Treatment drugs for post-COVID-19 complications

Abstract

Provided is a drug for treating aftereffects of a novel coronavirus infection. The drug for treating aftereffects of the novel coronavirus infection contains an acetylcholine receptor agonist as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a therapeutic agent for sequelae of coronavirus disease, a method for screening a therapeutic agent for sequelae of coronavirus disease, and a method for producing an animal model of sequelae of coronavirus disease.

Background Art

[0002] The acute respiratory disease COVID-19 (so-called coronavirus disease) caused by the SARS-CoV-2 virus is a serious infectious disease that has caused many infected people worldwide.

[0003] In addition to causing severe diseases in the acute phase and leading to death of patients, COVID-19 is known to frequently cause sequelae (hereinafter, "sequelae of coronavirus disease") in the recovery phase. Among the sequelae of coronavirus disease, fatigue, depressive symptoms, and olfactory disorders are said to be particularly frequent. In addition, since sequelae of coronavirus disease also occur in patients with less severe acute-phase symptoms, it is considered highly likely to occur even when the progression to severe cases is suppressed by a vaccine.

[0004] Since the sequelae of coronavirus disease are sequelae of virus infection as the name indicates, the growth of the causative SARS-CoV-2 virus does not occur at the time of the occurrence of the sequelae. Therefore, even if a drug capable of suppressing virus growth is administered after the occurrence of the sequelae of coronavirus disease, no effect on the sequelae can be expected. For this reason, it is considered that a therapeutic agent for sequelae of coronavirus disease needs to repair tissue damage caused by SARS-CoV-2 virus infection.

[0005] Regarding the treatment of sequelae of coronavirus disease, there is currently no effective one. For this reason, unsubstantiated folk remedies are rampant, and health hazards have also occurred.

Prior Art Documents

Non-Patent Documents

[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. [Overview of the project] [Problems that the invention aims to solve]

[0007] There is a need for treatments for the long-term effects of COVID-19. However, there are currently no effective treatments. In order to conduct research on the long-term effects of COVID-19 in order to develop treatments, animal models that exhibit the symptoms of long-term effects of COVID-19 are important. However, until now, animal models related to the novel coronavirus have mainly been used in research on infection prevention and acute symptoms, so the development of animal models suitable for research on long-term effects is insufficient.

[0008] One aspect of the present invention aims to provide a therapeutic agent for treating post-COVID-19 sequelae. [Means for solving the problem]

[0009] As a result of diligent research to solve the above problems, the inventors have for the first time discovered that the S1 region of the SARS-CoV-2 virus Spike protein is the causative protein of post-COVID-19 sequelae, and have completed an animal model exhibiting the symptoms of post-COVID-19 sequelae. As a result of research on post-COVID-19 sequelae using the post-COVID-19 sequelae model animal, the inventors have for the first time discovered that acetylcholine in the brain is reduced in post-COVID-19 sequelae, and that post-COVID-19 sequelae can be treated or prevented by receptor agonists.

[0010] In other words, one aspect of the present invention is a treatment for post-COVID-19 sequelae, comprising an acetylcholine receptor agonist as an active ingredient.

[0011] Furthermore, one aspect of the present invention includes an administration step of administering a test substance to a non-human mammal model animal expressing the SARS-CoV-2 S1 protein and exhibiting post-COVID-19 sequelae, The method for screening for the treatment of post-COVID-19 sequelae includes an evaluation step of evaluating changes in symptoms related to the novel coronavirus before and after administration of the test substance in the aforementioned animal model.

[0012] Furthermore, one aspect of the present invention is a method for producing a model animal of post-COVID-19 sequelae, 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. [Effects of the Invention]

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

[0014] [Figure 1]It is a schematic diagram showing the structure of the SARS-CoV-2 Spike protein. [Figure 2] It is a diagram showing the results of the examples and the results of measuring the intracellular calcium concentration in 3T3 cells and A549 cells transformed with the S1 protein expression plasmid or the control plasmid. [Figure 3] It is a diagram showing the results of the examples and the results of measuring the intracellular calcium concentration in 3T3 cells and A549 cells infected with the S1 protein expression adenovirus or the control adenovirus. [Figure 4] It is a diagram showing the results of the examples and the results of the 10% weighted forced swimming test in control mice and S1 protein-expressing mice (S1 mice). [Figure 5] It is a diagram showing the results of the examples and the results of the tail suspension test in control mice and S1 mice. [Figure 6] It is a diagram showing the results of the examples and the results of analyzing the expression level of the calbindin gene in the olfactory bulb after LPS administration in control mice and S1 mice. [Figure 7] It is a diagram showing the results of the examples and the results of analyzing the expression level of the calbindin gene in the brain after LPS administration in control mice and S1 mice. [Figure 8] It is a diagram showing the results of the examples and showing the impairment of cholinergic neurons in control mice and S1 mice. [Figure 9] It is a diagram showing the results of the examples and showing the impairment of cholinergic neurons in control mice and S1 mice. [Figure 10] It is a diagram for explaining the administration scheme of donepezil. [Figure 11] It is a diagram showing the results of the examples and the results of the 10% weighted forced swimming test after administration of donepezil in control mice and S1 mice. [Figure 12] It is a diagram showing the results of the examples and the results of the tail suspension test after administration of donepezil in control mice and S1 mice. [Figure 13]It is a diagram showing the results of the examples and presenting the analysis results of the gene expression levels of interleukin 6 (IL-6), tumor necrosis factor (TNFα), and chemokine CC motif ligand 2 (CCL2) after administration of donepezil in control mice and S1 mice. [Figure 14] It is a diagram showing the results of the examples and presenting the analysis results of the gene expression levels of interleukin 1 beta (IL-1β) and interleukin 6 (IL-6) after administration of PNU282987 in control mice and S1 mice. [Figure 15] It is a diagram showing the results of the examples and presenting the analysis results of the gene expression level of ZFP36 after administration of PNU282987 in control mice and S1 mice.

Mode for Carrying Out the Invention

[0015] <1. Therapeutic Agent> (Features) The therapeutic agent for post-COVID-19 sequelae (hereinafter referred to as "post-COVID-19 sequelae") according to one aspect of the present invention is a pharmaceutical composition used for the treatment or prevention of post-COVID-19 sequelae and contains an acetylcholine receptor agonist as an active ingredient. Thereby, post-COVID-19 sequelae can be treated or prevented in patients infected with the novel coronavirus. Therefore, it can contribute to Goal 3 of the Sustainable Development Goals (SDGs), "Ensure healthy lives and promote well-being for all". Hereinafter, for the sake of convenience of explanation, the therapeutic agent for post-COVID-19 sequelae according to one aspect of the present invention may be simply referred to as "therapeutic agent".

[0016] In this specification, "treatment" includes curing or alleviating the symptoms of post-COVID-19 sequelae and suppressing the aggravation of the symptoms of post-COVID-19 sequelae in a subject (hereinafter simply referred to as "administration subject") to whom the therapeutic agent according to one aspect of the present invention is administered. In the present invention, "prevention" includes suppressing or delaying the onset of the symptoms of post-COVID-19 sequelae in the administration subject.

[0017] If the recipient is experiencing symptoms of post-COVID-19 complications, the therapeutic agent according to one aspect of the present invention is used for the treatment of post-COVID-19 complications. If the recipient is not experiencing symptoms of post-COVID-19 complications, the therapeutic agent according to one aspect of the present invention is used for the prevention of post-COVID-19 complications.

[0018] In this specification, the definitions of "treatment" and "prevention" are as stated above, but in either case, the mechanism of action of the therapeutic agent according to one aspect of the present invention is the same. Therefore, "therapeutic agent" can be replaced with "preventive agent." That is, if a patient infected with the novel coronavirus does not develop symptoms of long-term COVID-19, "treatment" means "prevention."

[0019] Here, the term "post-COVID-19 symptoms" generally refers to a range of symptoms that appear during the recovery period after COVID-19 infection and persist for several weeks to several months, but its definition is not yet established. Furthermore, post-COVID-19 symptoms are sometimes referred to as "Long COVID." In this specification, "post-COVID-19 symptoms" refers to symptoms related to brain and nerve dysfunction, particularly those that occur frequently among the above-mentioned symptoms, such as fatigue, depression, olfactory dysfunction, memory impairment, decreased concentration, impaired thinking ability, and cognitive decline.

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

[0021] In this specification, "depressive symptoms related to COVID-19" refers to the symptoms of a disorder generally diagnosed as depression. These symptoms include not only those used to diagnose major depressive disorder according to the DSM-5, such as depressed mood, loss of interest or pleasure, loss of appetite, overeating, sleep disturbances, hypersomnia, psychomotor agitation or retardation, fatigue, feelings of worthlessness or guilt, decreased concentration or cognitive function, and suicidal ideation, but also depressive states exhibiting symptoms frequently seen in patients with depression, such as anxiety, memory impairment, aging, pain, and chronic pain. In other words, "depressive symptoms related to COVID-19" in this specification is a concept that includes not only major depressive disorder, but also stress-induced depression, depressive states in bipolar disorder, and negative-type depression, as well as symptoms in depressive states in other disorders.

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

[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 subjected to continuous physical or mental stress.

[0024] In this specification, "olfactory dysfunction related to the novel coronavirus" refers to symptoms or diseases that cause some kind of abnormality in the sense of smell, or the sense of odor. It is also called "olfactory abnormality." The main symptoms include olfactory attenuation, paresophagia, anosmia, and olfactory hypersensitivity.

[0025] In this specification, "memory impairment related to COVID-19" means a condition in which one or all of the four processes that constitute memory—encoding, retention, retrieval, and recognition—do not function properly.

[0026] In this specification, "symptoms" refers to phenomena and conditions (abnormalities) that appear in patients due to the effects of a disease involving the novel coronavirus. This concept includes subjective symptoms, which are abnormalities that the patient themselves can perceive 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 tests. In addition, in the animal models described later, behavioral changes and objective findings from examinations, etc., are collectively referred to as symptoms.

[0027] The inventors studied brain changes associated with post-COVID-19 sequelae using animal models of COVID-19. As a result, they were the first to discover that the number of cholinergic neurons, which are choline acetyltransferase (ChAT)-positive cells, is reduced in the septal area (MS) and Broca's diagonal zone (DB) of the basal forebrain in patients with post-COVID-19 sequelae.

[0028] These results suggest that acetylcholine levels are reduced in post-COVID-19 patients, and that acetylcholine receptor agonists are suitable as treatments for post-COVID-19 complications.

[0029] The inventors of this invention have, through research on post-COVID-19 sequelae using animal models of COVID-19 sequelae, discovered for the first time that administering acetylcholine receptor agonists improves depressive and fatigue symptoms expressed in these animal models. In other words, they have discovered for the first time that administering acetylcholine receptor agonists can treat or prevent post-COVID-19 sequelae.

[0030] Traditionally, it was common knowledge that administering acetylcholine receptor agonists increased acetylcholine levels in the brain, leading to the development of depressive symptoms (for example, 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] Furthermore, it was common technical knowledge that smoking exacerbates and worsens the symptoms of COVID-19, and that nicotine is the cause (for example, 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 acetylcholine levels in the brain through the administration of acetylcholine receptor agonists can improve depressive symptoms related to the novel coronavirus expressed in animal models of post-COVID-19 sequelae is completely contrary to the results that would be expected from conventional technological understanding of the effects of acetylcholine receptor agonists. Furthermore, this result suggests that depressive symptoms due to post-COVID-19 sequelae (i.e., "depressive symptoms related to the novel coronavirus") develop through a different mechanism than depression caused by elevated acetylcholine levels.

[0033] (Active ingredients) A therapeutic agent according to one aspect of the present invention contains an acetylcholine receptor agonist as an active ingredient. In this specification, "acetylcholine receptor agonist" refers to both indirect acetylcholine receptor agonists that increase the amount of acetylcholine in the brain by means of cholinesterase inhibition, etc., and direct acetylcholine receptor agonists that act by directly binding to receptors. In one aspect of the present invention, the acetylcholine receptor agonist may be a peripheral acetylcholine receptor agonist that acts via the parasympathetic nervous system, etc., or a central acetylcholine receptor agonist that acts on receptors in the brain. As mentioned above, post-COVID-19 symptoms are related to dysfunction of the brain and nerves, and since it has the advantage of being able to act directly on the site where the dysfunction occurs, it is preferable that the acetylcholine receptor agonist is a central acetylcholine receptor agonist that acts on acetylcholine receptors in the brain. "Acetylcholine receptor agonist" is also called a cholinergic agonist.

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

[0035] As a direct-acting acetylcholine receptor agonist, it is preferable that the substance can cross the blood-brain barrier to reach the brain and has the effect of activating acetylcholine receptors. More specifically, direct-acting acetylcholine receptor agonists include acetylcholine and its precursors, as well as agonists of acetylcholine receptors (muscarinic receptors or nicotinic receptors).

[0036] Indirect acetylcholine receptor agonists are preferably substances that can cross the blood-brain barrier to reach the brain and have acetylcholinesterase inhibitory activity. More specifically, examples of indirect acetylcholine receptor agonists include donepezil (2-[(1-benzyl-4-piperidinyl)methyl]-5,6-dimethoxyindan-1-one hydrochloride); rivastigmine (2,6-dioxo-4-phenyl-piperidine-3-carbonitride); metrifonate (O,O-dimethyl-2,2,2-trichloro-1-hydroxyethylphosphonate); tacrine (1,2,3,4-tetrahydro-9-aminoacridin); and galantamine (galantamine hydrobromide).

[0037] Furthermore, among the acetylcholine receptor agonists exemplified, donepezil is already used as a treatment for Alzheimer's disease, etc., making drug repositioning possible. Therefore, it has advantages such as (i) human safety and pharmacokinetic studies can be shortened or omitted, (ii) since side effects are known, it is possible to select an active ingredient that is less burdensome to patients, and (iii) since the method of producing the active ingredient is already established, the drug price can be kept low. Accordingly, in one embodiment of the present invention, donepezil is preferred as the acetylcholine receptor agonist.

[0038] Furthermore, acetylcholine receptor agonists may be in the form of "derivatives" or "pharmacologically acceptable salts," as long as they have the effect of increasing the amount of acetylcholine in the brain. In other words, in this specification, "acetylcholine receptor agonist" is a concept that includes its "derivatives" as well as "pharmacologically acceptable salts."

[0039] In this specification, "derivative" refers to a group of compounds resulting from the substitution of a portion of a particular compound's molecule with another functional group or atom. Examples of 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 other atoms include carbon atoms, hydrogen atoms, oxygen atoms, nitrogen atoms, sulfur atoms, phosphorus atoms, and halogen atoms.

[0040] As derivatives, prodrugs that exhibit the desired activity in vivo (under in vivo conditions) through hydrolysis, oxidation, or enzymatic reactions can also be used.

[0041] In this specification, "pharmaceutically acceptable salt" means a salt that is physiologically acceptable to administer to a subject as a pharmaceutical, and is not limited to specific examples. Examples of salts include alkali metal salts (such as potassium salts), alkaline earth metal salts (such as calcium salts and magnesium salts), ammonium salts, organic base salts (such as trimethylamine salts, triethylamine salts, pyridine salts, picoline salts, dicyclohexylamine salts, and N,N'-dibenzylethylenediamine salts), organic acid salts (such as acetate salts, maleate salts, tartrate salts, methanesulfonate salts, benzenesulfonate salts, formate salts, toluenesulfonate salts, and trifluoroacetate salts), and inorganic acid salts (such as hydrochloride salts, hydrobromide salts, sulfate salts, and phosphate salts).

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

[0043] (Other ingredients) A therapeutic agent according to one aspect of the present invention may contain components other than the aforementioned active ingredient (acetylcholine receptor agonist). The components other than the active ingredient may be any pharmaceutically acceptable components, such as buffers, pH adjusters, isotonic agents, preservatives, antioxidants, high molecular weight polymers, excipients, and solvents.

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

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

[0046] Examples of the aforementioned isotonic agents include ionic isotonic agents (such as sodium chloride, potassium chloride, calcium chloride, and magnesium chloride) and nonionic isotonic agents (such as glycerin, propylene glycol, sorbitol, and mannitol).

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

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

[0049] Examples of the aforementioned high molecular weight polymers include methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose acetate succinate, hydroxypropylmethylcellulose phthalate, carboxymethylethylcellulose, cellulose phthalate acetate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, and atelocollagen.

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

[0051] Examples of the aforementioned solvents include water, physiological saline solution, and alcohol.

[0052] A therapeutic agent according to one aspect of the present invention may contain a pharmacoactive ingredient having a desired effect (e.g., reduction of side effects) as one of the aforementioned components, and may be used in combination with a drug having a desired effect.

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

[0054] The amount of components other than the active ingredient in the therapeutic agent according to one aspect of the present invention is not particularly limited. For example, the amount of components other than the active ingredient may be 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, relative to the total weight of the therapeutic agent according to one aspect of the present invention.

[0055] (Target recipients) An example of a target population for the therapeutic agent according to one aspect of the present invention is a person suspected of having or confirmed to have contracted the novel coronavirus. Such a target population may be one who has not received a medical diagnosis for post-coronavirus complications.

[0056] The target recipients of the therapeutic agent according to one aspect of the present invention are not particularly limited and may be humans or non-human mammals (e.g., livestock, pets, and laboratory animals). Examples of non-human mammals include monkeys, chimpanzees, cattle, 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 can be administered to the target by any route of administration. Examples of routes of administration include oral administration, parenteral administration, transdermal administration, transmucosal administration, and intravenous administration. Therefore, the dosage form of the therapeutic agent according to one aspect of the present invention may be an oral drug, a topical drug, or an injectable drug. Oral administration is preferred as the route of administration of the therapeutic agent according to one aspect of the present invention because it is easy to administer and places little burden on the target. Therefore, the dosage form of the therapeutic agent according to one aspect of the present invention is preferably an oral drug.

[0058] (Formulations and prescriptions) Using an acetylcholine receptor agonist as the active ingredient and other components as raw materials, a therapeutic agent according to one aspect of the present invention can be formulated by known methods.

[0059] When administering a therapeutic agent according to one aspect of the present invention to a subject, there are no restrictions on the amount administered to the subject, as long as the desired effect is obtained. For example, in a therapeutic agent according to one aspect of the present invention, the amount of the active ingredient, an acetylcholine receptor agonist, may be administered at a dose of 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 to 10.0 mg / kg body weight, or 1.0 to 5.0 mg / kg body weight.

[0060] When administering a therapeutic agent according to one aspect of the present invention to a subject, there are no restrictions on the administration interval as long as the desired effect is obtained. 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 every 1 month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, or once every year. The administration interval may also be constant, but it may also be administered as needed (as-needed) when symptoms of post-COVID-19 sequelae appear severely.

[0061] A therapeutic agent according to one aspect of the present invention may be administered, for example, once a day, with a dose of the active ingredient, an acetylcholine receptor agonist, being 1.0 to 5.0 mg / kg body weight.

[0062] <2. Drug Screening Methods> (Features) A screening method for COVID-19 post-inflammatory drugs according to one aspect of the present invention is a method for screening COVID-19 post-inflammatory drugs that have therapeutic or preventive effects on COVID-19 post-inflammatory drugs using a COVID-19 post-inflammatory drug model animal in which the SARS-CoV-2 S1 protein is expressed in a non-human mammal, and includes an administration step of administering a test substance to the COVID-19 post-inflammatory drug model animal, and an evaluation step of evaluating changes in symptoms related to the novel coronavirus in the COVID-19 post-inflammatory drug model animal before and after administration of the test substance. This makes it possible to screen for COVID-19 post-inflammatory drugs. As a result, it will lead to the development of new drugs for treating COVID-19 post-inflammatory drugs. Therefore, it can contribute to Sustainable Development Goal (SDG) 3, "Good Health and Well-being." Hereinafter, for the sake of convenience of explanation, the screening method for COVID-19 post-inflammatory drugs according to one aspect of the present invention may be simply referred to as a "drug screening method."

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

[0064] In animal models of post-COVID-19 sequelae, the SARS-CoV-2 S1 protein may be expressed transiently or constitutively. Transgenic animals may also be used. It is preferable that the expression of the SARS-CoV-2 S1 protein is transient, and that the expression of the SARS-CoV-2 S1 protein in the animal models of post-COVID-19 sequelae has ended or decreased by the time of use in the administration step of this screening method, as this allows for detailed examination of the symptoms at the time the expression ceases or is reduced.

[0065] Furthermore, since it is possible to efficiently induce post-COVID-19 sequelae, it is preferable that the post-COVID-19 sequelae model animal is a non-human mammal model animal in which the SARS-CoV-2 S1 protein is expressed in at least one of the nasal cavity and surrounding area. Inflammation may be induced in the post-COVID-19 sequelae model animal before the step of administering the test substance.

[0066] The method for expressing the SARS-CoV-2 S1 protein in non-human mammals is not particularly limited. For example, the SARS-CoV-2 S1 protein can be expressed in non-human mammals by the method for producing COVID-19 sequela model animals described later, thereby producing COVID-19 sequela model animals.

[0067] (Administration process) The administration process involves administering the test substance to an animal model of COVID-19 sequelae. The dosage and method of administration of the test substance are not particularly limited. They 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 involves assessing changes in symptoms related to the novel coronavirus before and after administration of the test substance in animal models of COVID-19 sequelae.

[0069] In the evaluation process, the method for evaluating changes in symptoms related to the novel coronavirus in post-COVID-19 model animals is not particularly limited. For example, in the evaluation process, changes in symptoms related to the novel coronavirus in post-COVID-19 model animals can be evaluated by quantifying changes in the activity levels of the post-COVID-19 model animals.

[0070] As a method for quantifying changes in the activity levels of COVID-19 post-symptomatic model animals, behavioral experiments such as the 10% weighted forced swimming test and the tail suspension test, described in the examples, can be performed before and after administration of the test substance. This allows for the quantification of changes in the activity levels of COVID-19 post-symptomatic model animals before and after administration of the test substance.

[0071] Furthermore, in the evaluation process, for example, changes in symptoms related to the novel coronavirus in COVID-19 post-COVID-19 model animals may be confirmed by quantifying changes in the expression levels of inflammatory markers in the brains of the animal model animals. Examples of inflammatory markers include interleukin-6 (IL-6), tumor necrosis factor (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 sequela model animals, it is not possible to quantify the changes in inflammatory marker expression levels in the brains of COVID-19 sequela model animals before and after administration of the test substance in the same individual. Therefore, by comparing the measurement of inflammatory marker expression levels described above between the test substance administration group and the placebo administration group, it is possible to quantify the changes in inflammatory marker expression levels in the brains of COVID-19 sequela model animals before and after administration of the test substance.

[0073] In the evaluation process, the therapeutic or preventive effect of the test substance on post-COVID-19 sequelae should be evaluated based on the results of the evaluation of changes in symptoms related to the novel coronavirus. For example, in the evaluation process, if changes in symptoms related to the novel coronavirus in post-COVID-19 sequelae model animals are evaluated by quantifying changes in their activity levels, and fatigue or depressive symptoms related to the novel coronavirus are improved before and after administration of the test substance, then the test substance can be evaluated as having activity to treat or prevent post-COVID-19 sequelae.

[0074] Furthermore, for example, in the evaluation process, if the changes in the expression levels of inflammatory markers in the brains of COVID-19 sequelae model animals are quantified to evaluate changes in symptoms related to the novel coronavirus in these animals, and the expression levels of inflammatory markers in the brains are significantly reduced before and after administration of the test substance, then the test substance can be evaluated as having activity to treat or prevent COVID-19 sequelae.

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

[0076] In one embodiment of the present invention, a drug screening method can also be used to narrow down the number of effective test substances. For example, Example 6, described later, showed that acetylcholine was deficient in the brains of patients with post-COVID-19 sequelae. Therefore, acetylcholine receptor agonists can be designated as high-priority candidate drugs.

[0077] Furthermore, in Example 10, described later, it was shown that ventricular administration of PNU282987, an α7 nicotinic receptor agonist that does not cross the blood-brain barrier, has an effect of suppressing brain inflammation. This result suggests that α7 nicotinic receptor agonists should be given higher priority in future screenings. This indicates that even drugs that are not practical therapeutic candidates because they do not cross the blood-brain barrier can be used in ways such as suggesting methodologies for drug screening through methods such as intraventricular administration.

[0078] While it was known that α7 nicotinic receptor agonists have immunosuppressive functions, the mechanism was unclear. Example 11, described later, suggests that the mechanism involves increased expression of the immunosuppressive molecule ZFP36 by α7 nicotinic receptor agonists. This result suggests that clarifying the mechanism of a drug's therapeutic effect during the screening process can lead to the development of better efficacy assessment methods and the identification of drug target molecules.

[0079] <3. Method for manufacturing model animals> (Features) A method for producing a COVID-19 sequela model animal according to one aspect of the present invention 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 makes it possible to produce a COVID-19 sequela model animal in which the SARS-CoV-2 S1 protein is expressed in a non-human mammal. The COVID-19 sequela model animal can be used as an experimental animal in the development of methods for treating or preventing COVID-19 sequelae, particularly in the development of drugs such as therapeutic and preventive drugs. Furthermore, the COVID-19 sequela model animal can be used as an experimental animal in research on the causes of COVID-19 sequelae. Therefore, it can contribute to Sustainable Development Goal (SDG) 3, "Ensure healthy lives and promote well-being for all."

[0080] (Types of model animals) The COVID-19 sequela model animals produced by the method for producing COVID-19 sequela model animals according to one aspect of the present invention are not particularly limited in type, as long as they are non-human mammals (mammals other than humans) that can be used as experimental animals. Therefore, the type 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 being produced. Examples of non-human mammals targeted in the expression step include mice, rats, guinea pigs, dogs, rabbits, monkeys, chimpanzees, and the like.

[0081] While large animals such as monkeys are known to be effective in treating COVID-19 infections, 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, it is preferable that COVID-19 long-term disease model animals be small animal models such as mouse models.

[0082] (Expression process) The expression process involves 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, post-COVID-19 symptoms can be induced in the non-human mammal.

[0083] In general, viral infections trigger the production of inflammatory cytokines due to an immune response, which are known to cause fatigue and depressive symptoms during the acute phase of infection. However, in post-COVID-19 syndrome, fatigue and depressive symptoms are observed that are more severe than those seen with other viruses, and these symptoms persist as sequelae. For this reason, it is expected that the novel coronavirus possesses a protein with strong activity that causes neurological damage during infection. Therefore, the inventors diligently investigated to identify this protein, and as a result, they have for the first time discovered that the S1 region of the SARS-CoV-2 virus's Spike protein (1273 amino acids, GenBank accession number YP 009724390) is the causative protein of post-COVID-19 syndrome.

[0084] Handling model animals created by infecting them with the infectious SARS-CoV-2 virus itself requires advanced containment facilities such as P3 facilities. On the other hand, the method for producing COVID-19 sequelae model animals according to one aspect 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 causative protein of COVID-19 sequelae, in non-human mammals. Therefore, model animals produced by the method for producing COVID-19 sequelae model animals according to one aspect of the present invention can be used in normal experimental environments and are easy to handle.

[0085] Model animals created by infecting them with the infectious SARS-CoV-2 virus itself may not exhibit the same level of pathogenicity in acute infection and sequelae as humans. This can lead to low survival rates in model animals with acute symptoms and insufficient development of sequelae to serve as a model. Therefore, model animals produced by the method for producing COVID-19 sequelae model animals according to one aspect of the present invention are superior in that they efficiently and reliably provide model animals for COVID-19 sequelae.

[0086] In this specification, "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] The site in which the SARS-CoV-2 S1 protein is expressed in the body of the target non-human mammal is not particularly limited, but it is preferable to express the SARS-CoV-2 S1 protein in at least one of the nasal cavity and surrounding area of ​​the non-human mammal during the expression process, as this allows for efficient induction of post-COVID-19 sequelae. To express the SARS-CoV-2 S1 protein in at least one of the nasal cavity and surrounding area of ​​the non-human mammal, for example, the SARS-CoV-2 S1 protein expression vector may be administered into the nasal cavity of the non-human mammal during the expression process.

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

[0089] The SARS-CoV-2 S1 protein may be, for example, one of the following polypeptides: (a) or (b): (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 number YP_009724390; (b) A polypeptide having 80% or more sequence identity with the amino acid sequence (SEQ ID NO: 1) consisting of amino acids from the 1st to the 685th amino acids of the amino acid sequence shown in GeneBank accession number YP_009724390, and which has the activity to increase 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, as in (b) above, consisting of an amino acid sequence (SEQ ID NO: 1) comprising the amino acids from the 1st to the 685th amino acids of the amino acid sequence shown in GeneBank accession number YP_009724390, with a sequence identity of 80% or more (85% or more, 90% or more, 95% or more, 98% or more, 99% or more), and having the activity to increase intracellular calcium concentration when introduced into cells. In this specification, the amino acid sequence identity percentage is a value calculated using the genetic information processing software GENETYX Ver.7 (manufactured by Genetics).

[0092] Furthermore, the polypeptide in (b) may be a polypeptide having an amino acid sequence in which 100 or fewer amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence (SEQ ID NO: 1) consisting of the 1st to 685th amino acids of the amino acid sequence shown in GeneBank accession number YP_009724390, and which has the activity to increase intracellular calcium concentration when introduced into cells. In this specification, "100 or fewer amino acids substituted, deleted, inserted, and / or added" means that 100 or fewer (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) amino acids are substituted, deleted, inserted, and / or added by known mutant peptide production methods such as site-directed mutagenesis. Thus, the polypeptide in (b) can be said to be a variant of the polypeptide in (a). In this context, "mutation" primarily refers to mutations artificially introduced using known methods for producing mutant proteins, but it may also refer to similar mutant proteins isolated and purified from nature.

[0093] Mutant strains of the SARS-CoV-2 virus have been reported to date, and they are known to have mutations in the spike protein. The main mutations in the SARS-CoV-2 S1 protein in the mutant strains reported to date are as follows. The polypeptide in (b) preferably has these mutations. SARS-CoV-2 B.1.1.7 strain (so-called "alpha strain"): deletion69-70, deletion144-145, N501Y, A570D, and D614G, P681H • SARS-CoV-2 B.1.351 strain (so-called "beta strain"): D80A, D215G, Deletion241-243, K417N, E484K, N501Y, and D614G SARS-CoV-2 P.1 strain (so-called "gamma strains"): L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, and H655Y SARS-CoV-2 lineage B.1.617.2 (the so-called "Delta strain"): T19R, G142D, E156G, deletion157-158, L452R, T487K, E484Q, D614G, and P681R SARS-CoV-2 B.1.1.529 strain (the so-called "Omicron strain"): G142D, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, and P681H

[0094] The activity of a mutant SARS-CoV-2 S1 protein to increase intracellular calcium concentration can be confirmed by expressing the mutant protein in any cultured cell 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 the mutant protein-expressing cell is significantly higher than that in the parent cell, it can be determined that the mutant protein has the activity to increase intracellular calcium concentration.

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

[0096] Plasmid vectors or viral vectors can be any known vector commonly used in genetic engineering, as long as they are capable of expressing the SARS-CoV-2 S1 protein in non-human mammals; the type is not particularly limited. Viral vectors are preferably used because they have high infection efficiency and are easy to introduce into non-human mammals. Examples of viral vectors that are particularly preferably used include adenovirus vectors, which are commonly used for known purposes such as gene therapy and viral vaccines.

[0097] The promoter for expressing the SARS-CoV-2 S1 protein is not particularly limited in type, as long as it is capable of expressing mRNA in cells infected with SARS-CoV-2. A promoter that can produce SARS-CoV-2 S1 protein to the same extent as during SARS-CoV-2 infection is particularly preferable.

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

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

[0100] The method for obtaining the polynucleotide encoding the SARS-CoV-2 S1 protein is not particularly limited. For example, a method using amplification means such as PCR can be used. For instance, 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 PCR can be performed using these primers with genomic DNA (or cDNA) as a template. By amplifying the DNA region sandwiched between the two primers, a large amount of DNA fragments containing the polynucleotide encoding the SARS-CoV-2 S1 protein can be obtained. Alternatively, a polynucleotide having the base sequence of the polynucleotide encoding the SARS-CoV-2 S1 protein may be synthesized using known chemical synthesis methods based on gene sequence information. The base sequence may be optimized for the codon usage frequency of the animal being used.

[0101] In the expression process, the expression level of SARS-CoV-2 S1 protein should be adjusted considering 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 post-COVID-19 symptoms.

[0102] In the expression step of the method for producing a COVID-19 sequela model animal according to one aspect of the present invention, the SARS-CoV-2 S1 protein may be transiently expressed or constitutively expressed in a non-human mammal. In research on COVID-19 sequelae using COVID-19 sequela model animals, it is desirable to be able to examine the symptoms at the point when the expression of the SARS-CoV-2 S1 protein has ended or decreased. Therefore, in the expression step, it is preferable to transiently express the SARS-CoV-2 S1 protein in a non-human mammal.

[0103] (Inflammation induction process) A method for producing a COVID-19 sequela model animal according to one aspect of the present invention preferably further includes an inflammation induction step in which inflammation is induced 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 including an inflammation induction step in a method for producing a COVID-19 sequela model animal according to one aspect of the present invention, it is possible to produce a model animal that also takes into account the conditions in actual SARS-CoV-2 virus infection.

[0104] While there are no particular limitations on the method of inducing inflammation in non-human mammals, it is preferable to induce inflammation by drugs or other means rather than by viral infection, from the viewpoint of ease of handling the created 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 E. coli O111. LPS is known to have the biological activity of activating macrophages.

[0105] The inflammation induction step is performed after the expression step. Performing the inflammation induction step after the expression step allows for observation of the inflammation induction effect in animal model cells exhibiting post-COVID-19 sequelae. The inflammation induction step may also be performed before the expression step.

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

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

[0108] The degree of fatigue can be evaluated by performing a forced swimming test with a 10% weight, as shown in the examples described later. The degree of depressive symptoms can also be evaluated by performing a tail suspension test, as shown in the examples described later.

[0109] By performing the evaluation step, it can be confirmed that the model animal produced by the method for producing a COVID-19 sequela model animal according to one aspect of the present invention is indeed exhibiting symptoms of COVID-19 sequela. 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, and the degree of inflammation induced in the inflammation induction step can be appropriately adjusted.

[0110] <4. Kit for creating animal models of COVID-19 sequelae>

[0111] A kit for producing a COVID-19 sequela model animal according to one aspect 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 aspect of the present invention can be suitably used in a method for producing a COVID-19 sequela model animal according to one aspect of the present invention.

[0112] In the present invention, "kit" refers to a package containing a container (e.g., a bottle, plate, tube, dish, etc.) that contains specific materials. A kit according to one aspect of the present invention may be in a form in which each of the materials contained therein exists independently, or in a form in which multiple materials are mixed together (e.g., in the form of a composition). Preferably, the kit includes instructions for using each material.

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

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

[0115] <5. Animal models of COVID-19 sequelae> A COVID-19 sequela model animal produced by the method for producing a COVID-19 sequela model animal according to one aspect of the present invention is also included in the scope of the present invention. Since the COVID-19 sequela model animal according to one aspect of the present invention is produced by the method for producing a COVID-19 sequela model animal according to one aspect of the present invention, it does not require advanced containment facilities such as P3 facilities and can be used in a normal experimental environment, thus offering excellent handling.

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

[0117] <6. Treatment or prevention methods for long-term effects of COVID-19> A method for treating or preventing post-COVID-19 sequelae using a therapeutic agent for post-COVID-19 sequelae according to one aspect of the present invention is also included within the scope of the present invention.

[0118] In other words, a method for treating or preventing post-COVID-19 complications according to one aspect of the present invention is as follows: <16> A method for treating or preventing post-COVID-19 sequelae, comprising the step of administering a COVID-19 sequelae treatment drug containing an acetylcholine receptor agonist as an active ingredient to a subject (e.g., a human or a non-human animal). <17> The aforementioned acetylcholine receptor agonist is a centrally acting acetylcholine receptor agonist that acts on acetylcholine receptors in the brain. <16> The treatment or prevention methods for post-COVID-19 complications described herein. <18> The acetylcholine receptor agonist is donepezil. <16> or <17> The treatment or prevention methods for post-COVID-19 complications described herein. <19> The aforementioned post-COVID-19 symptoms are fatigue related to the novel coronavirus. <16> ~ <18> A method of treatment or prevention of post-COVID-19 complications described in any one of the following. <20> The aforementioned post-COVID-19 symptoms are depressive symptoms related to the novel coronavirus. <16> ~ <18> A method of treatment or prevention of post-COVID-19 complications described in any one of the following. <21> The aforementioned post-COVID-19 symptom is olfactory dysfunction related to the novel coronavirus. <16> ~ <18> A method of treatment or prevention of post-COVID-19 complications described in any one of the following. <22> The aforementioned post-COVID-19 syndrome is memory impairment related to the novel coronavirus. <16> ~ <18> A method of treatment or prevention of post-COVID-19 complications described in any one of the following.

[0119] The target population, route of administration, formulation, and prescription of the treatment for post-COVID-19 sequelae are as described for the treatment according to one aspect of the present invention and will not be repeated here. Oral administration of the treatment for post-COVID-19 sequelae is preferable because it is easy to administer and places little burden on the target population.

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

[0121] In other words, one aspect of the present invention is used as follows: <23> Use of acetylcholine receptor agonists for the manufacture of treatments for the long-term effects of COVID-19. <24> The aforementioned acetylcholine receptor agonist is a centrally acting acetylcholine receptor agonist that acts on acetylcholine receptors in the brain. <23> Use as described above. <25> The acetylcholine receptor agonist is donepezil. <23> or <24> Use as described above. <26> The aforementioned post-COVID-19 symptoms are fatigue related to the novel coronavirus. <23> ~ <25> Use as described in any one of the following. <27> The aforementioned post-COVID-19 symptoms are depressive symptoms related to the novel coronavirus. <23> ~ <25> Use as described in any one of the following. <28> The aforementioned post-COVID-19 symptom is olfactory dysfunction related to the novel coronavirus. <23> ~ <25> Use as described in any one of the following. <29> The aforementioned post-COVID-19 syndrome is memory impairment related to the novel coronavirus. <23> ~ <25> Use as described in any one of the following.

[0122] The present invention is not limited to the embodiments described above, 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. [Examples]

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

[0124] <Method> Construction of expression vectors for mammalian cells 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 incorporated 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 "S1 protein") is 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 number YP_009724390. The base sequence of the DNA fragment encoding the S1 protein is publicly available in GenBank accession number MN908947.

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

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

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

[0128] To measure the intracellular calcium concentration in cells expressing the S1 protein using the method described above, 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 ArrayScan XT (ThermoFisher).

[0129] <Result> As shown in Figure 1, the S1 protein used to construct the expression vector is a protein having the amino acid sequence (SEQ ID NO: 1) consisting of amino acids 1 through 685 of the amino acid sequence of the SARS-CoV-2 Spike protein, which is represented by 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 Figure 2. "Intensity / area" shown on the vertical axis of the graph in Figure 2 represents the fluorescence intensity per unit area. "Control" shown on the horizontal axis of the graph in Figure 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 bar shown in Figure 2 represents the median. ****: P < 0.0001.

[0131] As shown in Figure 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 expression adenovirus or the control adenovirus was measured. The results are shown in Figure 3. "Intensity / area" shown on the vertical axis of the graph in Figure 3 represents the fluorescence intensity per unit area. "Control" shown on the horizontal axis of the graph in Figure 3 represents the cells infected with the control adenovirus, and "S1 / Adv" represents the cells infected with the S1 protein expression adenovirus. Also, the horizontal bar shown in Figure 3 represents the median. ****: P < 0.0001.

[0133] As shown in Figure 3, the intracellular calcium concentration increased due to the expression of the S1 protein in all cells (Figure 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 (S1 mice) [Method] [Preparation of S1 protein-expressing mice (S1 mice)] C57BL / 6 mice aged 8-9 weeks were anesthetized with isoflurane, and 1 x 10⁶ mice were subjected to a smear test. 9 S1 mice (COVID-19 sequela model animals) were created by administering 25 μL of an ifu / mL S1 / Adv solution intranasally and allowing them to inhale naturally (expression step). They were then returned to their home cages and reared for one week. As a control, mice (control mice) were administered intranasally with an empty adenovirus vector (vector / Adv) that did not express anything.

[0136] <Fatigue Behavior Test> Six days after intranasal administration of S1 / Adv or vector / Adv, a 10% weighted forced swim test was performed as a fatigue behavior test. The method involved measuring the weight of S1 mice and control mice on the morning of the test day, and preparing a weight equivalent to approximately 10% of their body weight. This weight was attached to the tail of either the S1 or control mouse, and the mouse was placed in a water tank for the forced swim test. The time it took for the tip of the snout to sink underwater 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 (in seconds) from when the mouse was placed in the forced swimming test tank until its nose was submerged underwater for 10 seconds. The "control" on the horizontal axis of the graph in Figure 4 represents the control mouse, and "S1" represents the S1 mouse. ‡: P<0.1.

[0138] Compared to control mice, S1 mice tended to have shorter swimming times. This indicates that S1 mice tire more easily than control mice.

[0139] [Example 3] Expression of depression-like behavior in S1 mice <Method> S1 mice and control mice were prepared using the same method as in Example 2. A tail suspension test was performed to confirm depressive-like behavior. The method involved fixing the tails of S1 mice and control mice six days after intranasal administration of S1 / Adv or vector / Adv, suspending them for 10 minutes, recording the process, and analyzing the images using the image analysis software TailSuspScan (CleverSys Inc.) to measure the immobility time.

[0140] <Result> Figure 5 shows the plot of immobility time during the tail suspension test. In the graph in Figure 5, "Control" on the horizontal axis represents the control mouse, and "S1" represents the S1 mouse. *: P<0.05.

[0141] Compared to control mice, S1 mice showed a significantly increased immobility time, indicating that S1 mice exhibit depressive-like behaviors.

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

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

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

[0145] RT-qPCR analysis revealed a decreasing trend in calbindin expression in the olfactory bulb of S1 mice 30 minutes after LPS administration, and a significant decrease at 60 minutes after LPS administration. Since calbindin is a marker of mature neurons, it was therefore considered 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 was induced by LPS. <Method> S1 mice and control mice were prepared using the same method as in Example 2. Seven days after intranasal administration of S1 / Adv or vector / Adv, S1 mice and control mice were intraperitoneally administered 5 mg / kg of lipopolysaccharide derived from E. coli O111 (MERCK), and the brains excluding the olfactory bulb were collected 15 minutes later.

[0147] RNA was purified using the RNeasy Mini Kit (QIAGEN) from the collected brain tissue. cDNA was synthesized using the PrimeScript RT reagent Kit (Takara Bio) with the purified RNA as a template. Calbindin gene expression was analyzed using the synthesized cDNA via RT-qPCR. The 18S rRNA measurement results were used for standardization.

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

[0149] RT-qPCR analysis revealed a decreasing trend in calbindin expression in the brains of S1 mice 15 minutes after LPS administration. Therefore, it was considered that mature neurons in the brains of S1 mice were damaged when peripheral inflammation was induced.

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

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

[0152] These results indicate that S1 mice have impaired cholinergic neurons in their brains. Therefore, it is suggested that S1 mice may have reduced levels of acetylcholine in their brains.

[0153] [Example 7] Improvement of fatigue symptoms in S1 mice by administered donepezil <Method> <Administration of donepezil> Donepezil (Fujifilm Wako Pure Chemical Industries) was dissolved in water to a concentration of 32 mg / L and administered to mice via drinking water. This resulted in a donepezil dose of 4.0 mg / kg / day to the mice. Since many studies suggest a donepezil dose of 3.0–5.0 mg / kg / day, we adopted 4.0 mg / kg / day.

[0154] Figure 10 shows the administration scheme for donepezil. 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 swim test was performed as a fatigue behavior test. The method involved measuring the weight of S1 mice and control mice on the morning of the test day, and preparing a weight equivalent to approximately 10% of their body weight. This weight was attached to the tails of the S1 and control mice, and they were placed in a water tank for the forced swim test. The time it took for their noses to sink underwater 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 (in seconds) from when the mouse was placed in the forced swimming test tank until its nose was submerged underwater for 10 seconds. The "control" on the horizontal axis of the graph in Figure 11 represents the control mouse, and "S1" represents the S1 mouse. *: P<0.05.

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

[0158] [Example 8] Improvement of depressive-like behavior in S1 mice by donepezil administration <Method> In S1 mice and control mice that received donepezil via drinking water after intranasal administration of S1 / Adv or vector / Adv according to the administration scheme shown in Figure 10, a tail suspension test was performed to confirm depressive-like behavior. The method involved fixing the tails of S1 mice and control mice six days after intranasal administration of S1 / Adv or vector / Adv, suspending them for 10 minutes, recording the process, and analyzing the images using the image analysis software TailSuspScan (CleverSys Inc.) to measure immobility time.

[0159] <Result> Figure 12 shows the plot of immobility time during the tail suspension test. In the graph in Figure 12, "Control" on the horizontal axis represents the control mouse, and "S1" represents the S1 mouse. *: P<0.05, ***: P<0.001.

[0160] In the group not administered donepezil (marked "-" in Figure 12), S1 mice showed a significantly increased immobility time compared to control mice. In contrast, in the group administered donepezil (marked "donepezil" in Figure 12), S1 mice showed a significantly decreased immobility time compared to control mice. These results suggest that donepezil improves depression-like behaviors caused by S1 protein expression.

[0161] [Example 9] Improvement of brain inflammation in S1 mice by donepezil administration. <Method> In S1 mice and control mice that received donepezil via drinking water after intranasal administration of S1 / Adv or vector / Adv according to the administration scheme shown in Figure 10, the brain tissue excluding the olfactory bulb was collected on day 7 after intranasal administration of S1 / Adv or vector / Adv. RNA was purified from the collected brain tissue using the RNeasy Mini Kit (QIAGEN). cDNA was synthesized using the PrimeScript RT reagent Kit (Takara Bio) with the purified RNA as a template. Gene expression of interleukin-6 (IL-6), tumor necrosis factor (TNFα), and chemokine CC motif ligand 2 (CCL2) was analyzed by RT-qPCR using the synthesized cDNA. The 18S rRNA measurement results were used for standardization.

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

[0163] RT-qPCR analysis revealed that in the group not administered donepezil (marked "-" in Figure 13), IL-6 expression in the brains of S1 mice was significantly elevated, and the expression of other genes, TNFα and CCL2, also showed an upward trend. In contrast, in the group administered donepezil (marked "donepezil" in Figure 13), the expression of these genes in the brains of S1 mice showed a downward trend, and no difference was observed compared to the expression of these genes in the brains of control mice. Therefore, it was shown that brain inflammation is induced in S1 mice due to S1 protein expression, and that this brain inflammation is improved by the administration of donepezil.

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

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

[0166] <Result> The results of the RT-qPCR analysis are shown in Figure 14. In the graph in Figure 14, "Control" on the horizontal axis represents control mice, and "S1" represents S1 mice. **: P<0.01, ‡: P<0.1. The increased expression levels of inflammatory cytokine genes (IL-1β and IL-6) in the brain, which are thought to be the molecular mechanisms of fatigue and depression, observed in S1 mice, were suppressed by intracerebroventricular administration of PNU282987. This result suggests that PNU282987 and its derivatives may be candidate therapeutic agents for brain inflammation in S1 mice, and also suggests that α7 nicotinic acetylcholine receptor agonists should be prioritized when screening for therapeutic agents.

[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 encephalitis in Example 10, the effects of S1 and PNU282987 on the gene expression level of zinc finger protein 36 (Zfp36), a host protein with anti-inflammatory effects, were investigated. Specifically, Zfp36 gene expression was analyzed by RT-qPCR using cDNA synthesized in Example 10. The 18S rRNA measurement results were used for standardization.

[0168] <Result> The results of the RT-qPCR analysis are shown in Figure 15. In the graph in Figure 15, "Control" on the horizontal axis represents control mice, and "S1" represents S1 mice. *: P<0.05, **: P<0.01. As a result, S1 mice showed a decrease in ZFP36, and it was found that the decrease in ZFP36 was improved by administration of PNU282987. This suggests that the encephalitis in S1 mice is due to a decrease in ZFP36, which has anti-inflammatory effects, and that acetylcholine receptor agonists, especially α7 nicotinic acetylcholine receptor agonists, exert therapeutic effects against encephalitis by restoring ZFP36 expression. These results suggest that ZFP36 is a target for therapeutic drugs and that the expression level of the ZFP36 gene can serve as a biomarker for therapeutic drug screening.

[0169] The results above demonstrate that a model mouse for post-COVID-19 sequelae can be created by expressing the S1 protein in mice, that cholinergic neurons in the brain of this model mouse are impaired, and that the symptoms of post-COVID-19 sequelae in this model mouse are improved by the administration of donepezil. Furthermore, analysis of the mechanism of action of acetylcholine receptor agonists indicated that α7 nicotinic acetylcholine receptor agonists should be prioritized among acetylcholine receptor agonists when screening for treatments for post-COVID-19 sequelae. [Industrial applicability]

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

Claims

[Claim 1] It is a treatment for long-term effects of COVID-19, It contains an acetylcholine receptor agonist as an active ingredient, The acetylcholine receptor agonist is donepezil or PNU282987. The aforementioned post-COVID-19 symptoms are fatigue or depressive symptoms related to COVID-19. A drug used to treat long-term effects of COVID-19.

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