 2 (CCL2) in control mice and S1 mice after administration of donepezil. [Figure 14] FIG. 1 shows the results of an example, and is a diagram showing the results of analyzing the gene expression levels of interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) in control mice and S1 mice after administration of PNU282987. [Figure 15] FIG. 1 shows the results of an example, and is a diagram showing the results of analyzing the expression levels of the ZFP36 gene in control mice and S1 mice after administration of PNU282987. DETAILED DESCRIPTION OF THE INVENTION

[0015] <1. Therapeutic drug> (Features) A therapeutic agent for COVID-19 sequelae (hereinafter, "COVID-19 sequelae") according to one embodiment of the present invention is a pharmaceutical composition used for treating or preventing COVID-19 sequelae, and contains an acetylcholine receptor agonist as an active ingredient. This allows for the treatment or prevention of COVID-19 sequelae in patients infected with the novel coronavirus. This can contribute to achieving Goal 3 of the Sustainable Development Goals (SDGs), "Ensure good health and well-being for all." For ease of explanation, the therapeutic agent for COVID-19 sequelae according to one embodiment of the present invention may be referred to simply as the "therapeutic agent."

[0016] As used herein, "treatment" includes curing or alleviating the symptoms of COVID-19 sequelae and suppressing the worsening of the symptoms of COVID-19 sequelae in a subject to whom a therapeutic agent according to one embodiment of the present invention is administered (hereinafter simply referred to as "subject to administration"). As used herein, "prevention" includes suppressing or delaying the onset of symptoms of COVID-19 sequelae in a subject to administration.

[0017] When a subject is experiencing symptoms of COVID-19 sequelae, a therapeutic agent according to one embodiment of the present invention is used to treat the COVID-19 sequelae.When a subject is not experiencing symptoms of COVID-19 sequelae, a therapeutic agent according to one embodiment of the present invention is used to prevent the COVID-19 sequelae.

[0018] In this specification, the definitions of "treatment" and "prevention" are as described above, but in both cases, the mechanism of action of a therapeutic agent according to one embodiment of the present invention is the same. Therefore, the term "therapeutic agent" can be substituted for a "prophylactic agent." In other words, when a patient infected with the novel coronavirus does not show symptoms of COVID-19 sequelae, "treatment" means "prevention."

[0019] Here, COVID-19 sequelae generally refers to a range of symptoms that appear during the recovery phase after infection with the novel coronavirus and persist for weeks to months thereafter, but the definition is unclear. COVID-19 sequelae are also sometimes referred to as Long COVID. In this specification, "COVID-19 sequelae" refers to symptoms related to brain and nerve dysfunction, such as fatigue, depression, loss of smell, memory loss, impaired concentration, impaired thinking ability, and impaired cognitive function, which are particularly common among the symptoms listed above.

[0020] One aspect of the "COVID-19 sequelae" herein is fatigue associated with the novel coronavirus. Another aspect of the "COVID-19 sequelae" herein is depressive symptoms associated with the novel coronavirus. Another aspect of the "COVID-19 sequelae" herein is olfactory dysfunction associated with the novel coronavirus. Another aspect of the "COVID-19 sequelae" herein is memory impairment associated with the novel coronavirus.

[0021] As used herein, "COVID-19-associated depressive symptoms" refers to symptoms of illnesses commonly diagnosed as depression. Symptoms include depressed mood, loss of interest and pleasure, loss of appetite, overeating, sleep disorders, hypersomnia, psychomotor agitation or retardation, fatigue, feelings of worthlessness or guilt, impaired concentration and thinking, and suicidal ideation, all of which are used to diagnose major depression according to the DSM-5. In addition, symptoms frequently seen in patients with depression, such as anxiety, memory loss, aging, pain, and chronic pain, are also considered. In other words, "COVID-19-associated depressive symptoms" as used herein are not limited to major depression, but also encompass symptoms of the above-mentioned depressive symptoms, such as stress-related depression, depressive states associated with bipolar disorder, and negative depression, as well as depressive states associated with other illnesses.

[0022] In this specification, "COVID-19-related fatigue" refers to a persistent or chronic phenomenon associated with diseases of the central nervous system or other organs, characterized by pathological fatigue characterized by loss of interest and pleasure, sleep disturbances, psychomotor agitation or retardation, easy fatigability, and decreased concentration and thinking ability as its main symptoms. "Fatigue" can also be described as a feeling of tiredness or malaise.

[0023] In this specification, "pathological fatigue" refers to persistent or chronic fatigue associated with COVID-19. It is distinct from "physiological fatigue," which refers to a temporary qualitative or quantitative decline in physical and mental work capacity observed in healthy individuals when they are subjected to continuous physical or mental stress.

[0024] In this specification, "novel coronavirus-associated olfactory dysfunction" refers to a condition or disease that causes some abnormality in the sense of smell, or "smell." It is also called "dysosmia." The main symptoms include hyposmia, olfactory agnosia, anosmia, and hyperosmia.

[0025] In this specification, "memory disorder associated with the new coronavirus" refers to a condition in which any or all of the four processes that make up memory - encoding, retention, recall, and recognition - do not function normally.

[0026] In this specification, the term "symptoms" refers to phenomena and conditions (abnormalities) that occur in patients due to the effects of a disease involving the novel coronavirus, and is a concept that includes subjective symptoms, which are abnormalities that the patient himself or herself perceives as symptoms of the disease, and objective symptoms, which are abnormalities that can be objectively confirmed as symptoms of the disease through a doctor's examination or examination, etc. In the animal models described below, behavioral changes and objective findings obtained through examination, etc. are collectively referred to as symptoms.

[0027] The present inventors studied brain changes associated with COVID-19 sequelae using an animal model of COVID-19 sequelae, and found for the first time that the number of cholinergic neurons, which are choline acetyltransferase (ChAT)-positive cells, in the medial septal area (MS) and diagonal band of Broca (DB) of the basal forebrain was reduced in the brains of patients with COVID-19 sequelae.

[0028] These results suggest that the amount of acetylcholine is reduced in the aftereffects of COVID-19, and suggest that acetylcholine receptor agonists may be suitable as treatments for the aftereffects of COVID-19.

[0029] As a result of research into COVID-19 sequelae using COVID-19 sequelae animal model, the present inventors have found for the first time that administration of an acetylcholine receptor agonist improves the symptoms of depression and fatigue exhibited in COVID-19 sequelae animal model. In other words, they have found for the first time that administration of an acetylcholine receptor agonist can treat or prevent COVID-19 sequelae.

[0030] It has traditionally been common knowledge that increasing the amount of acetylcholine in the brain through the administration of acetylcholine receptor agonists leads to the onset of depressive symptoms (e.g., Reference 1: Cholinergic regulation of mood: from basic and clinical studies to emerging therapeutics. Stephanie C Dulawa and David S Janowsky. Mol Psychiatry. 2019 May;24(5):694-709. and Reference 2: Maintenance treatment of depression in old age: a randomized, double-blind, placebo-controlled evaluation of the efficacy and safety of donepezil combined with antidepressant pharmacotherapy. Charles F Reynolds 3rd, Meryl A Butters, Oscar Lopez. et.al. Arch Gen Psychiatry. 2011;68(1):51-60.).

[0031] It was also common knowledge that smoking worsens and progresses the symptoms of COVID-19, and that nicotine is the cause of this (e.g., Reference 3: Tobacco smoking and COVID-19 infection. Richard N van Zyl-Smit, Guy Richards, Frank T Leone. Lancet Respir Med. 2020 Jul;8(7):664-665.; Reference 4: Smoking Is Associated With COVID-19 Progression: A Meta-analysis. Roengrudee Patanavanich, Stanton A Glantz. Nicotine Tob Res. 2020 Aug 24;22(9):1653-1656.; Reference 5: The Role of Smoking and Nicotine in the Transmission and Pathogenesis of COVID-19. Ali Ehsan Sifat, Saeideh Nozohouri, Heidi Villalba, Bhuvaneshwar Vaidya, Thomas J Abbruscato J Pharmacol Exp Ther. 2020 Dec;375(3):498-509.;Reference 6: The Effect of Smoking on COVID-19 Symptom Severity: Systematic Review and Meta-Analysis Askin Gulsen 1, Burcu Arpinar Yigitbas 2, Berat Uslu 2, Daniel Dromann 1, Oguz Kilinc Pulm Med. 2020 Sep 8;2020:7590207.).

[0032] In contrast, the finding that increasing the amount of acetylcholine in the brain by administering an acetylcholine receptor agonist can improve the novel coronavirus-related depressive symptoms manifested in COVID-19 sequelae animal model is completely opposite to the results expected from conventional technical knowledge as the effects of acetylcholine receptor agonists. Furthermore, this result suggests that depressive symptoms due to the sequelae of COVID-19 (i.e., "novel coronavirus-related depressive symptoms") develop through a different mechanism than depression caused by increased acetylcholine levels.

[0033] (active ingredient) A therapeutic agent according to one embodiment of the present invention contains an acetylcholine receptor agonist as an active ingredient. As used herein, the term "acetylcholine receptor agonist" refers to both indirect acetylcholine receptor agonists, which increase the amount of acetylcholine in the brain by, for example, inhibiting cholinesterase, and direct acetylcholine receptor agonists, which act by directly binding to the receptor. In one embodiment of the present invention, the acetylcholine receptor agonist may be a peripheral acetylcholine receptor agonist that acts via the parasympathetic nervous system, or a central acetylcholine receptor agonist that acts on receptors in the brain. As mentioned above, COVID-19 sequelae are symptoms related to brain and nerve dysfunction. Because of the advantage of being able to act directly on the site of dysfunction, the acetylcholine receptor agonist is preferably a central acetylcholine receptor agonist that acts on acetylcholine receptors in the brain. "Acetylcholine receptor agonists" are also referred to as cholinergic agonists.

[0034] More specifically, receptors in the brain on which central acetylcholine receptor agonists act include the olfactory bulb, hippocampus, septal area, and / or olfactory tubercle, but it is known that there are many sites on which acetylcholine receptor agonists act, and they are not limited to those exemplified here.

[0035] The direct acetylcholine receptor agonist is preferably a substance that can reach the brain by crossing the blood-brain barrier and has the effect of activating acetylcholine receptors. More specifically, direct acetylcholine receptor agonists include acetylcholine and its precursors, and agonists of acetylcholine receptors (muscarinic receptors or nicotinic receptors).

[0036] The indirect acetylcholine receptor agonist is preferably a substance that can cross the blood-brain barrier to reach the brain and has an acetylcholinesterase inhibitory effect. More specifically, the indirect acetylcholine receptor agonist includes donepezil (2-[(1-benzyl-4-piperidinyl)methyl]-5,6-dimethoxyindan-1-one hydrochloride); rivastigmine (2,6-dioxo-4-phenyl-piperidine-3-carbonitrile); metrifonate (O,O-dimethyl-2,2,2-trichloro-1-hydroxyethylphosphonate ester); tacrine (1,2,3,4-tetrahydro-9-aminoacridine); galantamine (galantamine hydrobromide), etc.

[0037] Furthermore, among the exemplified acetylcholine receptor agonists, donepezil is already used as a therapeutic drug for Alzheimer's disease, etc., and therefore drug repositioning is possible. Therefore, it has advantages such as (i) human safety tests and pharmacokinetic tests can be shortened or omitted, (ii) side effects are known, so an active ingredient that places less burden on patients can be selected, and (iii) a manufacturing method for the active ingredient has already been established, so the drug price can be kept low. Therefore, in one aspect of the present invention, the acetylcholine receptor agonist is preferably donepezil.

[0038] As long as the acetylcholine receptor agonist has the effect of increasing the amount of acetylcholine in the brain, it may be in the form of a "derivative" or a "pharmacologically acceptable salt." That is, in this specification, the term "acetylcholine receptor agonist" is a concept that includes both its "derivative" and its "pharmacologically acceptable salt."

[0039] As used herein, the term "derivative" refers to a group of compounds resulting from the substitution of a portion of a molecule of a specific compound with another functional group or another atom. Examples of such other functional groups include alkyl groups, alkoxy groups, alkylthio groups, aryl groups, aryloxy groups, arylthio groups, arylalkyl groups, arylalkoxy groups, arylalkylthio groups, arylalkenyl groups, arylalkynyl groups, allyl groups, amino groups, substituted amino groups, silyl groups, substituted silyl groups, silyloxy groups, substituted silyloxy groups, arylsulfonyloxy groups, alkylsulfonyloxy groups, and nitro groups. Examples of such other atoms include carbon atoms, hydrogen atoms, oxygen atoms, nitrogen atoms, sulfur atoms, phosphorus atoms, and halogen atoms.

[0040] As the derivative, a prodrug that exhibits a desired activity by hydrolysis, oxidation, enzymatic reaction, or the like in a living body (in vivo conditions) can also be used.

[0041] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is physiologically acceptable for administration to a subject as a pharmaceutical, and specific examples thereof are not limited thereto. Examples of salts include alkali metal salts (potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), ammonium salts, organic base salts (trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, etc.), organic acid salts (acetate salt, maleate salt, tartrate salt, methanesulfonate salt, benzenesulfonate salt, formate salt, toluenesulfonate salt, trifluoroacetate salt, etc.), and inorganic acid salts (hydrochloride salt, hydrobromide salt, sulfate salt, phosphate salt, etc.).

[0042] In one aspect of the present invention, the acetylcholine receptor agonist may activate or inhibit a receptor other than an acetylcholine receptor, but from the viewpoint of suppressing unintended side effects and the like, it is preferable that the acetylcholine receptor agonist is a compound that selectively (specifically) activates an acetylcholine receptor.

[0043] (Other ingredients) The therapeutic agent according to one embodiment of the present invention may contain ingredients other than the active ingredient (acetylcholine receptor agonist). The ingredients other than the active ingredient may be any pharmaceutically acceptable ingredients, such as a buffering agent, a pH adjuster, an isotonicity agent, a preservative, an antioxidant, a high molecular weight polymer, an excipient, a solvent, etc.

[0044] Examples of the buffering agent include phosphoric acid or phosphoric acid salts, boric acid or boric acid salts, citric acid or citrate salts, acetic acid or acetate salts, carbonic acid or carbonate salts, tartaric acid or tartrate salts, ε-aminocaproic acid, trometamol, etc. Examples of the phosphate salts include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, etc. Examples of the borates include borax, sodium borate, potassium borate, etc. Examples of the citrate salts include sodium citrate, disodium citrate, trisodium citrate, etc. Examples of the acetate salts include sodium acetate, potassium acetate, etc. Examples of the carbonate salts include sodium carbonate, sodium bicarbonate, etc. Examples of the tartrate salts include sodium tartrate, potassium tartrate, etc.

[0045] Examples of the pH adjuster include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, and potassium hydroxide.

[0046] Examples of the isotonic agent include ionic isotonic agents (sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc.) and non-ionic isotonic agents (glycerin, propylene glycol, sorbitol, mannitol, etc.).

[0047] Examples of the preservatives include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.

[0048] Examples of the antioxidant include ascorbic acid, tocopherol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.

[0049] Examples of the high molecular weight polymer include methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, carboxymethyl ethyl cellulose, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, and atelocollagen.

[0050] Examples of the excipient include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose.

[0051] Examples of the solvent include water, physiological saline, alcohol, and the like.

[0052] A therapeutic agent according to one embodiment of the present invention may contain, as the other component described above, a medicinal ingredient having a desired effect (e.g., reduction of side effects), or may be used in combination with a drug having the desired effect.

[0053] (Content of active ingredients and other ingredients) The amount of the active ingredient in the therapeutic agent according to one embodiment of the present invention is not particularly limited. For example, the amount of the active ingredient may be 0.001% by weight to 100% by weight, 0.01% by weight to 100% by weight, 0.1% by weight to 100% by weight, 0.1% by weight to 95% by weight, 0.1% by weight to 90% by weight, 0.1% by weight to 80% by weight, 0.1% by weight to 70% by weight, 0.1% by weight to 60% by weight, 0.1% by weight to 50% by weight, 0.1% by weight to 40% by weight, 0.1% by weight to 30% by weight, 0.1% by weight to 20% by weight, or 0.1% by weight to 10% by weight, relative to the total weight of the therapeutic agent according to one embodiment of the present invention.

[0054] The amount of components other than the active ingredient in a therapeutic agent according to an embodiment of the present invention is not particularly limited. For example, the amount of components other than the active ingredient may be, relative to the total weight of the therapeutic agent according to an embodiment of the present invention, 0% to 99.999% by weight, 0% to 99.99% by weight, 0% to 99.9% by weight, 5% to 99.9% by weight, 10% to 99.9% by weight, 20% to 99.9% by weight, 30% to 99.9% by weight, 40% to 99.9% by weight, 50% to 99.9% by weight, 60% to 99.9% by weight, 70% to 99.9% by weight, 80% to 99.9% by weight, or 90% to 99.9% by weight.

[0055] (Target of administration) An example of a subject to which the therapeutic agent according to one embodiment of the present invention is administered is a subject suspected of or confirmed to be infected with the novel coronavirus, and such a subject may not have been diagnosed by a doctor with coronavirus sequelae.

[0056] The subject to which the therapeutic agent according to one embodiment of the present invention is administered is not particularly limited, and may be a human or a non-human mammal (e.g., livestock, pets, and laboratory animals). Examples of non-human mammals include monkeys, chimpanzees, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats.

[0057] (Route of administration) A therapeutic agent according to one aspect of the present invention may be administered to a subject via any administration route. Examples of administration routes include oral administration, parenteral administration, transdermal administration, transmucosal administration, and intravenous administration. Therefore, the dosage form of a therapeutic agent according to one aspect of the present invention may be an oral medication, an external medication, an injection, or the like. Because administration is simple and places less strain on the subject, oral administration is preferred as the administration route of a therapeutic agent according to one aspect of the present invention. Therefore, the dosage form of a therapeutic agent according to one aspect of the present invention is preferably an oral medication.

[0058] (Preparation and formulation) The therapeutic agent according to one aspect of the present invention can be formulated using the acetylcholine receptor agonist, which is the active ingredient, and other ingredients as raw materials by known methods.

[0059] When a therapeutic agent according to one embodiment of the present invention is administered to a subject, there is no limitation on the dosage to the subject as long as the desired effect is obtained. For example, a therapeutic agent according to one embodiment of the present invention may be administered so that the dosage of the acetylcholine receptor agonist, which is the active ingredient, is 0.1 mg to 1000.0 mg / kg body weight, 0.1 mg to 500.0 mg / kg body weight, 1.0 mg to 500.0 mg / kg body weight, 1.0 mg to 300.0 mg / kg body weight, 1.0 mg to 100.0 mg / kg body weight, 1.0 mg to 50.0 mg / kg body weight, 1.0 mg to 10.0 mg / kg body weight, 1.0 mg to 10.0 mg / kg body weight, or 1.0 to 5.0 mg / kg body weight.

[0060] When a therapeutic agent according to one embodiment of the present invention is administered to a subject, there is no limit to the interval at which it is administered to the subject, as long as the desired effect is achieved. The administration interval may be, for example, once every hour, once every 1 to 6 hours, once every 6 to 12 hours, once every 12 hours to 1 day, once every 1 to 3 days, once every 1 to 5 days, once every 1 to 7 days, once every 7 to 14 days, once every 14 to 21 days, once per month, once per 2 months, once per 3 months, once per 4 months, once per 5 months, once per 6 months, or once per year. The administration interval may be constant, or the agent may be administered whenever severe symptoms of COVID-19 sequelae appear (as needed).

[0061] The therapeutic agent according to one embodiment of the present invention may be administered, for example, once a day so that the dose of the acetylcholine receptor agonist, which is the active ingredient, is 1.0 to 5.0 mg / kg body weight.

[0062] 2. Drug screening method (Features) One embodiment of the present invention relates to a screening method for drugs for treating COVID-19 sequelae that uses a COVID-19 sequelae animal model in which SARS-CoV-2 S1 protein is expressed in a non-human mammal. The method screens for drugs effective in treating or preventing COVID-19 sequelae. The method includes an administration step of administering a test substance to the COVID-19 sequelae animal model and an evaluation step of assessing changes in novel coronavirus-related symptoms in the COVID-19 sequelae animal model before and after administration of the test substance. This allows for screening of drugs for treating COVID-19 sequelae. This will lead to the development of new drugs for treating COVID-19 sequelae. This will contribute to Goal 3 of the Sustainable Development Goals (SDGs), "Ensure good health and promote well-being for all." Hereinafter, for ease of explanation, the screening method for drugs for treating COVID-19 sequelae according to one embodiment of the present invention will sometimes be simply referred to as the "drug screening method."

[0063] (COVID-19 sequelae animal model) The COVID-19 sequelae model animal used in the screening method for drugs for treating COVID-19 sequelae according to one embodiment of the present invention is a non-human mammal in which SARS-CoV-2 S1 protein is expressed.

[0064] In COVID-19 sequelae animal models, the SARS-CoV-2 S1 protein may be expressed transiently or constitutively. Transgenic animals may also be used. Since symptoms can be closely examined when expression has ceased or attenuated, it is preferable that the expression of SARS-CoV-2 S1 protein is transient and that expression of SARS-CoV-2 S1 protein has ceased or attenuated in the COVID-19 sequelae animal models by the time of use in the administration step of this screening method.

[0065] Furthermore, since COVID-19 sequelae can be efficiently developed in the model animal, the COVID-19 sequelae animal is preferably a model animal in which the SARS-CoV-2 S1 protein is expressed in at least one of the nasal cavity and the peri-nasal cavity of a non-human mammal. In the COVID-19 sequelae animal model, inflammation may be induced before the step of administering the test substance.

[0066] The method for expressing the SARS-CoV-2 S1 protein in a non-human mammal is not particularly limited. For example, a COVID-19 sequelae model animal can be produced by expressing the SARS-CoV-2 S1 protein in a non-human mammal using the method for producing a COVID-19 sequelae model animal described below.

[0067] (Administration step) The administration step is a step of administering a test substance to a COVID-19 sequelae model animal. The dose and administration method of the test substance are not particularly limited and can be appropriately determined depending on the concentration of the active ingredient in the test substance, the dosage form of the test substance, etc.

[0068] (Evaluation process) The evaluation process is a process of evaluating changes in symptoms associated with the novel coronavirus in a COVID-19 sequelae model animal before and after administration of the test substance.

[0069] In the evaluation step, the method for evaluating changes in novel coronavirus-associated symptoms in the COVID-19 sequelae animal model is not particularly limited. For example, in the evaluation step, changes in novel coronavirus-associated symptoms in the COVID-19 sequelae animal model can be evaluated by quantifying changes in the activity level of the COVID-19 sequelae animal model.

[0070] As a method for quantifying changes in activity in COVID-19 sequelae model animals, behavioral experiments such as a 10% weighted forced swimming test and a tail suspension test, which will be described later in the Examples, can be performed before and after administration of a test substance, thereby quantifying changes in activity in COVID-19 sequelae model animals before and after administration of a test substance.

[0071] Furthermore, for example, in the evaluation step, changes in the novel coronavirus-related symptoms in the COVID-19 sequelae model animal may be confirmed by quantifying changes in the expression levels of inflammatory markers in the brain of the COVID-19 sequelae model animal. Examples of inflammatory markers include interleukin 6 (IL-6), tumor necrosis factor α (TNFα), and chemokine CC motif ligand 2 (CCL2).

[0072] When quantifying changes in the expression levels of inflammatory markers in the brains of COVID-19 sequelae model animals, it is not possible to quantify changes in the expression levels of inflammatory markers in the brains of the same COVID-19 sequelae model animals before and after administration of a test substance. Therefore, by comparing the above-mentioned measurements of the expression levels of inflammatory markers between the test substance administration group and the placebo administration group, it is possible to quantify changes in the expression levels of inflammatory markers in the brains of COVID-19 sequelae model animals before and after administration of the test substance.

[0073] In the evaluation step, the therapeutic or preventive effect of the test substance on COVID-19 sequelae may be evaluated based on the results of evaluation of changes in symptoms associated with the novel coronavirus. For example, in the evaluation step, if changes in symptoms associated with the novel coronavirus in a COVID-19 sequelae model animal are evaluated by quantifying changes in the activity of the COVID-19 sequelae model animal, and if the results show improvement in fatigue or depression symptoms associated with the novel coronavirus before and after administration of the test substance, the test substance can be evaluated as having activity to treat or prevent COVID-19 sequelae.

[0074] Furthermore, for example, in the evaluation process, if the changes in the expression levels of inflammatory markers in the brain of a COVID-19 sequelae model animal are quantified to evaluate changes in symptoms associated with the novel coronavirus in the COVID-19 sequelae model animal, and the results show a significant decrease in the expression levels of inflammatory markers in the brain before and after administration of the test substance, the test substance can be evaluated as having activity to treat or prevent COVID-19 sequelae.

[0075] Screening by quantifying changes in behavioral activity in COVID-19 sequelae model animals and screening by quantifying changes in the expression levels of inflammatory markers in the brains of COVID-19 sequelae model animals can also be used in combination as appropriate.

[0076] The drug screening method according to one embodiment of the present invention can also narrow down the effective test substances. For example, Example 6 below shows that acetylcholine is deficient in the brains of patients with COVID-19 sequelae. Therefore, acetylcholine receptor agonists can be prioritized as candidate drugs.

[0077] Furthermore, in Example 10 described below, it was shown that among acetylcholine receptor agonists, ventricular administration of PNU282987, an α7 nicotinic receptor agonist that does not cross the blood-brain barrier, has the effect of suppressing brain inflammation. This result suggests that α7 nicotinic receptor agonists should be prioritized in future screening. This indicates that even drugs that are not realistic therapeutic candidates because they do not cross the blood-brain barrier can be used to suggest the feasibility of drug screening methods by methods such as intraventricular administration.

[0078] Although it was known that α7 nicotinic receptor agonists have immunosuppressive functions, the mechanism was unclear. Example 11, which will be described later, suggests that the mechanism is that α7 nicotinic receptor agonists increase the expression of the immunosuppressive molecule ZFP36. This result suggests that by clarifying the mechanism of the therapeutic effect of a drug in the screening process, it is possible to develop better efficacy assessment methods and identify drug target molecules.

[0079] 3. Method for producing model animals (Features) One embodiment of the present invention provides a method for producing a COVID-19 sequelae animal model, which includes an expression step of expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector. This allows for the production of a COVID-19 sequelae animal model in which the SARS-CoV-2 S1 protein is expressed in a non-human mammal. The COVID-19 sequelae animal model can be used as an experimental animal in the development of treatments or prevention methods for COVID-19 sequelae, particularly in the development of drugs such as therapeutic agents and prophylactic agents. The COVID-19 sequelae animal model can also be used as an experimental animal in research into the causes of COVID-19 sequelae. This contributes to the achievement of Goal 3 of the Sustainable Development Goals (SDGs), "Good health and well-being for all."

[0080] (Type of model animal) The COVID-19 sequelae animal model produced by the method for producing a COVID-19 sequelae animal model according to one embodiment of the present invention is not particularly limited to any particular species, as long as it is a non-human mammal (a mammal other than a human) that can be used as an experimental animal. Therefore, the species of non-human mammal targeted in the expression step is not particularly limited and can be appropriately selected depending on the intended use of the model animal to be produced. Examples of non-human mammals targeted in the expression step include mice, rats, guinea pigs, dogs, rabbits, monkeys, and chimpanzees.

[0081] Although it is known that large animals such as monkeys are effective in treating novel coronavirus infection, animal models using small animals such as mice are considered necessary for the development of treatment or prevention methods, particularly for the development of therapeutic and preventive drugs. For this reason, small animal models such as mice are preferred as COVID-19 sequelae model animals.

[0082] (Expression process) The expression step is a step of expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector. By expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector, COVID-19 sequelae can be expressed in the non-human mammal.

[0083] Viral infections generally result in the production of inflammatory cytokines through an immune response, which is known to cause fatigue and depression during the acute phase of infection. However, COVID-19 sequelae are known to cause severe fatigue and depression not seen with other viruses, and these symptoms persist as sequelae. Therefore, it is predicted that the novel coronavirus possesses a protein with strong activity that causes neurological damage during infection. Therefore, the inventors conducted extensive research to identify the protein, and as a result, they discovered for the first time that the S1 region of the SARS-CoV-2 virus Spike protein (1273 amino acids, GenBank accession number YP 009724390) is the protein responsible for COVID-19 sequelae.

[0084] Handling model animals generated by infection with the infectious SARS-CoV-2 virus itself requires a highly confined facility, such as a P3 facility. In contrast, a method for producing a COVID-19 sequelae animal model according to one embodiment of the present invention uses a SARS-CoV-2 S1 protein expression vector, rather than the SARS-CoV-2 virus itself, to express the SARS-CoV-2 S1 protein, the protein responsible for COVID-19 sequelae, in a non-human mammal. Therefore, model animals produced by a method for producing a COVID-19 sequelae animal model according to one embodiment of the present invention can be used in a standard experimental environment, making them easy to handle.

[0085] In animal models produced by infection with the infectious SARS-CoV-2 virus itself, the pathogenicity of acute infection and the pathogenicity of sequelae may not be comparable to those in humans, and therefore the survival rate of model animals due to acute symptoms may be low, and sequelae sufficient for use as a model may not occur. Therefore, model animals produced by the method for producing a COVID-19 sequelae model animal according to one embodiment of the present invention are advantageous in that they can efficiently and reliably produce model animals of COVID-19 sequelae.

[0086] As used herein, "expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector" means expressing the SARS-CoV-2 S1 protein in the body of a target non-human mammal using a SARS-CoV-2 S1 protein expression vector.

[0087] Although the site where the SARS-CoV-2 S1 protein is expressed in the body of the target non-human mammal is not particularly limited, it is preferable to express the SARS-CoV-2 S1 protein in at least one of the nasal cavity and the peri-nasal region of the non-human mammal in the expression step, since this can efficiently cause COVID-19 sequelae. To express the SARS-CoV-2 S1 protein in at least one of the nasal cavity and the peri-nasal region of the non-human mammal, for example, in the expression step, a SARS-CoV-2 S1 protein expression vector may be administered to the nasal cavity of the non-human mammal.

[0088] The structure of the SARS-CoV-2 Spike protein is shown in Figure 1. The S1 region is a region having an amino acid sequence consisting of amino acids 1 to 685 of the amino acid sequence of the SARS-CoV-2 Spike protein. The S1 region contains a signal peptide sequence (SP), an N-terminal domain (NTD), and a receptor-binding domain (RBD). A polypeptide containing the S1 region of the Spike protein of the SARS-CoV-2 virus is referred to herein as the "SARS-CoV-2 S1 protein" or simply as the "S1 protein."

[0089] The SARS-CoV-2 S1 protein may be, for example, a polypeptide of (a) or (b) below: (a) a polypeptide having an amino acid sequence (SEQ ID NO: 1) consisting of amino acids 1 to 685 of the amino acid sequence shown in GeneBank Accession No. YP_009724390; (b) A polypeptide having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence consisting of amino acids 1 to 685 (SEQ ID NO: 1) of the amino acid sequence shown in GeneBank accession number YP_009724390, and that has the activity of increasing intracellular calcium concentration when introduced into cells.

[0090] The SARS-CoV-2 S1 protein is a polypeptide consisting of 685 amino acids with a molecular weight of approximately 76.7 kDa.

[0091] The SARS-CoV-2 S1 protein may be a polypeptide, such as the polypeptide (b) described above, that has an amino acid sequence with 80% or more sequence identity (85% or more, 90% or more, 95% or more, 98% or more, or 99% or more) to the amino acid sequence consisting of amino acids 1 to 685 (SEQ ID NO: 1) of the amino acid sequence set forth in GeneBank Accession Number YP_009724390, and that has the activity of increasing intracellular calcium concentration when introduced into cells. Herein, the percent identity of the amino acid sequence is a value calculated using genetic information processing software GENETYX Ver. 7 (manufactured by Genetyx).

[0092] Alternatively, the polypeptide (b) may be a polypeptide comprising an amino acid sequence consisting of amino acids 1 to 685 of the amino acid sequence shown in GeneBank Accession No. YP_009724390 (SEQ ID NO: 1), in which 100 or fewer amino acids have been substituted, deleted, inserted, and / or added, and which has the activity of increasing intracellular calcium concentration when introduced into cells. As used herein, "in which 100 or fewer amino acids have been substituted, deleted, inserted, and / or added" means that 100 or fewer amino acids (90 or fewer, 80 or fewer, 70 or fewer, 60 or fewer, 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 15 or fewer, 10 or fewer, 7 or fewer, 5 or fewer, or 2 or fewer) have been substituted, deleted, inserted, and / or added by known methods for producing mutant peptides, such as site-directed mutagenesis. Thus, the polypeptide (b) can be said to be a variant of the polypeptide (a). The term "mutation" as used herein primarily refers to a mutation artificially introduced by a known method for producing mutant proteins, but may also refer to an isolated and purified version of a similar mutant protein that exists in nature.

[0093] Mutant strains of the SARS-CoV-2 virus have been reported to date, and are known to have mutations in the spike protein. The main mutations in the SARS-CoV-2 S1 protein in mutant strains reported to date are as follows. The polypeptide of (b) preferably has these mutations. SARS-CoV-2 B.1.1.7 lineage (also known as the "alpha strain"): deletions 69-70, 144-145, N501Y, A570D, and D614G, P681H SARS-CoV-2 B.1.351 lineage (so-called "beta strain"): D80A, D215G, Deletions 241-243, K417N, E484K, N501Y, and D614G SARS-CoV-2 P.1 lineage (so-called "gamma strain"): L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, and H655Y SARS-CoV-2 B.1.617.2 lineage (the so-called "Delta strain"): T19R, G142D, E156G, deletions 157-158, L452R, T487K, E484Q, D614G, and P681R SARS-CoV-2 B.1.1.529 lineage (also known as the "Omicron strain"): G142D, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, and P681H

[0094] Whether a mutant protein of SARS-CoV-2 S1 protein has the activity of increasing intracellular calcium concentration can be confirmed by expressing the mutant protein in any cultured cells and measuring the intracellular calcium concentration. The intracellular calcium concentration can be measured by the method described in the Examples below. If the calcium concentration in cells expressing the mutant protein is significantly increased compared to parent cells, it can be determined that the mutant protein has the activity of increasing intracellular calcium concentration.

[0095] The SARS-CoV-2 S1 protein expression vector is an expression vector obtained by introducing a polynucleotide encoding the SARS-CoV-2 S1 protein into a known plasmid vector or viral vector.

[0096] The plasmid vector or viral vector can be any known vector commonly used in genetic engineering, as long as it is capable of expressing the SARS-CoV-2 S1 protein in the body of a non-human mammal, and the type is not particularly limited. Viral vectors are preferred because of their high infection efficiency and ease of introduction into non-human mammals. Adenoviral vectors, which are commonly used for known gene therapy, viral vaccines, and other purposes, are particularly preferred.

[0097] The promoter for expressing the SARS-CoV-2 S1 protein is not particularly limited as long as it is capable of expressing mRNA in cells infected with SARS-CoV-2. Promoters that can produce the SARS-CoV-2 S1 protein at the same level as during SARS-CoV-2 infection are particularly suitable.

[0098] The polynucleotide encoding the SARS-CoV-2 S1 protein may be, for example, a polynucleotide encoding the polypeptide (a) or (b) described above.

[0099] As an example, the entire genome sequence of SARS-CoV-2 S1, including the nucleotide sequence of a polynucleotide encoding the polypeptide (a), is published under GenBank accession number MN908947. The polynucleotide encoding the polypeptide (a) has a nucleotide sequence (SEQ ID NO: 2) consisting of nucleotides 21563 to 23617 of the nucleotide sequence shown in GeneBank accession number MN908947. The polynucleotide encoding the polypeptide (a) has a size of 2055 base pairs (approximately 2 kbp).

[0100] The method for obtaining a polynucleotide encoding the SARS-CoV-2 S1 protein is not particularly limited. Examples include methods using amplification techniques such as PCR. For example, primers can be prepared from the 5' and 3' sequences (or their complementary sequences) of the cDNA sequence of the gene encoding the SARS-CoV-2 Spike protein, and these primers can be used to perform PCR or other procedures using genomic DNA (or cDNA) as a template to amplify the DNA region between the two primers, thereby obtaining large quantities of a DNA fragment containing a polynucleotide encoding the SARS-CoV-2 S1 protein. Based on gene sequence information, a polynucleotide having the nucleotide sequence of a polynucleotide encoding the SARS-CoV-2 S1 protein can be synthesized using known chemical synthesis techniques. The nucleotide sequence can be optimized for the codon usage of the animal being used.

[0101] In the expression process, the expression amount of SARS-CoV-2 S1 protein can be adjusted taking into consideration the species and body weight of the target non-human mammal so that a sufficient amount of SARS-CoV-2 S1 protein can be expressed to induce COVID-19 sequelae.

[0102] In the expression step of the method for producing a COVID-19 sequelae animal model according to one embodiment of the present invention, the SARS-CoV-2 S1 protein may be transiently or constitutively expressed in a non-human mammal. In research into COVID-19 sequelae using a COVID-19 sequelae animal model, it is desirable to be able to closely examine symptoms when expression of the SARS-CoV-2 S1 protein has terminated or attenuated. Therefore, in the expression step, the SARS-CoV-2 S1 protein is preferably transiently expressed in a non-human mammal.

[0103] (Inflammation induction process) A method for producing a COVID-19 sequelae animal model according to one embodiment of the present invention preferably further comprises an inflammation induction step inducing inflammation in a non-human mammal. In COVID-19 sequelae, the SARS-CoV-2 S1 protein is expressed in an inflammatory state caused by SARS-CoV-2 virus infection. Therefore, by further comprising an inflammation induction step, a method for producing a COVID-19 sequelae animal model according to one embodiment of the present invention can produce a model animal that takes into account the conditions of actual SARS-CoV-2 virus infection.

[0104] Although there are no particular limitations on the method for inducing inflammation in non-human mammals, it is preferable to induce inflammation using drugs rather than viral infection, considering the ease of handling of the model animals. For example, inflammation can be induced in non-human mammals by intraperitoneal administration of lipopolysaccharide (LPS) derived from Gram-negative bacteria such as Escherichia coli O111. LPS is known to have the biological activity of activating macrophages.

[0105] The inflammation induction step is performed after the expression step. By performing the inflammation induction step after the expression step, the inflammation induction effect can be observed in a model animal exhibiting COVID-19 sequelae. The inflammation induction step may also be performed before the expression step.

[0106] (Evaluation process) The method for producing a COVID-19 sequelae model animal according to one embodiment of the present invention may further include an evaluation step of evaluating the severity of symptoms of COVID-19 sequelae in the non-human mammal after the expression step.

[0107] Typical symptoms of COVID-19 sequelae are fatigue and depression. Therefore, the severity of COVID-19 sequelae in non-human mammals after the onset process can be assessed by evaluating the severity of fatigue or depression through behavioral experiments and their quantification.

[0108] The degree of fatigue can be evaluated by performing a 10% weighted forced swimming test as described in the Examples below, and the degree of depressive symptoms can be evaluated by performing a tail suspension test as described in the Examples below.

[0109] By performing the evaluation step, it is possible to confirm that the model animal produced by the method for producing a COVID-19 sequelae animal model according to one embodiment of the present invention indeed exhibits symptoms of COVID-19 sequelae. Depending on the results of the evaluation step, the amount of SARS-CoV-2 S1 protein expressed in the expression step, the type of expression vector, the degree of inflammation induced in the inflammation induction step, and the like can be adjusted as appropriate.

[0110] <4. COVID-19 Sequelae Animal Model Creation Kit>

[0111] A kit for producing a COVID-19 sequelae animal model according to one embodiment of the present invention (hereinafter simply referred to as the "kit") includes an expression vector capable of expressing the SARS-CoV-2 S1 protein in the cells of a non-human mammal. The kit according to one embodiment of the present invention can be suitably used in the method for producing a COVID-19 sequelae animal model according to one embodiment of the present invention.

[0112] In the present invention, the term "kit" refers to a package containing containers (e.g., bottles, plates, tubes, dishes, etc.) containing specific materials. The kit according to one embodiment of the present invention may be in a form in which each material contained therein exists independently, or in a form in which multiple materials are mixed (e.g., in the form of a composition). The kit preferably includes instructions for using each material.

[0113] A kit according to one embodiment of the present invention need only include materials for carrying out a method for producing a COVID-19 sequelae model animal according to one embodiment of the present invention, and the specific configuration, materials, equipment, etc. of the kit other than the expression vector capable of expressing the SARS-CoV-2 S1 protein in the cells of a non-human mammal are not particularly limited.

[0114] In addition to an expression vector capable of expressing the SARS-CoV-2 S1 protein in the cells of a non-human mammal, a kit according to one embodiment of the present invention may further comprise lipopolysaccharide (LPS) for use in the inflammation induction process.

[0115] <5. COVID-19 Sequelae Animal Model> The present invention also encompasses a COVID-19 sequelae animal model produced by the method for producing a COVID-19 sequelae animal model according to one embodiment of the present invention. Because the COVID-19 sequelae animal model according to one embodiment of the present invention is produced by the method for producing a COVID-19 sequelae animal model according to one embodiment of the present invention, it does not require a highly confined facility such as a P3 facility and can be used in a normal experimental environment, making it easy to handle.

[0116] <Additional Notes> As described above, one aspect of the present invention is as follows. <1> A drug for treating the aftereffects of COVID-19, containing an acetylcholine receptor agonist as its active ingredient. <2> The acetylcholine receptor agonist is a central acetylcholine receptor agonist that acts on acetylcholine receptors in the brain. <1> A drug for treating the aftereffects of COVID-19 described above. <3> The acetylcholine receptor agonist is donepezil. <1> or <2> A drug for treating the aftereffects of COVID-19 described above. <4> The COVID-19 infection sequelae are fatigue associated with the COVID-19 infection, <1> ~ <3> A drug for treating sequelae of COVID-19 infection described in any one of the above. <5> The aftereffects of the novel coronavirus infection are depressive symptoms associated with the novel coronavirus, <1> ~ <3> A drug for treating sequelae of COVID-19 infection described in any one of the above. <6> The novel coronavirus infection sequelae is olfactory dysfunction associated with the novel coronavirus, <1> ~ <3> A drug for treating sequelae of COVID-19 infection described in any one of the above. <7> The novel coronavirus infection sequelae are memory disorders associated with the novel coronavirus, <1> ~ <3> A drug for treating sequelae of COVID-19 infection described in any one of the above. <8> an administration step of administering a test substance to a model animal of sequelae of COVID-19, in which SARS-CoV-2 S1 protein is expressed in a non-human mammal; A screening method for drugs to treat sequelae of COVID-19 infection, comprising an evaluation step of evaluating changes in symptoms associated with COVID-19 in the model animal before and after administration of the test substance. <9> The model animal is a model animal in which SARS-CoV-2 S1 protein is expressed in at least one of the nasal cavity and the surrounding area of ​​the nasal cavity of the non-human mammal. <8> A method for screening a therapeutic drug according to the above. <10> The model animal is produced by a method for producing a model animal for sequelae of COVID-19, which method includes an expression step of expressing the SARS-CoV-2 S1 protein in the non-human mammal using a SARS-CoV-2 S1 protein expression vector. <8> or <9> A method for screening a therapeutic drug according to the above. <11> The method for producing the novel coronavirus infection sequelae model animal further comprises an inflammation induction step of inducing inflammation in the non-human mammal. <10> A method for screening a therapeutic drug according to the above. <12> A method for producing a model animal suffering from the sequelae of COVID-19, which comprises an expression step of expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector. <13> In the expression step, SARS-CoV-2 S1 protein is expressed in at least one of the nasal cavity and the perinasal cavity of the non-human mammal. <12> A method for producing a model animal for COVID-19 sequelae described in the above. <14> Further comprising an inflammation inducing step of inducing inflammation in the non-human mammal. <12> or <13> A method for producing a model animal for COVID-19 sequelae described in the above. <15> The SARS-CoV-2 S1 protein is a polypeptide of the following (a) or (b): <12> ~ <14> A method for producing a model animal for sequelae of novel coronavirus infection according to any one of the above: (a) a polypeptide having an amino acid sequence (SEQ ID NO: 1) consisting of amino acids 1 to 685 of the amino acid sequence shown in GeneBank Accession No. YP_009724390; (b) A polypeptide having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence consisting of amino acids 1 to 685 (SEQ ID NO: 1) of the amino acid sequence shown in GeneBank accession number YP_009724390, and that has the activity of increasing intracellular calcium concentration when introduced into cells.

[0117] <6. Methods for treating or preventing COVID-19 aftereffects> A method for treating or preventing COVID-19 sequelae using a therapeutic agent for COVID-19 sequelae according to one embodiment of the present invention is also included in the scope of the present invention.

[0118] That is, a method for treating or preventing COVID-19 sequelae according to one embodiment of the present invention is as follows. <16> A method for treating or preventing COVID-19 sequelae, comprising administering to a subject (e.g., a human or non-human animal) a drug for treating COVID-19 sequelae, which drug contains an acetylcholine receptor agonist as an active ingredient. <17> The acetylcholine receptor agonist is a central acetylcholine receptor agonist that acts on acetylcholine receptors in the brain. <16> A method for treating or preventing COVID-19 sequelae described in the above. <18> The acetylcholine receptor agonist is donepezil. <16> or <17> A method for treating or preventing COVID-19 sequelae described in the above. <19> The COVID-19 sequelae are fatigue associated with the new coronavirus, <16> ~ <18> A method for treating or preventing COVID-19 sequelae described in any one of the above. <20> The COVID-19 sequelae are depressive symptoms associated with the new coronavirus. <16> ~ <18> A method for treating or preventing COVID-19 sequelae described in any one of the above. <21> The COVID-19 sequelae are olfactory disorders associated with the new coronavirus, <16> ~ <18> A method for treating or preventing COVID-19 sequelae described in any one of the above. <22> The COVID-19 sequelae are memory disorders associated with the new coronavirus. <16> ~ <18> A method for treating or preventing COVID-19 sequelae described in any one of the above.

[0119] The subjects, administration routes, formulations, and formulations of the therapeutic agent for COVID-19 sequelae are the same as those described for the therapeutic agent according to one embodiment of the present invention, and will not be repeated here. Oral administration of the therapeutic agent for COVID-19 sequelae is preferred because it is easy to administer and places less strain on the recipient.

[0120] <7.Other> The use of an acetylcholine receptor agonist for the manufacture of a therapeutic agent for COVID-19 sequelae according to one embodiment of the present invention is also within the scope of the present invention.

[0121] That is, the use according to one aspect of the present invention is as follows. <23> Use of an acetylcholine receptor agonist for the manufacture of a drug for the treatment of COVID-19 sequelae. <24> The acetylcholine receptor agonist is a central acetylcholine receptor agonist that acts on acetylcholine receptors in the brain. <23> Use as described in. <25> The acetylcholine receptor agonist is donepezil. <23> or <24> Use as described in. <26> The COVID-19 sequelae are fatigue associated with the new coronavirus, <23> ~ <25> 1. Use according to any one of the preceding items. <27> The COVID-19 sequelae are depressive symptoms associated with the new coronavirus. <23> ~ <25> 1. Use according to any one of the preceding items. <28> The COVID-19 sequelae are olfactory disorders associated with the new coronavirus, <23> ~ <25> 1. Use according to any one of the preceding items. <29> The COVID-19 sequelae are memory disorders associated with the new coronavirus. <23> ~ <25> 1. Use according to any one of the preceding items.

[0122] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Example]

[0123] Example 1 The neurotoxicity of SARS-CoV-2 S1 protein was examined using the increase in intracellular calcium as an indicator.

[0124] <Method> Construction of mammalian cell expression vectors A DNA fragment (2055 base pairs) encoding the S1 region (685 amino acids) of the SARS-CoV-2 Spike protein (1273 amino acids, GenBank accession number YP_009724390) was inserted into the mammalian cell expression vector pFlag-CMV-5a to construct the S1 expression vector plasmid (S1 / pFlag). The S1 region of the SARS-CoV-2 Spike protein (hereinafter simply referred to as the "S1 protein") is a polypeptide having the amino acid sequence (SEQ ID NO: 1) consisting of amino acids 1 through 685 of the amino acid sequence shown in GeneBank accession number YP_009724390. The nucleotide sequence of the DNA fragment encoding the S1 protein has been published under GenBank accession number MN908947.

[0125] Furthermore, to construct an S1-expressing adenoviral vector, we used the Adenovirus Dual Expression Kit (TaKaRa). A DNA fragment encoding the S1 region was inserted into the cosmid vector pAxCAwtit2, and an adenoviral vector (S1 / Adv) expressing S1 under the control of the CAG promoter was constructed.

[0126] <Intracellular calcium measurement> To express the S1 protein in mouse and human cells, we used the mouse skin fibroblast cell line 3T3 and the human lung basal epithelial adenocarcinoma cell line A549. S1 / pFlag was transfected into these cells using the CalPhos Mammalian Transfection Kit (TaKaRa) to transiently express the S1 protein. The empty vector plasmid pFlag-CMV-5a was used as a control.

[0127] In the case of adenoviral vectors, these cells were directly infected with S1 / Adv to transiently express the S1 protein, and an empty adenoviral vector was used as a control.

[0128] To measure the intracellular calcium concentration in cells expressing S1 protein using the above method, calcium ions were bound to a fluorescent probe using Calcium Kit II - Fluo 4 (DOJINDO), and the fluorescence intensity of each cell was measured using an ArrayScan XT (ThermoFisher).

[0129] <Result> As shown in Figure 1, the S1 protein used to construct the expression vector is a protein having an amino acid sequence (SEQ ID NO: 1) consisting of amino acids 1 to 685 of the amino acid sequence of the SARS-CoV-2 Spike protein shown in GeneBank accession number YP_009724390.

[0130] 3T3 cells and A549 cells were transformed with the S1 protein expression plasmid or the control plasmid, and the intracellular calcium concentration was measured. The results are shown in Fig. 2. "Intensity / area" shown on the vertical axis of the graph in Fig. 2 represents the fluorescence intensity per unit area. "Control" shown on the horizontal axis of the graph in Fig. 2 represents the cells transformed with the control plasmid, and "S1 / pFlag" represents the cells transformed with the S1 protein expression plasmid. Also, the horizontal bars shown in Fig. 2 represent the median. ****: P<0.0001.

[0131] As shown in Fig. 2, the intracellular calcium concentration increased due to the expression of the S1 protein in all cells.

[0132] Furthermore, the intracellular calcium concentration of 3T3 cells and A549 cells infected with the S1 protein-expressing adenovirus or the control adenovirus was measured. The results are shown in Fig. 3. "Intensity / area" shown on the vertical axis of the graph in Fig. 3 represents the fluorescence intensity per unit area. "Control" shown on the horizontal axis of the graph in Fig. 3 represents the cells infected with the control adenovirus, and "S1 / Adv" represents the cells infected with the S1 protein-expressing adenovirus. Also, the horizontal bars shown in Fig. 3 represent the median. ****: P<0.0001.

[0133] As shown in Fig. 3, the intracellular calcium concentration increased due to the expression of the S1 protein in all cells (Fig. 3).

[0134] Therefore, it was shown that the intracellular calcium concentration increased due to the expression of the S1 protein in mouse and human cells. That is, when the S1 protein is expressed in nerve cells, it is suggested that the intracellular calcium concentration increases and induces nerve cell death.

[0135] [Example 2] Expression of fatigue in S1 protein-expressing mice (S-null mice) [Method] [Preparation of S1 protein-expressing mice (S-null mice)] Eight- to nine-week-old C57BL / 6 mice were anesthetized with isoflurane and given 1 × 10 9 25 μL of S1 / Adv solution (100 μg / mL) was administered into the nasal cavity and allowed to breathe naturally (expression step). S1 mice (COVID-19 sequelae model animals) were then generated. The mice were then returned to their home cages and housed for one week. As a control, mice (control mice) were administered an empty adenoviral vector (vector / Adv) that does not express anything.

[0136] <Fatigue behavior test> Six days after intranasal administration of S1 / Adv or vector / Adv, a 10% weighted forced swimming test was performed as a fatigue behavior test. The weights of S1 and control mice were measured in the morning of the test, and weights equivalent to approximately 10% of the mice's body weight were prepared. The weights were attached to the tails of S1 or control mice, which were then placed in a tank for the forced swimming test. The time it took for the mice's nose to sink below the water surface for 10 seconds was measured.

[0137] <Result> The results of the 10% weighted forced swimming test are shown in Figure 4. The "swimming time" on the vertical axis of the graph in Figure 4 represents the time (seconds) from when the mouse was placed in the tank for the forced swimming test until its nose was submerged under the water for 10 seconds. Also, "Control" on the horizontal axis of the graph in Figure 4 represents control mice, and "S1" represents S1 mice. ‡: P<0.1.

[0138] Compared with control mice, S1 mice tended to have a shorter swimming time, indicating that they tire more easily than control mice.

[0139] Example 3 Expression of depression-like behavior in S1 mice <Method> S1 mice and control mice were generated using the same method as in Example 2. To confirm depression-like behavior, a tail suspension test was performed. Six days after intranasal administration of S1 / Adv or vector / Adv, S1 mice and control mice were suspended by their tails for 10 minutes, videotaped, and analyzed using image analysis software TailSuspScan (CleverSys Inc.) to measure the immobility time.

[0140] <Result> The results of plotting the immobility time in the tail suspension test are shown in Figure 5. "Control" on the horizontal axis of the graph in Figure 5 represents control mice, and "S1" represents S1 mice. *: P<0.05.

[0141] Compared with control mice, S1 mice had significantly increased immobility time, revealing that S1 mice exhibited depression-like behavior.

[0142] Example 4 Olfactory bulb nerve damage in mice expressing S1 protein and inducing inflammation with LPS <Method> S1 mice and control mice were generated by the same method as in Example 2. Seven days after nasal administration of S1 / Adv or vector / Adv, S1 mice and control mice were intraperitoneally administered 5 mg / kg of Escherichia coli O111-derived lipopolysaccharide (MERCK), and the olfactory bulbs were collected 30 and 60 minutes later.

[0143] RNA was purified from the collected olfactory bulb using the RNeasy Mini Kit (QIAGEN). cDNA was synthesized using the purified RNA as a template using the PrimeScript RT reagent Kit (Takara Bio). Calbindin gene expression was analyzed by RT-qPCR using the synthesized cDNA. 18S rRNA measurement results were used for normalization.

[0144] <Result> The results of the RT-qPCR analysis are shown in Figure 6. "Control" on the horizontal axis of the graph in Figure 6 represents control mice, and "S1" represents S1 mice. ‡: P<0.1, **: P<0.01.

[0145] RT-qPCR analysis showed that calbindin expression in the olfactory bulb of S1 mice tended to decrease 30 minutes after LPS administration and was significantly decreased 60 minutes after LPS administration. Because calbindin is a marker of mature neurons, it was suggested that mature neurons in the olfactory bulb of S1 mice were damaged when peripheral inflammation was induced.

[0146] Example 5 Neuronal damage in mice expressing S1 protein and in which inflammation is induced by LPS <Method> S1 mice and control mice were generated by the same method as in Example 2. Seven days after nasal administration of S1 / Adv or vector / Adv, E. coli O111-derived lipopolysaccharide (MERCK) was intraperitoneally administered to the S1 mice and control mice at 5 mg / kg, and the brains excluding the olfactory bulbs were collected 15 minutes later.

[0147] RNA was purified from the collected brains using the RNeasy Mini Kit (QIAGEN). cDNA was synthesized using the purified RNA as a template using the PrimeScript RT Reagent Kit (Takara Bio). Calbindin gene expression was analyzed by RT-qPCR using the synthesized cDNA. 18S rRNA measurement results were used for normalization.

[0148] <Result> The results of the RT-qPCR analysis are shown in Figure 7. "Control" on the horizontal axis of the graph in Figure 7 represents control mice, and "S1" represents S1 mice. ‡: P<0.1.

[0149] RT-qPCR analysis showed that calbindin expression in the brains of S1 mice tended to decrease 15 minutes after LPS administration, suggesting that mature neurons in the brains of S1 mice are damaged when peripheral inflammation is induced.

[0150] Example 6 Cholinergic neuronal damage in mice expressing S1 protein <Method> Brains were removed from S1 and control mice 7 days after intranasal administration of S1 / Adv or vector / Adv and fixed in 10% neutral formalin. The fixed brains were embedded in paraffin and coronal sections (brain sections) were prepared at a position (Bregma 0.62 mm) where the septal area and Broca's diagonal band could be observed. After deparaffinization, the brain sections were subjected to antigen retrieval and immunofluorescence staining with anti-choline acetyltransferase antibody (Abcam).

[0151] <Result> The results of immunofluorescence staining are shown in Figure 8. Figure 8 suggests that the number of cholinergic neurons, which are choline acetyltransferase (ChAT)-positive cells, in the medial septal area (MS) and diagonal band of Broca (DB) of the basal forebrain is reduced in S1 mice compared to control mice. Therefore, the number of ChAT-positive cells in the MS / DB region was counted. The results are shown in Figure 9. Figure 9 indicates that the number of ChAT-positive cells in the MS / DB region is significantly reduced in S1 mice. **: P<0.01.

[0152] These results indicated that cholinergic neurons in the brain of S1 mice were impaired, suggesting that the amount of acetylcholine in the brain may be reduced.

[0153] Example 7 Donepezil administration improves fatigue symptoms in S1 mice <Method> <Administration of donepezil> Donepezil (Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in water to a concentration of 32 mg / L and administered to mice via drinking water. This resulted in a dose of 4.0 mg / kg / day of donepezil. Because most literature suggests a dose of 3.0 to 5.0 mg / kg / day, we adopted 4.0 mg / kg / day.

[0154] The administration scheme of donepezil is shown in Figure 10. The donepezil solution was administered to S1 mice and control mice immediately after intranasal administration of S1 / Adv or vector / Adv, and the drinking water containing donepezil was changed every two days. Donepezil was administered in the same manner in the following examples.

[0155] <Fatigue behavior test> Six days after intranasal administration of S1 / Adv or vector / Adv, a 10% weighted forced swimming test was performed as a fatigue behavior test. The weights of S1 and control mice were measured in the morning of the test, and weights equivalent to approximately 10% of their body weight were prepared. The weights were attached to the tails of S1 and control mice and placed in a tank for the forced swimming test. The time it took for the tip of the mouse's nose to sink below the water surface for 10 seconds was measured.

[0156] <Result> The results of the 10% weighted forced swimming test are shown in Figure 11. The "swimming time" on the vertical axis of the graph in Figure 11 represents the time (seconds) from when the mouse was placed in the tank for the forced swimming test until its nose was submerged under the water for 10 seconds. Also, "Control" on the horizontal axis of the graph in Figure 11 represents control mice, and "S1" represents S1 mice. *: P<0.05.

[0157] In the group not administered donepezil ("-" in Figure 11), the swimming time of S1 mice was significantly shorter than that of control mice. In contrast, in the group administered donepezil ("donepezil" in Figure 11), no difference was observed between the swimming time of control mice and that of S1 mice. This result was considered to indicate that donepezil improves fatigue-like behavior caused by S1 protein expression.

[0158] Example 8 Donepezil treatment improves depression-like behavior in S1 mice <Method> After intranasal administration of S1 / Adv or vector / Adv, S1 mice and control mice that had received donepezil in their drinking water according to the administration scheme shown in Figure 10 were subjected to a tail suspension test to confirm depression-like behavior. Six days after intranasal administration of S1 / Adv or vector / Adv, S1 mice and control mice were suspended by their tails for 10 minutes, videotaped, and analyzed using image analysis software TailSuspScan (CleverSys Inc.) to measure the duration of immobility.

[0159] <Result> The results of plotting the immobility time in the tail suspension test are shown in Figure 12. "Control" on the horizontal axis of the graph in Figure 12 represents control mice, and "S1" represents S1 mice. *: P<0.05, ***: P<0.001.

[0160] In the group not administered donepezil ("-" in Figure 12), the immobility time of S1 mice was significantly increased compared to the control mice. In contrast, in the group administered donepezil ("donepezil" in Figure 12), the immobility time of S1 mice was significantly decreased compared to the control mice. This result was considered to indicate that donepezil improves depression-like behavior caused by S1 protein expression.

[0161] Example 9 Donepezil administration improves brain inflammation in S1 mice <Method> After intranasal administration of S1 / Adv or vector / Adv, S1 mice and control mice were administered donepezil in their drinking water according to the administration scheme shown in Figure 10. Seven days after intranasal administration of S1 / Adv or vector / Adv, brains other than the olfactory bulb were collected. RNA was purified from the collected brains using the RNeasy Mini Kit (QIAGEN). cDNA was synthesized using the purified RNA as a template using the PrimeScript RT Reagent Kit (Takara Bio). The synthesized cDNA was used for RT-qPCR analysis of gene expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNFα), and chemokine CC motif ligand 2 (CCL2). 18S rRNA measurement results were used for normalization.

[0162] <Result> The results of the RT-qPCR analysis are shown in Figure 13. "Control" on the horizontal axis of the graph in Figure 13 represents control mice, and "S1" represents S1 mice. *: P<0.05, ‡: P<0.1.

[0163] RT-qPCR analysis showed that in the group not administered donepezil ("-" in Figure 13), IL-6 expression in the brains of S1 mice was significantly elevated, and the expression of TNFα and CCL2 also tended to increase. In contrast, in the group administered donepezil ("donepezil" in Figure 13), the expression of these genes in the brains of S1 mice tended to decrease, and no difference was observed between the expression of these genes in the brains of control mice. Therefore, it was demonstrated that brain inflammation is induced in S1 mice due to S1 protein expression, and that administration of donepezil ameliorates brain inflammation.

[0164] Example 10 Efficacy assessment of acetylcholine receptor agonists other than donepezil <Method> Because donepezil exerts its effects by increasing the amount of acetylcholine in the brain, screening for drugs that act more selectively on acetylcholine receptors is necessary to develop more effective therapeutic agents. Therefore, we investigated the therapeutic effect of PNU282987, an α7 nicotinic acetylcholine receptor-specific agonist, intracerebroventricularly administered at 400 nmol / mouse to S1 and control mice 7 days after intranasal administration of S1 / Adv or vector / Adv.

[0165] One hour after intracerebroventricular administration of PNU282987, the brain was collected except for the olfactory bulb, and RNA was purified using the RNeasy Mini Kit (QIAGEN). cDNA was synthesized using the purified RNA as a template using the PrimeScript RT reagent Kit (Takara Bio). The synthesized cDNA was used for RT-qPCR analysis of interleukin-1β (IL-1β) and interleukin-6 (IL-6) gene expression. 18S rRNA measurement results were used for normalization.

[0166] <Result> The results of RT-qPCR analysis are shown in Figure 14. The horizontal axis of the graph in Figure 14 indicates control mice, and "S1" indicates S1 mice. **: P<0.01, ‡: P<0.1. Increased expression of inflammatory cytokine genes (IL-1β and IL-6) in the brain, which is thought to be a molecular mechanism behind fatigue and depression, was observed in S1 mice. Intracerebroventricular administration of PNU282987 suppressed this increase. These results suggest that PNU282987 and its derivatives may be potential therapeutic candidates for brain inflammation in S1 mice. These results also suggest that α7 nicotinic acetylcholine receptor agonists should be prioritized for therapeutic drug screening.

[0167] Example 11 Examples of mechanisms of action of acetylcholine receptor agonists <Method> To elucidate the molecular mechanism of the therapeutic effect of PNU282987 on brain inflammation in Example 10, we investigated the effects of S1 and PNU282987 on the gene expression level of zinc finger protein 36 (Zfp36), a host protein with anti-inflammatory effects. Specifically, RT-qPCR analysis of Zfp36 gene expression was performed using the cDNA synthesized in Example 10. 18S rRNA measurement results were used for normalization.

[0168] <Result> The results of RT-qPCR analysis are shown in Figure 15. The horizontal axis of the graph in Figure 15 indicates control mice, and "S1" indicates S1 mice. *: P<0.05, **: P<0.01. The results showed that S1 mice exhibited reduced ZFP36 expression, and administration of PNU282987 ameliorated this reduction. This suggests that brain inflammation in S1 mice is due to a reduction in ZFP36, which has anti-inflammatory properties, and that acetylcholine receptor agonists, especially α7 nicotinic acetylcholine receptor agonists, exert their therapeutic effects on brain inflammation by restoring ZFP36 expression. These results suggest that ZFP36 is a therapeutic target and that the expression level of the gene, ZFP36, can be used as a biomarker for therapeutic drug screening.

[0169] These results demonstrate that expressing S1 protein in mice can produce a mouse model of COVID-19 sequelae, that cholinergic neurons in the brain of these model mice are damaged, and that the symptoms of COVID-19 sequelae in these model mice are improved by administration of donepezil. Furthermore, analysis of the mechanism of action of acetylcholine receptor agonists indicates that, among acetylcholine receptor agonists, α7 nicotinic acetylcholine receptor agonists should be prioritized when screening for drugs to treat COVID-19 sequelae. [Industrial Applicability]

[0170] A drug for treating COVID-19 sequelae according to one embodiment of the present invention can contribute to the treatment or prevention of COVID-19 sequelae in patients infected with the novel coronavirus. Furthermore, a method for screening drugs for treating COVID-19 sequelae according to one embodiment of the present invention can contribute to the development of new drugs for treating COVID-19 sequelae. Furthermore, a COVID-19 sequelae model animal produced by a method for producing a COVID-19 sequelae model animal according to one embodiment of the present invention can be used in drug development, basic research on COVID-19 sequelae, and the like.

Claims

1. an administration step of administering a test substance to a model animal of sequelae of COVID-19, in which SARS-CoV-2 S1 protein is expressed in a non-human mammal; and an evaluation step of evaluating changes in symptoms associated with the novel coronavirus before and after administration of the test substance in the novel coronavirus infection sequelae model animal, The novel coronavirus infection sequelae animal model is produced by a method for producing a novel coronavirus infection sequelae animal model, the method comprising an expression step of expressing the SARS-CoV-2 S1 protein in the non-human mammal using a SARS-CoV-2 S1 protein expression vector. Screening method for drugs to treat sequelae of COVID-19.

2. The method for screening for a therapeutic drug for the sequelae of novel coronavirus infection according to claim 1, wherein the model animal for novel coronavirus infection sequelae is a model animal in which SARS-CoV-2 S1 protein is expressed in at least one of the nasal cavity and the surrounding area of ​​the nasal cavity of the non-human mammal.

3. The method for screening for a therapeutic drug for the sequelae of COVID-19 according to claim 2, wherein the method for producing the model animal for COVID-19 sequelae further comprises an inflammation induction step of inducing inflammation in the non-human mammal.

4. A method for producing a model animal with sequelae of COVID-19, comprising an expression step of expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector.

5. The method for producing a novel coronavirus infection sequelae model animal according to claim 4, wherein the expression step expresses SARS-CoV-2 S1 protein in at least one of the nasal cavity and the peri-nasal cavity of the non-human mammal.

6. The method for producing a novel coronavirus infection sequelae model animal according to claim 4, further comprising an inflammation induction step of inducing inflammation in the non-human mammal.

7. The method for producing a novel coronavirus infection sequelae model animal according to any one of claims 4 to 6, wherein the SARS-CoV-2 S1 protein is a polypeptide of the following (a) or (b): (a) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1; (b) A polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and having the activity of increasing intracellular calcium concentration when introduced into a cell.

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