WO2022163852A1 - Composition for inhibiting expression of angiotensin converting enzyme 2 (ace2) and/or tmprss2 - Google Patents

Composition for inhibiting expression of angiotensin converting enzyme 2 (ace2) and/or tmprss2 Download PDF

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WO2022163852A1
WO2022163852A1 PCT/JP2022/003677 JP2022003677W WO2022163852A1 WO 2022163852 A1 WO2022163852 A1 WO 2022163852A1 JP 2022003677 W JP2022003677 W JP 2022003677W WO 2022163852 A1 WO2022163852 A1 WO 2022163852A1
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composition
insulin
foxo1
inhibitor
gly
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Japanese (ja)
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直樹 漆畑
勝幸 隠岐
誠一 吉原
啓介 大西
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株式会社 バイオミメティクスシンパシーズ
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Priority claimed from JP2021017835A external-priority patent/JP6906127B1/en
Priority claimed from JP2021068032A external-priority patent/JP6953047B1/en
Application filed by 株式会社 バイオミメティクスシンパシーズ filed Critical 株式会社 バイオミメティクスシンパシーズ
Priority to CN202280011900.9A priority Critical patent/CN117999099A/en
Publication of WO2022163852A1 publication Critical patent/WO2022163852A1/en

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    • AHUMAN NECESSITIES
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    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47Quinolines; Isoquinolines
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    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47Quinolines; Isoquinolines
    • A61K31/47064-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
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    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Definitions

  • ACE2 angiotensin converting enzyme 2
  • TMPRSS2 Transmembraneprotease, serine 2
  • COVID-19 The infectious disease (COVID-19) caused by the new coronavirus (SARS-CoV2), which is said to have started infecting humans from the end of 2019, is raging all over the world, and research and development of vaccines against the virus and its therapeutic drugs are being carried out in various countries. is actively carried out.
  • SARS-CoV2 new coronavirus
  • COVID-19 develops, it triggers an excessive inflammatory reaction called cytokine storm, which was initially thought to cause death by causing abnormal pneumonia symptoms. Since then, it has become clear that symptoms are observed not only in the lungs but also in the blood vessels, the cranial nervous system, the liver, the kidneys, and other parts of the body.
  • Non-Patent Document 1 summarizes the knowledge about SARS-CoV2 so far.
  • an object of the present disclosure is to provide a composition for suppressing the gene expression level (mRNA level) of ACE2 and/or TMPRSS2, which is also related to the treatment/prevention of COVID-19.
  • invention 1 A composition for inhibiting the expression of angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2, comprising: A composition, wherein the composition comprises a FoxO1 inhibitor.
  • invention 2 The composition according to Invention 1, wherein the FoxO1 inhibitor is a compound represented by the following formula or a salt thereof.
  • R1 is NH or CH2 ;
  • R5 is S or O;
  • R6 is a carboxyl group,
  • R7 is NH2 or CH3 , n is 1-11.
  • Invention 3 The composition of Invention 1, wherein the inhibitor is a compound represented by the following formula or a salt thereof.
  • R 1 is NH;
  • R 2 is NR 8 (R 8 is an alkyl group of C1 to C3);
  • R3 is F , Cl, Br, or I; each R4 is independently H or CH3 ;
  • R5 is O;
  • R6 is a carboxyl group,
  • R7 is NH2 ;
  • n is 1-11.
  • (Invention 4) The composition of invention 1, wherein said inhibitor is AS1842856 or AS1708727.
  • the composition of invention 1, wherein said FoxO1 inhibitor is an insulin-based compound.
  • Invention 6) The composition of Invention 5, wherein said insulin-based compound is wild-type insulin.
  • invention 7) The composition of invention 5, wherein said insulin-based compound is a modified insulin.
  • invention 8 The composition of invention 7, wherein the modified insulin is one or more selected from ultra-rapid-acting insulin, fast-acting insulin, intermediate-acting insulin, long-acting soluble insulin, mixed-acting insulin, and combined soluble insulin. .
  • invention 9 The composition of invention 7, wherein said modified insulin has an amino acid sequence that is 90% or more identical to human wild-type insulin.
  • invention 10 The composition of invention 7, wherein said modified insulin has an amino acid sequence that is 95% or more identical to human wild-type insulin.
  • invention 11 11.
  • the composition of invention 9 or 10 wherein said modified insulin A chain has an amino acid sequence that is 100% identical to that of human wild-type insulin.
  • invention 12 The composition of invention 1, wherein said FoxO1 inhibitor is a growth factor and/or a nucleic acid encoding same.
  • invention 13 The composition of invention 12, wherein said growth factor has 90% or more amino acid sequence identity with any selected from: IGF1, IGF2, EGF, FGF2, FGF14, VEGF, HGF.
  • invention 14 The composition of invention 13, wherein said growth factor has 95% or more amino acid sequence identity with any selected from: IGF1, IGF2, EGF, FGF2, FGF14, VEGF, HGF.
  • invention 15 The composition of invention 14, wherein said growth factor has 100% or more amino acid sequence identity with any selected from: IGF1, IGF2, EGF, FGF2, FGF14, VEGF, HGF.
  • invention 16 The composition of Invention 1, wherein the FoxO1 inhibitor is a FoxO1-acting inhibitor.
  • invention 17 The composition of invention 1, wherein said FoxO1 inhibitor is a PI3K/PDK1/Akt acting inhibitor.
  • invention 18 The composition of Invention 1, wherein the FoxO1 inhibitor is a FoxO1 gene expression inhibitor.
  • invention 19 19. The composition according to any one of Inventions 1 to 18, which is a composition for preventing or treating coronavirus infection (excluding diabetic patients).
  • Invention 20 A composition for preventing or treating a coronavirus infection, the composition comprising a FoxO1 inhibitor (excluding diabetic patients).
  • a composition of the present disclosure comprises a FoxO1 inhibitor. This can reduce the expression level of the ACE2 and/or TMPRSS2 gene.
  • Fig. 3 is a graph showing the relative expression levels of ACE2 and TMPRSS2 genes in HepG2 cells with the addition of a forkhead transcription factor FoxO1 inhibitor AS1842856 (concentrations shown below the graph). Furthermore, it is a schematic diagram of the expression induction mechanism of ACE2 and TMPRSS2 genes by FoxO1. Schematic representation of the cellular uptake assay of the recombinant protein (RBD-mFc) of the ACE2 binding domain (RBD) in the SARS-CoV2 surface protein spike protein.
  • RBD-mFc recombinant protein
  • RBD ACE2 binding domain
  • the RBD is fused with a mouse IgG-derived Fc sequence (mFc), and is incorporated into cells by immunostaining using a fluorescence-labeled antibody that recognizes mouse IgG (for example, Alexa Fluor (R) 488 antibody). It is possible to quantify the amount of RBD added.
  • mFc mouse IgG-derived Fc sequence
  • R Alexa Fluor 488 antibody
  • Recombinant protein (RBD-mFc) of the incorporated RBD protein was quantified using flow cytometry, and graphs showing relative mean fluorescence intensity.
  • FIG. 3 is a schematic diagram showing the relationship between the suppression of ACE2 and/or TMPRSS2 expression by the forkhead transcription factor FoxO1 inhibitor AS1842856 and the suppression of binding and uptake with spike protein.
  • Fig. 10 is a graph showing relative expression levels of ACE2 and TMPRSS2 genes in HepG2 cells to which wild-type insulin was added.
  • FIG. 10 is a graph showing the relative expression levels of ACE2 and TMPRSS2 genes in HepG2 cells with the addition of modified insulin.
  • FIG. Figure 10 is a graph showing the relative mean fluorescence intensity of S1 and RBD protein uptake quantified using flow cytometry.
  • FIG. 2 is a schematic diagram showing the relationship between suppression of ACE2 and/or TMPRSS2 expression by insulin and suppression of binding/uptake to spike protein.
  • 2 is a graph showing relative expression levels of ACE2 and TMPRSS2 genes in HepG2 cells to which various growth factors were added.
  • 2 is a graph showing relative expression levels of ACE2 and TMPRSS2 genes in HepG2 cells with the addition of HGF.
  • FIG. 10 is a graph showing the relative expression levels of ACE2 and TMPRSS2 genes in HepG2 cells with the addition of siRNA.
  • the term "to inhibit the expression of angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2" encompasses the purpose of ameliorating a particular disease associated with ACE2 and/or TMPRSS2.
  • the term is not limited to therapeutic purposes.
  • the term encompasses the purpose of preventing the development of a particular disease even if one does not have that disease.
  • FluoroxO1 inhibitor described in this specification has a broad meaning and a narrow meaning, but is interpreted in the former meaning unless otherwise specified.
  • the term means an inhibitor that acts by directly binding to FoxO1 (herein, in the narrow sense, it is also referred to as a "FoxO1-acting inhibitor").
  • the term means an inhibitor that directly or indirectly acts on FoxO1 to inhibit the activity of FoxO1 as a transcription factor.
  • the term includes inhibitors that act on signaling pathways upstream of FoxO1.
  • a drug that directly acts on Akt and PDK1 upstream of FoxO1 to activate them.
  • Another example is a ligand for a receptor that affects the activity of FoxO1 (more specifically triggers a signaling pathway that suppresses the activity of FoxO1).
  • Receptors include insulin receptors, growth factor receptors, and the like.
  • the term encompasses insulin receptor ligands, growth factor receptor ligands.
  • the term further includes substances that inhibit the expression of "FoxO1" itself.
  • Typical examples include nucleic acids, such as antisense nucleic acids, shRNA, siRNA, miRNA and the like.
  • the definition of the term “inhibits the activity of FoxO1 as a transcription factor” can be determined by an analysis method known in the art. For example, performing a luciferase assay using a consensus sequence (GTAAA(T/C)AA) known to bind to FoxO1, and discriminating based on whether there is a significant difference in fluorescence intensity, etc. can be done. Alternatively, a commercially available ELISA-type activity detection assay (Active Motif, TransAMRFKHR (FOXO1), product number: 46396) may be used.
  • the addition of the test substance reduced the average value of the index of FOXO1 activity to 60%, compared with the value indicating the activity of FOXO1 in the control conditions in which water or a solution used as a solvent (DMSO, etc.) was added. % or less is defined as "inhibiting the activity of FoxO1 as a transcription factor.”
  • compositions for suppressing the expression level of angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2 used the present disclosure provides A composition, said composition relating to a composition comprising a FoxO1 inhibitor.
  • any one or more of the following FoxO1 inhibitors may be included as active ingredients in the composition.
  • the disclosure relates to the use of any one or more of these active ingredients for the manufacture of pharmaceutical compositions.
  • ⁇ Insulin compounds ⁇ Growth factors or nucleic acids encoding them ⁇ FoxO1-acting inhibitors and/or PI3K/PDK1/Akt-acting inhibitors ⁇ FoxO1 gene expression inhibitors do.
  • compositions for Inhibiting Angiotensin Converting Enzyme 2 (ACE2) and/or TMPRSS2 Expression Using Insulin-Based Compounds comprise an insulin-based compound (eg, human insulin). This inhibits transcription of the gene for ACE2 and/or TMPRSS2 and reduces the amount of mRNA.
  • an insulin-based compound eg, human insulin
  • the "insulin-based compound” described herein means an agent that activates the insulin signaling pathway inside cells. Therefore, the “insulin-based compound” described herein includes not only the so-called “wild-type insulin” expressed in the human body, but also the “modified insulin” produced using genetic modification technology. Further, it can contain an agent that acts on pancreatic ⁇ -cells in vivo to promote the expression of insulin.
  • a preferred "insulin-based compound” is wild-type insulin or modified insulin. Examples of modified insulins include ultra-rapid-acting insulins, fast-acting insulins, intermediate-acting insulins, long-acting soluble insulins, mixed-acting insulins, combined soluble insulins.
  • the modified insulin may have an amino acid sequence identical to that of human wild-type insulin to some extent. For example, 90% or more, preferably 95% or more (eg, identity determined using blastp or the like). Wild-type insulin is a heterodimer of A chain and B chain. Therefore, the identity described here means the identity when the A chain and the B chain are linked and compared as one amino acid sequence. More preferably, the A-chain of the modified insulin has 100% amino acid sequence identity with the A-chain of human wild-type insulin.
  • Insulin acts as a factor that inhibits the transcription of the angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2 genes. Therefore, the expression of these genes can be suppressed by adding insulin. This makes it possible to reduce the uptake of viruses into cells using the spike protein.
  • ACE2 angiotensin converting enzyme 2
  • TMPRSS2 TMPRSS2
  • compositions for Suppressing the Expression Level of Angiotensin Converting Enzyme 2 (ACE2) and/or TMPRSS2 Using Growth Factors comprises a growth factor. This inhibits transcription of the gene for ACE2 and/or TMPRSS2 and reduces the amount of mRNA.
  • growth factor means an entity that satisfies all of the following conditions. - At least partially containing a peptide or protein - Suppressing the activity of the intracellular FoxO1 transcription factor
  • the peptide or protein may be a naturally occurring substance or an artificially produced substance.
  • chemically modified naturally occurring proteins or peptides are also included.
  • Peptides or proteins may be in dimeric or higher forms as well as in monomeric form.
  • Peptides or proteins may be non-linear, eg cyclic.
  • an artificial linker may be appropriately provided. For example, according to a paper by Kenichiro Ito et al. (Nat Commun.
  • HGF HGF It has been reported that it functions as an agonist of the receptor.
  • growth factor receptor agonists that have not existed in the past may be developed. are also intended to be included in the term "growth factor" as used herein.
  • the species of the growth factor is not particularly limited, it is preferably human type.
  • growth factors include, but are not limited to, any one or more of the following: IGF1, IGF2, EGF, FGF2, FGF14, VEGF, HGF.
  • another growth factor may have an amino acid sequence that is somewhat identical to any of the growth factors listed above. For example, 90% or more, preferably 95% or more, and most preferably 100% (eg, identity determined using blastp or the like).
  • nucleic acids encoding the above growth factors may be incorporated into the composition.
  • Nucleic acids can be DNA and can be RNA (eg, mRNA).
  • the compositions of the present disclosure can contain the DNA described above in any form.
  • the coding region of DNA described above may be incorporated into a plasmid, vector, or the like.
  • compositions for suppressing the expression level of angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2 using a FoxO1-acting inhibitor and/or a PI3K/PDK1/Akt-acting inhibitor include FoxO1-acting inhibitors and/or PI3K/PDK1/Akt-acting inhibitors. This inhibits transcription of the gene for ACE2 and/or TMPRSS2 and reduces the amount of mRNA.
  • PI3K/PDK1/Akt-acting inhibitor means an agent that satisfies all of the following three conditions. ⁇ Permeate the cell membrane to increase intracellular PIP 3 and/or act directly on at least one of PI3K, PDK1 and Akt Changing at least one activity ⁇ As a result of changing the activity, suppressing the activity of the intracellular FoxO1 transcription factor
  • PI3K/PDK1/Akt-acting inhibitor means a substance that exhibits an activating effect on PI3K/PDK1/Akt, although it is called an “inhibitor.” It is called an “inhibitor” in the sense that it consequently suppresses the activity of FoxO1.
  • Whether or not the transcription factor activity of FoxO1 is suppressed can be determined by a desired assay, as described in "1. Definition" above.
  • PIP3 is a second messenger generated by signals triggered by binding of receptors and ligands.
  • PIP 3 is generated by PI3K.
  • the generated PIP3 activates PDK1.
  • activated PDK1 activates Akt.
  • Activated Akt inhibits the activity of FoxO1. Therefore , by increasing intracellular PIP3 and/or directly acting on and activating at least one of PI3K, PDK1, and Akt, the activity of FoxO1 as a transcription factor is inhibited. can be done. This inhibits transcription of the gene for ACE2 and/or TMPRSS2 and reduces the amount of mRNA.
  • the action inhibits the transcription of the ACE2 and/or TMPRSS2 gene and reduces the amount of mRNA.
  • the FoxO1-acting inhibitor and/or the PI3K/PDK1/Akt-acting inhibitor preferably acts on each in the following manner. ⁇ Action to inhibit the activity of FoxO1 ⁇ Action to increase PIP3 , ⁇ Action to promote the activity of at least one of PI3K, PDK1, and Akt
  • the drug having an activity-inhibiting effect on FoxO1 includes, for example, compounds represented by the following formulas or salts thereof.
  • R1 is NH or CH2 ;
  • R 2 is NR 8 or CH-R 8 (R 8 : C1-C3 alkyl group),
  • R3 is F , Cl, Br, or I;
  • each R4 is independently H or CH3 ;
  • R5 is S or O;
  • R6 is a carboxyl group,
  • R7 is NH2 or CH3 , n is 1-11.
  • examples of agents that directly bind to and inhibit FoxO1 include compounds represented by the following formulas or salts thereof.
  • R 1 is NH
  • R 2 is NR 8 (R 8 is an alkyl group of C1 to C3)
  • R3 is F , Cl, Br, or I
  • each R4 is independently H or CH3
  • R5 is O
  • R6 is a carboxyl group
  • R7 is NH2
  • n is 1-11.
  • a specific example of the compound represented by the above formula or a salt thereof is AS1842856 (CAS No. 836620-48-5).
  • AS1708727 is an example of a drug that has the effect of inhibiting the activity of FoxO1.
  • agents having an activity-inhibiting effect on FoxO1 include KIS-154, chalcones, triterpenes, flavones, monoterpenoids, lignans, coumarins, and FOXO1 activity inhibitors consisting of phytochemicals. (Re-Table 2018/079715) and the like.
  • examples of drugs having an activity-promoting effect on PI3K/PDK1/Akt include the following compounds.
  • VO-Ohpic trihydrate SF1670 bpV (HOpic) These compounds are known to have the effect of indirectly promoting the activity of PI3K/PDK1/Akt by inhibiting PTEN, which acts oppositely to PI3K.
  • compositions for Suppressing the Expression Level of Angiotensin Converting Enzyme 2 (ACE2) and/or TMPRSS2 Using FoxO1 Gene Expression Inhibitor comprises a FoxO1 gene expression inhibitor. This reduces the expression level of FoxO1 itself. Then, the transcription of the ACE2 and/or TMPRSS2 gene is decreased, and the amount of mRNA is decreased.
  • FoxO1 gene expression inhibitors include inhibition of FoxO1 gene expression using siRNA (small interference RNA), miRNA (microRNA), or the like.
  • compositions of the present disclosure can be used to treat and/or prevent diseases involving angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2. Specifically, they can be used for treatment and/or prevention of infectious diseases in which they are involved.
  • infectious diseases include coronavirus infections, eg infections by viruses such as SARS-CoV or SARS-CoV2. Preferably, it is an infection caused by SARS-CoV2 (COVID19). These viruses bind their spike proteins to the ACE2 receptors of human cells and use this as a springboard to enter cells.
  • RNA viruses including SARS-CV2 are prone to mutation.
  • proteins on the surface of viruses for example, spike proteins
  • vaccines there is a problem that the effects of vaccines can be avoided due to mutations in these proteins.
  • this approach has the advantage that it is less directly affected by mutations.
  • composition of the present disclosure can contribute not only to the treatment of infection but also to the prevention of infection.
  • approaches to treatment of infection based on anti-inflammatory effects but these approaches are countermeasures after infection has occurred and cannot contribute to prevention.
  • the compositions of the present disclosure can also serve as therapeutic agents in the sense that, once infection has occurred and the virus has propagated, it prevents further infection of other cells.
  • it not only that, but even before infection, by administering it in advance, it suppresses the expression of genes that are a stepping stone for virus infection, thereby preventing cell infection. It can also be used as a medicine.
  • compositions of the present disclosure may be formulated with suitable additional ingredients depending on the dosage form.
  • compositions of the present disclosure may include ingredients commonly included in pharmaceuticals. Examples of such ingredients include preservatives, preservatives, fragrances, pH adjusters, antioxidants, antifungal agents, excipients, binders, disintegrants, lubricants, and the like.
  • the FoxO1 inhibitors described herein are not intended for administration to diabetic patients.
  • Example 4-1 Example 1 (HepG2 cell culture) HepG2 cells were purchased from ATCC® (American Type Culture Collection, HB-8065). The cells were subcultured and maintained using a serum medium of DMEM (SIGMA, D8900) + 10% FBS (SIGMA, F7524).
  • SIGMA serum medium of DMEM
  • FBS F7524
  • Example 2 (Effect of FoxO1 inhibitor AS1842856) 4-2-1. Preparation of RNA HepG2 cells were seeded into each well of a 12-well plate (Corning (registered trademark), 3336) at 50,000 cells/cm 2 and cultured overnight. The next day (16-24 hours after seeding), the FoxO1 inhibitor AS1842856 was added at multiple concentrations (1 nM, 10 nM, 50 nM, 100 nM, 1 ⁇ M) or DMSO, which was used as its solvent, as a control. time) cultured. After culturing, total RNA was extracted from HepG2 cells using ReliaPrep RNA Miniprep system (Promega (registered trademark), Z6012).
  • cDNA synthesis (PrimeScript RT Master Mix; Takara (registered trademark), RR036A) was performed using 500 ng of RNA, and quantitative PCR (Thunderbird (registered trademark) Sybr qPCR Mix; TOYOBO (registered trademark), QPS -201X5) was performed.
  • a mixture for cDNA synthesis was prepared according to the following composition. 2 ⁇ l of 5x PrimeScript RT Master Mix (final concentration 1x) Total RNA 500 ng Adjusted to 10 ⁇ l total of RNase free H 2 O
  • the above mixture was processed using a Veriti 96 well Thermal Cycler manufactured by Applied Biosystems (registered trademark) under the following conditions. 37°C 15 minutes ⁇ 85°C 5 seconds ⁇ 4°C ⁇
  • the synthesized cDNA (10 ⁇ l) was diluted 10-fold with 90 ⁇ l of TE (10 mM Tris-HCl pH 8.0 + 1 mM EDTA pH 8.0). The dilutions were subjected to quantitative PCR.
  • cDNA was amplified from the above mixture using Bio-Rad (registered trademark) CFX-Connect.
  • the PCR cycle conditions were as follows. 1.95°C 1 minute (initial denaturation) 2.95°C 15 seconds (denaturation) 3.60°C 30 seconds (elongation) (Repeat steps 2-3 40 times, detect fluorescence signal each time step 3 is completed) 4. Raise the temperature from 65°C to 95°C in increments of 0.5°C, hold the temperature for 5 seconds, and then detect the fluorescence signal.
  • GAPDH Glyceraldehyde 3- phosphate dehydrogenase
  • the primer sequences for detecting each gene were as follows.
  • the expression level of each gene indicates the standardized value obtained by dividing the expression level of each gene obtained by quantitative PCR by the expression level of GAPDH. Furthermore, the expression level under the control condition (DMSO treatment) without FoxO1 inhibitor treatment is normalized to "1". It was confirmed by a melting curve that the PCR products amplified using the above primers were all single (that is, multiple types of sequences were not amplified with the same primers).
  • the incorporated mFc was stained, and the fluorescence intensity of Alexa Fluor (registered trademark) 488 per cell was measured and quantified using flow cytometry (Beckman Coulter (registered trademark), FC500). Kaluza Analysis 2.1 (Beckman Coulter (registered trademark)) was used for the analysis. Photographs of typical cells were taken using a fluorescence microscope (Keyence, BX-700).
  • FIG. 4 shows the difference in fluorescence intensity.
  • the fluorescence intensity exhibited by the cells when mFc, which is a negative control, was added was taken up only by mFc without mediated by ACE2, was nonspecifically bound by the secondary antibody, and autofluorescence of cells, which is considered to be a background signal. Subtracting the fluorescence intensity at the mFc therefore allows an accurate comparison of signal differences from more accurately incorporated RBDs.
  • FIG. 5 summarizes the mechanism of action of the FoxO1 inhibitor AS1842856 of the present disclosure, which is shown by the above experimental results, to inhibit the uptake of the novel coronavirus into cells.
  • AS1842856 acts on intracellular FoxO1 to suppress gene expression of ACE2 and/or TMPRSS2. This reduces the amount of ACE2 and/or TMPRSS2 on the cell surface. Then, the binding between the spike protein present on the virus surface and ACE2 is inhibited. In addition, the effect of TMPRSS2 to promote virus entry into cells is also suppressed. As a result, it provides preventive or therapeutic effects for infectious diseases, particularly coronaviruses, and other diseases mediated by ACE2 and/or TMPRSS2.
  • Example 3 Effect of insulin
  • HepG2 cells were seeded in each well of a 12-well plate (Corning (registered trademark), 3336) at 50,000 cells/cm 2 and cultured overnight. The next day (16-24 hours after seeding), wild-type and modified insulin were added at 10 ⁇ g/ml, or PBS( ⁇ ) was added as a control, and cultured for 3 days (about 72 hours). After culturing, total RNA extraction, cDNA synthesis, and quantitative PCR were performed as described in Example 2 above.
  • Insulin is categorized into modified insulins such as fast-acting insulin, rapid-acting insulin, intermediate-acting insulin, etc., depending on when mutations are introduced into amino acids by genetic engineering and how long the effect appears after administration and how long it lasts. It is manufactured and used properly according to the condition of the diabetic patient. Therefore, using these modified insulins, it was examined whether or not reduction in expression of ACE2 and TMPRSS2 mRNAs by wild-type insulin was observed.
  • modified insulins such as fast-acting insulin, rapid-acting insulin, intermediate-acting insulin, etc.
  • Wild-type insulin consists of a total of 51 amino acid sequences in which the A chain and B chain are linked.
  • the amino acid sequence of insulin mentioned above is shown below.
  • Isophane insulin also known as NPH insulin
  • NPH insulin is a product obtained by binding and crystallizing wild-type (regular) insulin and protamine.
  • Protamine crystalline insulin aspart is a combination and crystallization of protamine and insulin aspart.
  • Example 4 (Effect of insulin on spike protein uptake into cells) Next, we investigated whether suppression of ACE2 expression by insulin could inhibit the binding of SARS-CoV2 to host cells and their uptake into cells.
  • the Fc region of mouse IgG is fused to the S1 domain of the spike protein of SARS- CoV2 , and to the region RBD (Receptor Binding Domain, R319 to F541 ) necessary and sufficient for binding to ACE2 in S1.
  • Recombinant protein S1-mFc (Sino Biological, 40592-V05H1)
  • RBD-mFc (Sino Biological, 40592-V05H2) were prepared.
  • this result indicates that insulin inhibited the uptake of S1 and RBD into HepG2 cells, and that insulin-induced suppression of mRNA expression levels such as ACE2 inhibited the uptake of S1 and RBD into HepG2 cells. It suggests that it is suppressed to a level that can be suppressed.
  • FIG. 9 summarizes the mechanism of action by which the insulin-based compound of the present disclosure inhibits the cellular uptake of the novel coronavirus in one embodiment, as shown by the above experimental results.
  • Insulin-based compounds act on cells to suppress the expression of the ACE2 and/or TMPRSS2 genes. This reduces the amount of ACE2 and/or TMPRSS2 on the cell surface. Then, the binding between the spike protein present on the virus surface and ACE2 is inhibited. In addition, the effect of TMPRSS2 to promote virus entry into cells is also suppressed. As a result, it provides preventive or therapeutic effects for infectious diseases, particularly coronaviruses, and other diseases mediated by ACE2 and/or TMPRSS2.
  • Example 5 (Effects of growth factors) Experiments similar to Examples 1-4 were conducted. However, instead of various insulin compounds, the following growth factors were used at a concentration of 10 ng/ml. These are known to suppress FoxO1 as a downstream signal pathway. IGF1 (PeproTech, product number AF-100-11), IGF2 (PeproTech, product number AF-100-12), EGF (PeproTech, product number AF-100-15), FGF2 (PeproTech, product number AF-100-18B), FGF14 (BioVision®, product number 7347), VEGF165 (PeproTech, product number AF-100-20),
  • amino acid sequences of the above growth factors are as follows.
  • VEGF 165 https://www.peprotech.com/en/recombinant-human-vegfsub165sub-2 Ala Pro Met Ala Glu Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu
  • Example 6 Effect of HGF
  • HGF R&D systems, product number 294-HGN-005/CF
  • HGF is widely known to repress FoxO1 as a downstream intracellular signaling pathway after binding to its receptor.
  • HGF is highly effective in preventing or treating infections, especially coronaviruses, and other diseases mediated by ACE2 and/or TMPRSS2.
  • the amino acid sequence of HGF is as follows. https://www.uniprot.org/uniprot/P14210 Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe Gly His Glu Phe Asp Leu Tyr Glu Asn Ly
  • Example 7 (Effect of siRNA) Experiments similar to Examples 1-4 were conducted. However, siRNA was used to suppress the gene expression of FoxO1. As a control, siRNAs that do not suppress anything (Cell Signaling (registered trademark), product number 6568S) and those targeting the human FoxO1 gene (#1; Cell Signaling (registered trademark), product number 6242S, and # 2; Cell Signaling (registered trademark), product number 6256S, sequence information including controls not disclosed) was used. A transfection reagent (Horizon Discovery, DharmaFECT1, product number T2001-01) was used to introduce siRNA into cells. Three days (approximately 72 hours) after introduction of various siRNAs, cells were harvested and quantified by qPCR as described above.
  • FoxO1 inhibitors in general, compounds that function as FoxO1 inhibitors, even though the method of inhibition is greatly different, such as via a signal transduction pathway or in the case of direct inhibition (inhibitors, siRNA, etc.) It has been shown that it is effective as an ACE2 or TMPRSS2 expression inhibitor or a coronavirus infection inhibitor depending on the content of .

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Abstract

The purpose of the present invention is to provide a composition for inhibiting the expression of angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2. Provided is a composition for inhibiting the expression of angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2, the composition containing a FoxO1 inhibitor.

Description

アンジオテンシン転換酵素2(ACE2)及び/又はTMPRSS2発現を阻害するための組成物Compositions for inhibiting angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2 expression
 本開示は、アンジオテンシン転換酵素2(ACE2)及び/又はTMPRSS2(Transmembraneprotease, serine 2)の発現を抑制するための組成物に関する。 The present disclosure relates to compositions for suppressing the expression of angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2 (Transmembraneprotease, serine 2).
 2019年の終わりから人への感染が始まったとされる新型コロナウィルス(SARS-CoV2)による感染症(COVID-19)は世界中で猛威を振るい、その治療薬並びにウイルスに対するワクチンの研究開発が各国で盛んに行われている。COVID-19は発症するとサイトカインストームと呼ばれる過剰な炎症反応が引き起こされ、当初は異常な肺炎症状を引き起こすことにより死に至らしめると考えられていた。その後、肺のみならず、血管、脳神経系、肝臓、腎臓など全身に症状が見られることが明らかになりつつある。これまでの研究成果から、SARS-CoV2は全ての細胞に感染するというより、アンジオテンシン転換酵素2(ACE2)を細胞表面に発現している細胞に特異的に感染することが示唆されている。さらに最近ではTMPRSS2(Transmembrane Protease, Serine2)というタンパク質分解酵素によって、SARS-CoV2の表面にあるスパイクタンパク質(Spike protein)が切断されることでさらに感染力が高まる、などの報告も相次いでいる。非特許文献1では、これまでのSARS-CoV2に関する知見に関してまとめられている。 The infectious disease (COVID-19) caused by the new coronavirus (SARS-CoV2), which is said to have started infecting humans from the end of 2019, is raging all over the world, and research and development of vaccines against the virus and its therapeutic drugs are being carried out in various countries. is actively carried out. When COVID-19 develops, it triggers an excessive inflammatory reaction called cytokine storm, which was initially thought to cause death by causing abnormal pneumonia symptoms. Since then, it has become clear that symptoms are observed not only in the lungs but also in the blood vessels, the cranial nervous system, the liver, the kidneys, and other parts of the body. Previous research results suggest that SARS-CoV2 specifically infects cells expressing angiotensin-converting enzyme 2 (ACE2) on the cell surface, rather than infecting all cells. More recently, there have been a series of reports that a proteolytic enzyme called TMPRSS2 (Transmembrane Protease, Serine 2) cleaves the spike protein on the surface of SARS-CoV2, further increasing its infectivity. Non-Patent Document 1 summarizes the knowledge about SARS-CoV2 so far.
 SARS-CoV2の感染拡大に伴い、SARS-CoV2に対する治療法及び予防法の開発が全世界で期待されている。SARS-CoV2はACE2を介して感染することは広く知られており、このACE2に対する中和抗体の作成などが急ピッチで進められているものの、いまだ実用化に至ってはいない。そこで、本開示は、COVID-19の治療・予防にも関係する、ACE2及び/又はTMPRSS2の遺伝子発現量(mRNA量)を抑制するための組成物を提供することを目的とする。 With the spread of SARS-CoV2 infection, the development of treatment and prevention methods for SARS-CoV2 is expected worldwide. It is widely known that SARS-CoV2 infects via ACE2, and although the production of neutralizing antibodies against ACE2 is being rapidly advanced, it has not yet been put to practical use. Therefore, an object of the present disclosure is to provide a composition for suppressing the gene expression level (mRNA level) of ACE2 and/or TMPRSS2, which is also related to the treatment/prevention of COVID-19.
 発明者が鋭意検討したところ、FoxO1のシグナル経路を調節することで、ACE2並びにTMPRSS2のmRNA量を減少させることを明らかにした。具体的には、FoxO1に直接作用する阻害剤、FoxO1の上流に存在するPDK1に直接作用する阻害剤、インスリン系化合物(受容体のリガンドであり、下流にシグナル経路でFoxO1に影響を及ぼす)、成長因子(受容体のリガンドであり、下流にシグナル経路でFoxO1に影響を及ぼす)などで処理することで、ACE2、及び/又はTMPRSS2の発現量に有意な変化が起こることを見出した。 As a result of the inventor's intensive studies, it was revealed that the amount of ACE2 and TMPRSS2 mRNA decreased by regulating the FoxO1 signal pathway. Specifically, inhibitors that directly act on FoxO1, inhibitors that directly act on PDK1 present upstream of FoxO1, insulin-based compounds (receptor ligands and downstream influences FoxO1 through a signaling pathway), It was found that treatment with a growth factor (which is a receptor ligand and affects FoxO1 in the downstream signal pathway) or the like causes a significant change in the expression level of ACE2 and/or TMPRSS2.
 さらに、上述のACE2及び/又はTMPRSS2の発現量の減少が、新型コロナウィルスのスパイクタンパク質の細胞内の取り込みの減少につながることを見出した。 Furthermore, it was found that the decrease in the expression level of ACE2 and/or TMPRSS2 described above leads to a decrease in the intracellular uptake of the novel coronavirus spike protein.
 本開示の発明は、上記知見に基づいて完成され、一側面において、以下の発明を包含する。

(発明1)
 アンジオテンシン転換酵素2(ACE2)及び/又はTMPRSS2の発現を阻害するための組成物であって、
 前記組成物は、FoxO1阻害剤を含む、組成物。
(発明2)
 前記FoxO1阻害剤は、以下の式で表される化合物又はその塩である、発明1の組成物。
Figure JPOXMLDOC01-appb-C000003
{ただし、
1は、NH又はCH2であり、
2は、N-R8又はCH-R8 (R8:C1~C3までのアルキル基)であり、
3は、F、Cl、Br、又はIであり、
4は、それぞれ独立して、H又はCH3であり、
5は、S又はOであり、
6は、カルボキシル基であり、
7は、NH2、又はCH3であり、
nは、1-11である。

(発明3)
 前記阻害剤が、以下の式で表される化合物又はその塩である、発明1の組成物。
Figure JPOXMLDOC01-appb-C000004
{ただし、
1は、NHであり、
2は、N-R8 (R8:C1~C3までのアルキル基)であり、
3は、F、Cl、Br、又はIであり、
4は、それぞれ独立して、H又はCH3であり、
5は、Oであり、
6は、カルボキシル基であり、
7は、NH2であり、
nは、1-11である。

(発明4)
 前記阻害剤が、AS1842856又はAS1708727である、発明1の組成物。
(発明5)
 前記FoxO1阻害剤は、インスリン系化合物である、発明1の組成物。
(発明6)
 前記インスリン系化合物が、野生型インスリンである、発明5の組成物。
(発明7)
 前記インスリン系化合物が、改変型インスリンである、発明5の組成物。
(発明8)
 前記改変型インスリンが、超速効型インスリン、速効型インスリン、中間型インスリン、持効型溶解性インスリン、混合型インスリン、及び配合溶解性インスリンから選択される1種以上である、発明7の組成物。
(発明9)
 前記改変型インスリンが、ヒト野生型インスリンと90%以上同一のアミノ酸配列を有する、発明7の組成物。
(発明10)
 前記改変型インスリンが、ヒト野生型インスリンと95%以上同一のアミノ酸配列を有する、発明7の組成物。
(発明11)
 前記改変型インスリンのA鎖が、ヒト野生型インスリンのA鎖と100%同一のアミノ酸配列を有する、発明9又は10の組成物。
(発明12)
 前記FoxO1阻害剤は、成長因子及び/又はこれをコードする核酸である、発明1の組成物。
(発明13)
 前記成長因子が、以下から選択されるいずれかと、90%以上同一のアミノ酸配列を有する、発明12の組成物:
 IGF1、IGF2、EGF、FGF2、FGF14、VEGF、HGF。
(発明14)
 前記成長因子が、以下から選択されるいずれかと、95%以上同一のアミノ酸配列を有する、発明13の組成物:
 IGF1、IGF2、EGF、FGF2、FGF14、VEGF、HGF。
(発明15)
 前記成長因子が、以下から選択されるいずれかと、100%以上同一のアミノ酸配列を有する、発明14の組成物:
 IGF1、IGF2、EGF、FGF2、FGF14、VEGF、HGF。
(発明16)
 前記FoxO1阻害剤は、FoxO1作用型阻害剤である、発明1の組成物。
(発明17)
 前記FoxO1阻害剤は、PI3K/PDK1/Akt作用型阻害剤である、発明1の組成物。
(発明18)
 前記FoxO1阻害剤は、FoxO1の遺伝子発現抑制剤である、発明1の組成物。
(発明19)
 コロナウィルス感染症を予防又は治療するための組成物である、発明1~18いずれか1つに記載の組成物(投与対象として、糖尿病患者を除く)。
(発明20)
 コロナウィルス感染症を予防又は治療するための組成物であって、FoxO1阻害剤を含む、組成物(投与対象として、糖尿病患者を除く)。
The invention of the present disclosure has been completed based on the above findings, and includes the following inventions in one aspect.

(Invention 1)
A composition for inhibiting the expression of angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2, comprising:
A composition, wherein the composition comprises a FoxO1 inhibitor.
(Invention 2)
The composition according to Invention 1, wherein the FoxO1 inhibitor is a compound represented by the following formula or a salt thereof.
Figure JPOXMLDOC01-appb-C000003
{however,
R1 is NH or CH2 ;
R 2 is NR 8 or CH-R 8 (R 8 : C1-C3 alkyl group),
R3 is F , Cl, Br, or I;
each R4 is independently H or CH3 ;
R5 is S or O;
R6 is a carboxyl group,
R7 is NH2 or CH3 ,
n is 1-11.
}
(Invention 3)
The composition of Invention 1, wherein the inhibitor is a compound represented by the following formula or a salt thereof.
Figure JPOXMLDOC01-appb-C000004
{however,
R 1 is NH;
R 2 is NR 8 (R 8 is an alkyl group of C1 to C3);
R3 is F , Cl, Br, or I;
each R4 is independently H or CH3 ;
R5 is O;
R6 is a carboxyl group,
R7 is NH2 ;
n is 1-11.
}
(Invention 4)
The composition of invention 1, wherein said inhibitor is AS1842856 or AS1708727.
(Invention 5)
The composition of invention 1, wherein said FoxO1 inhibitor is an insulin-based compound.
(Invention 6)
The composition of Invention 5, wherein said insulin-based compound is wild-type insulin.
(Invention 7)
The composition of invention 5, wherein said insulin-based compound is a modified insulin.
(Invention 8)
The composition of invention 7, wherein the modified insulin is one or more selected from ultra-rapid-acting insulin, fast-acting insulin, intermediate-acting insulin, long-acting soluble insulin, mixed-acting insulin, and combined soluble insulin. .
(Invention 9)
The composition of invention 7, wherein said modified insulin has an amino acid sequence that is 90% or more identical to human wild-type insulin.
(Invention 10)
The composition of invention 7, wherein said modified insulin has an amino acid sequence that is 95% or more identical to human wild-type insulin.
(Invention 11)
11. The composition of invention 9 or 10, wherein said modified insulin A chain has an amino acid sequence that is 100% identical to that of human wild-type insulin.
(Invention 12)
The composition of invention 1, wherein said FoxO1 inhibitor is a growth factor and/or a nucleic acid encoding same.
(Invention 13)
The composition of invention 12, wherein said growth factor has 90% or more amino acid sequence identity with any selected from:
IGF1, IGF2, EGF, FGF2, FGF14, VEGF, HGF.
(Invention 14)
The composition of invention 13, wherein said growth factor has 95% or more amino acid sequence identity with any selected from:
IGF1, IGF2, EGF, FGF2, FGF14, VEGF, HGF.
(Invention 15)
The composition of invention 14, wherein said growth factor has 100% or more amino acid sequence identity with any selected from:
IGF1, IGF2, EGF, FGF2, FGF14, VEGF, HGF.
(Invention 16)
The composition of Invention 1, wherein the FoxO1 inhibitor is a FoxO1-acting inhibitor.
(Invention 17)
The composition of invention 1, wherein said FoxO1 inhibitor is a PI3K/PDK1/Akt acting inhibitor.
(Invention 18)
The composition of Invention 1, wherein the FoxO1 inhibitor is a FoxO1 gene expression inhibitor.
(Invention 19)
19. The composition according to any one of Inventions 1 to 18, which is a composition for preventing or treating coronavirus infection (excluding diabetic patients).
(Invention 20)
A composition for preventing or treating a coronavirus infection, the composition comprising a FoxO1 inhibitor (excluding diabetic patients).
 本開示の組成物は、FoxO1阻害剤を含む。これによって、ACE2及び/又はTMPRSS2遺伝子の発現量を減少させることができる。 A composition of the present disclosure comprises a FoxO1 inhibitor. This can reduce the expression level of the ACE2 and/or TMPRSS2 gene.
フォークヘッド型転写因子FoxO1阻害剤AS1842856(濃度はグラフ下に示す)の添加によるHepG2細胞におけるACE2及びTMPRSS2遺伝子の発現量を相対化したグラフである。更には、FoxO1によるACE2及びTMPRSS2遺伝子の発現誘導メカニズムの模式図である。Fig. 3 is a graph showing the relative expression levels of ACE2 and TMPRSS2 genes in HepG2 cells with the addition of a forkhead transcription factor FoxO1 inhibitor AS1842856 (concentrations shown below the graph). Furthermore, it is a schematic diagram of the expression induction mechanism of ACE2 and TMPRSS2 genes by FoxO1. SARS-CoV2表面タンパク質であるスパイクタンパク質中の、ACE2結合領域(RBD)の組換えタンパク質(RBD-mFc)の細胞内への取り込みアッセイの模式図である。RBDにはマウスIgG由来のFc配列(mFc)が融合しており、マウスIgGを認識する蛍光標識抗体(例えば、Alexa Fluor(R) 488 antibody)を用いた免疫染色法により、細胞内に取り込まれたRBDの量を定量化することが可能である。Schematic representation of the cellular uptake assay of the recombinant protein (RBD-mFc) of the ACE2 binding domain (RBD) in the SARS-CoV2 surface protein spike protein. The RBD is fused with a mouse IgG-derived Fc sequence (mFc), and is incorporated into cells by immunostaining using a fluorescence-labeled antibody that recognizes mouse IgG (for example, Alexa Fluor (R) 488 antibody). It is possible to quantify the amount of RBD added. SARS-CoV2表面タンパク質であるスパイクタンパク質の中の、ACE2結合領域(RBD)の組換えタンパク質(RBD-mFc)のHepG2細胞内への取り込みアッセイにおける、実際に取り込まれたRBDタンパク質の典型的な顕微鏡画像である。A typical micrograph of the actual internalized RBD protein in the HepG2 cell uptake assay of the recombinant protein (RBD-mFc) of the ACE2 binding domain (RBD) in the SARS-CoV2 surface protein spike protein. It is an image. 取り込まれたRBDタンパク質の組換えタンパク質(RBD-mFc)を、フローサイトメトリーを用いて定量し、平均蛍光強度を相対化したグラフである。Recombinant protein (RBD-mFc) of the incorporated RBD protein was quantified using flow cytometry, and graphs showing relative mean fluorescence intensity. フォークヘッド型転写因子FoxO1阻害剤AS1842856によるACE2及び/又はTMPRSS2の発現抑制と、スパイクタンパク質との結合・取り込み抑制の関係を表した模式図である。FIG. 3 is a schematic diagram showing the relationship between the suppression of ACE2 and/or TMPRSS2 expression by the forkhead transcription factor FoxO1 inhibitor AS1842856 and the suppression of binding and uptake with spike protein. 野生型インスリンの添加によるHepG2細胞におけるACE2及びTMPRSS2遺伝子の発現量を相対化したグラフである。Fig. 10 is a graph showing relative expression levels of ACE2 and TMPRSS2 genes in HepG2 cells to which wild-type insulin was added. 改変型インスリンの添加によるHepG2細胞におけるACE2及びTMPRSS2遺伝子の発現量を相対化したグラフである。FIG. 10 is a graph showing the relative expression levels of ACE2 and TMPRSS2 genes in HepG2 cells with the addition of modified insulin. FIG. 取り込まれたS1およびRBDタンパク質を、フローサイトメトリーを用いて定量し、平均蛍光強度を相対化したグラフである。Figure 10 is a graph showing the relative mean fluorescence intensity of S1 and RBD protein uptake quantified using flow cytometry. インスリンによるACE2及び/又はTMPRSS2の発現抑制と、スパイクタンパク質との結合・取り込み抑制の関係を表した模式図である。FIG. 2 is a schematic diagram showing the relationship between suppression of ACE2 and/or TMPRSS2 expression by insulin and suppression of binding/uptake to spike protein. 各種成長因子の添加によるHepG2細胞におけるACE2及びTMPRSS2遺伝子の発現量を相対化したグラフである。2 is a graph showing relative expression levels of ACE2 and TMPRSS2 genes in HepG2 cells to which various growth factors were added. HGFの添加によるHepG2細胞におけるACE2及びTMPRSS2遺伝子の発現量を相対化したグラフである。2 is a graph showing relative expression levels of ACE2 and TMPRSS2 genes in HepG2 cells with the addition of HGF. siRNAの添加によるHepG2細胞におけるACE2及びTMPRSS2遺伝子の発現量を相対化したグラフである。FIG. 10 is a graph showing the relative expression levels of ACE2 and TMPRSS2 genes in HepG2 cells with the addition of siRNA. FIG.
 以下、本開示の発明を実施するための具体的な実施形態について説明する。以下の説明は、発明の理解を促進するためのものである。即ち、本発明の範囲を限定することを意図するものではない。 Specific embodiments for carrying out the invention of the present disclosure will be described below. The following description is intended to facilitate understanding of the invention. it is not intended to limit the scope of the invention.
1. 定義
 本明細書で記載される用語「アンジオテンシン転換酵素2(ACE2)及び/又はTMPRSS2の発現を阻害するため」は、ACE2及び/又はTMPRSS2に関する特定の疾患を改善するという目的を包含する。更には、当該用語は、治療目的に限定されない。例えば、当該用語は、特定の疾患を有さないとしても、その疾患を発症することを予防する目的を包含する。
1. Definitions As used herein, the term "to inhibit the expression of angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2" encompasses the purpose of ameliorating a particular disease associated with ACE2 and/or TMPRSS2. Furthermore, the term is not limited to therapeutic purposes. For example, the term encompasses the purpose of preventing the development of a particular disease even if one does not have that disease.
 本明細書で記載される用語「FoxO1阻害剤」は、広義の意味と狭義の意味を有するが、特記しない限り前者の意味で解釈する。 The term "FoxO1 inhibitor" described in this specification has a broad meaning and a narrow meaning, but is interpreted in the former meaning unless otherwise specified.
 狭義の意味の場合には、上記用語は、FoxO1に直接結合して作用する阻害剤を意味する(本明細書においては、狭義の意味の場合には、「FoxO1作用型阻害剤」とも称する)。一方で、広義の意味の場合には、上記用語は、直接又は間接的にFoxO1に作用して、FoxO1の転写因子としての活性を阻害する阻害剤を意味する。間接的に作用する場合には、上記用語は、FoxO1の上流のシグナル経路に作用する阻害剤を包含する。一例としては、FoxO1の上流にあるAktやPDK1に直接作用してこれらを活性化させる薬剤が挙げられる。別の例としては、FoxO1の活性に影響を及ぼす(より具体的には、FoxO1の活性を抑制するシグナル経路のトリガーとなる)受容体のリガンドが挙げられる。受容体としては、インスリン受容体、成長因子受容体などが挙げられる。換言すれば、上記用語は、インスリン受容体のリガンド、成長因子受容体のリガンドを包含する。また、広義の意味の場合には、上記用語は、更に、「FoxO1」自体の発現を阻害する物質も含む。典型的な例としては、核酸を含み、例えば、アンチセンス核酸、shRNA、siRNA、miRNA等が挙げられる。 In the narrow sense, the term means an inhibitor that acts by directly binding to FoxO1 (herein, in the narrow sense, it is also referred to as a "FoxO1-acting inhibitor"). . On the other hand, in a broader sense, the term means an inhibitor that directly or indirectly acts on FoxO1 to inhibit the activity of FoxO1 as a transcription factor. When acting indirectly, the term includes inhibitors that act on signaling pathways upstream of FoxO1. One example is a drug that directly acts on Akt and PDK1 upstream of FoxO1 to activate them. Another example is a ligand for a receptor that affects the activity of FoxO1 (more specifically triggers a signaling pathway that suppresses the activity of FoxO1). Receptors include insulin receptors, growth factor receptors, and the like. In other words, the term encompasses insulin receptor ligands, growth factor receptor ligands. In a broader sense, the term further includes substances that inhibit the expression of "FoxO1" itself. Typical examples include nucleic acids, such as antisense nucleic acids, shRNA, siRNA, miRNA and the like.
 具体的に、「FoxO1の転写因子としての活性を阻害する」という言葉の定義についてであるが、これは当分野で公知の解析手法よって判別することができる。例えば、FoxO1と結合することが知られているコンセンサス配列(GTAAA(T/C)AA)を用いたルシフェラーゼアッセイを実施して、蛍光強度などで有意な差が生じるかどうかに基づいて判別することができる。あるいは、市販のELISA型活性検出アッセイ(Active Motif社,TransAMRFKHR(FOXO1),製品番号:46396)なども用いられる。いずれの実験条件の場合においても、水、あるいは溶媒として用いる溶液(DMSOなど)を添加したコントロール条件におけるFOXO1の活性の指標となる値と比較して、試験物質の添加によって指標の平均値が60%以下に抑制できる試験物質を、「FoxO1の転写因子としての活性を阻害する」ものとして定義する。 Specifically, regarding the definition of the term "inhibits the activity of FoxO1 as a transcription factor", this can be determined by an analysis method known in the art. For example, performing a luciferase assay using a consensus sequence (GTAAA(T/C)AA) known to bind to FoxO1, and discriminating based on whether there is a significant difference in fluorescence intensity, etc. can be done. Alternatively, a commercially available ELISA-type activity detection assay (Active Motif, TransAMRFKHR (FOXO1), product number: 46396) may be used. In all experimental conditions, the addition of the test substance reduced the average value of the index of FOXO1 activity to 60%, compared with the value indicating the activity of FOXO1 in the control conditions in which water or a solution used as a solvent (DMSO, etc.) was added. % or less is defined as "inhibiting the activity of FoxO1 as a transcription factor."
2.用いたアンジオテンシン転換酵素2(ACE2)及び/又はTMPRSS2の発現量を抑制するための組成物
 一実施形態において、本開示は、アンジオテンシン転換酵素2(ACE2)及び/又はTMPRSS2の発現を阻害するための組成物であって、前記組成物は、FoxO1阻害剤を含む、組成物に関する。
2. In one embodiment of the composition for suppressing the expression level of angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2 used , the present disclosure provides A composition, said composition relating to a composition comprising a FoxO1 inhibitor.
 更なる一実施形態において、FoxO1阻害剤として、以下のいずれか1種以上が上記組成物内に有効成分として含まれてもよい。別の一側面において、本開示は、医薬組成物を製造するためのこれらのいずれか1種以上の有効成分の使用に関する。
・インスリン系化合物
・成長因子又はこれをコードする核酸
・FoxO1作用型阻害剤、及び/又は、PI3K/PDK1/Akt作用型阻害剤
・FoxO1の遺伝子発現抑制剤
 以下では、各々の阻害剤について詳述する。
In a further embodiment, any one or more of the following FoxO1 inhibitors may be included as active ingredients in the composition. In another aspect, the disclosure relates to the use of any one or more of these active ingredients for the manufacture of pharmaceutical compositions.
・Insulin compounds ・Growth factors or nucleic acids encoding them ・FoxO1-acting inhibitors and/or PI3K/PDK1/Akt-acting inhibitors ・FoxO1 gene expression inhibitors do.
2-1.インスリン系化合物を用いたアンジオテンシン転換酵素2(ACE2)及び/又はTMPRSS2の発現量を抑制するための組成物
 一実施形態においては、こうした組成物は、インスリン系化合物(例えば、ヒトインスリン)を含む。これにより、ACE2及び/又はTMPRSS2の遺伝子の転写が阻害され、mRNA量が減少する。
2-1. Compositions for Inhibiting Angiotensin Converting Enzyme 2 (ACE2) and/or TMPRSS2 Expression Using Insulin-Based Compounds In one embodiment, such compositions comprise an insulin-based compound (eg, human insulin). This inhibits transcription of the gene for ACE2 and/or TMPRSS2 and reduces the amount of mRNA.
 また、本明細書で記載される「インスリン系化合物」は、細胞内部でインスリンシグナル伝達経路を活性化する剤を意味する。従って、本明細書で記載される「インスリン系化合物」は、ヒト生体内で発現している、いわゆる「野生型インスリン」だけではなく、遺伝子改変技術を用いて製造された「改変型インスリン」を含むことができ、更には、生体内で膵臓のβ細胞に作用してインスリンの発現を促進する剤を含むことができる。好ましい「インスリン系化合物」は、野生型インスリンあるいは改変型インスリンである。改変型インスリンの例として、超速効型インスリン、速効型インスリン、中間型インスリン、持効型溶解インスリン、混合型インスリン、配合溶解性インスリンが挙げられる。 In addition, the "insulin-based compound" described herein means an agent that activates the insulin signaling pathway inside cells. Therefore, the "insulin-based compound" described herein includes not only the so-called "wild-type insulin" expressed in the human body, but also the "modified insulin" produced using genetic modification technology. Further, it can contain an agent that acts on pancreatic β-cells in vivo to promote the expression of insulin. A preferred "insulin-based compound" is wild-type insulin or modified insulin. Examples of modified insulins include ultra-rapid-acting insulins, fast-acting insulins, intermediate-acting insulins, long-acting soluble insulins, mixed-acting insulins, combined soluble insulins.
 また、改変型インスリンは、ヒト野生型インスリンとある程度同一のアミノ酸配列を有してもよい。例えば、90%以上であり、好ましくは、95%以上である(例えば、blastpなどを利用して判定された同一性)。なお、野生型インスリンは、A鎖とB鎖のヘテロダイマーである。従って、ここで述べる同一性は、A鎖とB鎖を連結して1つのアミノ酸配列として比較したときの同一性を意味する。更に好ましくは、改変型インスリンのA鎖が、ヒト野生型インスリンのA鎖と100%同一のアミノ酸配列を有する。 In addition, the modified insulin may have an amino acid sequence identical to that of human wild-type insulin to some extent. For example, 90% or more, preferably 95% or more (eg, identity determined using blastp or the like). Wild-type insulin is a heterodimer of A chain and B chain. Therefore, the identity described here means the identity when the A chain and the B chain are linked and compared as one amino acid sequence. More preferably, the A-chain of the modified insulin has 100% amino acid sequence identity with the A-chain of human wild-type insulin.
 インスリンは、アンジオテンシン転換酵素2(ACE2)及び/又はTMPRSS2の遺伝子の転写を阻害する因子として作用する。従って、インスリンを添加することで、これらの遺伝子の発現を抑制することができる。これによって、ウイルスがスパイクタンパク質を利用して細胞内に取り込まれることを低減することができる。 Insulin acts as a factor that inhibits the transcription of the angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2 genes. Therefore, the expression of these genes can be suppressed by adding insulin. This makes it possible to reduce the uptake of viruses into cells using the spike protein.
2-2.成長因子を用いたアンジオテンシン転換酵素2(ACE2)及び/又はTMPRSS2の発現量を抑制するための組成物
 一実施形態においては、組成物は、成長因子を含む。これにより、ACE2及び/又はTMPRSS2の遺伝子の転写が阻害され、mRNA量が減少する。
2-2. Compositions for Suppressing the Expression Level of Angiotensin Converting Enzyme 2 (ACE2) and/or TMPRSS2 Using Growth Factors In one embodiment, the composition comprises a growth factor. This inhibits transcription of the gene for ACE2 and/or TMPRSS2 and reduces the amount of mRNA.
 本明細書で記載される用語「成長因子」は、以下の条件をすべて満たす物を意味する。
・ペプチド又はタンパク質を少なくとも部分的に含むこと
・細胞内のFoxO1の転写因子の活性を抑制すること
As used herein, the term "growth factor" means an entity that satisfies all of the following conditions.
- At least partially containing a peptide or protein - Suppressing the activity of the intracellular FoxO1 transcription factor
 上述した「ペプチド又はタンパク質を少なくとも部分的に含むこと」について、ペプチド又はタンパク質は天然に存在する物であってもよく、人工的に作られた物であってもよい。例えば、天然に存在するタンパク質又はペプチドに化学修飾された物も包含される。ペプチド又はタンパク質は、モノマー形態だけではなく、2量体以上の形態であってもよい。ペプチド又はタンパク質は、直鎖上ではなく、例えば、環状であってもよい。また、2量体以上の形態である場合には適宜人工的なリンカーを設けてもよい。例えば、Kenichiro Itoら(Nat Commun. 2015 Mar 11;6:6373.doi: 10.1038/ncomms7373.)の論文によれば、環状のアミノ酸配列2つをリンカーで結合したユニークな構造を有する物質が、HGF受容体のアゴニストとして機能することを報告している。 Regarding "at least partially including a peptide or protein" mentioned above, the peptide or protein may be a naturally occurring substance or an artificially produced substance. For example, chemically modified naturally occurring proteins or peptides are also included. Peptides or proteins may be in dimeric or higher forms as well as in monomeric form. Peptides or proteins may be non-linear, eg cyclic. In addition, in the case of a dimer or higher form, an artificial linker may be appropriately provided. For example, according to a paper by Kenichiro Ito et al. (Nat Commun. 2015 Mar 11;6:6373.doi: 10.1038/ncomms7373.), a substance having a unique structure in which two cyclic amino acid sequences are linked by a linker is HGF It has been reported that it functions as an agonist of the receptor.
 本件の出願日以降も、従来存在しなかった様々なタイプの新たな成長因子受容体のアゴニストが開発される可能性があるが、上述した条件を充足する限りは、こうした成長因子受容体のアゴニストも、本明細書で記載される用語「成長因子」に含まれることを意図する。 Even after the filing date of the present application, various types of new growth factor receptor agonists that have not existed in the past may be developed. are also intended to be included in the term "growth factor" as used herein.
 また、成長因子の種(species)については特に限定されないが、好ましくはヒト型である。 In addition, although the species of the growth factor is not particularly limited, it is preferably human type.
 限定されるものではないが、成長因子の一例として以下のいずれか1種以上が挙げられる:IGF1、IGF2、EGF、FGF2、FGF14、VEGF、HGF。 Examples of growth factors include, but are not limited to, any one or more of the following: IGF1, IGF2, EGF, FGF2, FGF14, VEGF, HGF.
 さらには、別の成長因子は、上記列挙した成長因子のいずれかとある程度同一のアミノ酸配列を有してもよい。例えば、90%以上であり、好ましくは、95%以上であり、最も好ましくは100%である(例えば、blastpなどを利用して判定された同一性)。 Furthermore, another growth factor may have an amino acid sequence that is somewhat identical to any of the growth factors listed above. For example, 90% or more, preferably 95% or more, and most preferably 100% (eg, identity determined using blastp or the like).
 さらには、上記のHGFは、環状ペプチドを用いた代替ペプチドが開発されており(https://www.nature.com/articles/ncomms7373)、HGFの機能と同様の機能を有していることが報告されている。このような、成長因子の機能を代替するようにして開発された中~高分子化合物も、今回の目的に使用することは十分に可能である。 Furthermore, for the above HGF, an alternative peptide using a cyclic peptide has been developed (https://www.nature.com/articles/ncomms7373), and it is said to have a function similar to that of HGF. It has been reported. Medium to high-molecular-weight compounds developed to replace the functions of growth factors can also be used for the present purpose.
 また、上記成長因子に加えて、或いは、これに代えて、上述した成長因子(上述した同一性のアミノ酸配列を有する物も含む)をコードする核酸が、組成物に配合されてもよい。核酸は、DNAであってもよく、そして、RNA(例えば、mRNA)であってもよい。好ましくは、本開示の組成物は、上述したDNAを任意の形態で含むことができる。例えば、上述したDNAのコーティング領域は、プラスミド、ベクター等に組み込んでもよい。これらの核酸は最終的に成長因子を産生し、上述した作用により、ACE2及び/又はTMPRSS2の遺伝子の転写が阻害され、mRNA量が減少する。 In addition to or instead of the above growth factors, nucleic acids encoding the above growth factors (including those having the same amino acid sequence as above) may be incorporated into the composition. Nucleic acids can be DNA and can be RNA (eg, mRNA). Preferably, the compositions of the present disclosure can contain the DNA described above in any form. For example, the coding region of DNA described above may be incorporated into a plasmid, vector, or the like. These nucleic acids eventually produce growth factors, and the above-mentioned action inhibits the transcription of the ACE2 and/or TMPRSS2 gene and reduces the amount of mRNA.
2-3.FoxO1作用型阻害剤、及び/又は、PI3K/PDK1/Akt作用型阻害剤を用いたアンジオテンシン転換酵素2(ACE2)及び/又はTMPRSS2の発現量を抑制するための組成物
 一実施形態においては、組成物は、FoxO1作用型阻害剤、及び/又は、PI3K/PDK1/Akt作用型阻害剤を含む。これにより、ACE2及び/又はTMPRSS2の遺伝子の転写が阻害され、mRNA量が減少する。
2-3. A composition for suppressing the expression level of angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2 using a FoxO1-acting inhibitor and/or a PI3K/PDK1/Akt-acting inhibitor In one embodiment, the composition Articles include FoxO1-acting inhibitors and/or PI3K/PDK1/Akt-acting inhibitors. This inhibits transcription of the gene for ACE2 and/or TMPRSS2 and reduces the amount of mRNA.
 本明細書で記載される用語「FoxO1作用型阻害剤」の意味については、上述した狭義の「FoxO1阻害剤」で説明したとおりである。また、本明細書で記載される用語「PI3K/PDK1/Akt作用型阻害剤」は、以下の3つの条件をすべて満たす物を意味する。
・細胞膜を透過して、細胞内のPIP3を増加させるか、及び/又は、PI3K、PDK1、Aktのうち少なくともいずれか1つに直接作用すること
・作用した結果、PI3K、PDK1及びAktのうち少なくともいずれか1つの活性を変化させること
・活性を変化させた結果、細胞内のFoxO1の転写因子の活性を抑制すること
The meaning of the term “FoxO1-acting inhibitor” described in this specification is as explained in the above narrowly defined “FoxO1 inhibitor”. Also, the term "PI3K/PDK1/Akt-acting inhibitor" described herein means an agent that satisfies all of the following three conditions.
・Permeate the cell membrane to increase intracellular PIP 3 and/or act directly on at least one of PI3K, PDK1 and Akt Changing at least one activity ・As a result of changing the activity, suppressing the activity of the intracellular FoxO1 transcription factor
 従って、語弊を避けるために補足すると、用語「PI3K/PDK1/Akt作用型阻害剤」は、「阻害剤」と称するものの、PI3K/PDK1/Aktに対して活性化作用を示す物を意味する。そして、結果的にFoxO1の活性を抑制するという意味で、「阻害剤」と称している。 Therefore, to avoid misinterpretation, the term "PI3K/PDK1/Akt-acting inhibitor" means a substance that exhibits an activating effect on PI3K/PDK1/Akt, although it is called an "inhibitor." It is called an "inhibitor" in the sense that it consequently suppresses the activity of FoxO1.
 なお、FoxO1の転写因子の活性を抑制するかどうかについては、上記「1.定義」で述べたように、所望のアッセイにより判別可能である。 Whether or not the transcription factor activity of FoxO1 is suppressed can be determined by a desired assay, as described in "1. Definition" above.
 PIP3は、受容体とリガンドの結合をトリガーとするシグナルにより生成されるセカンドメッセンジャーである。PI3KによってPIP3が生成される。生成されたPIP3は、PDK1を活性化させる。また、活性化されたPDK1は、Aktを活性化させる。活性化されたAktは、FoxO1の活性を阻害する。従って、細胞内のPIP3を増加させるか、及び/又は、PI3K、PDK1、Aktのうち少なくともいずれか1つに直接作用して活性化させることで、FoxO1の転写因子としての活性を阻害することができる。これにより、ACE2及び/又はTMPRSS2の遺伝子の転写が阻害され、mRNA量が減少する。 PIP3 is a second messenger generated by signals triggered by binding of receptors and ligands. PIP 3 is generated by PI3K. The generated PIP3 activates PDK1. In addition, activated PDK1 activates Akt. Activated Akt inhibits the activity of FoxO1. Therefore , by increasing intracellular PIP3 and/or directly acting on and activating at least one of PI3K, PDK1, and Akt, the activity of FoxO1 as a transcription factor is inhibited. can be done. This inhibits transcription of the gene for ACE2 and/or TMPRSS2 and reduces the amount of mRNA.
 また、FoxO1に直接抑制的に作用する場合であれば、当該作用によって、ACE2及び/又はTMPRSS2の遺伝子の転写が阻害され、mRNA量が減少する。 In addition, if it directly suppresses FoxO1, the action inhibits the transcription of the ACE2 and/or TMPRSS2 gene and reduces the amount of mRNA.
 こうした観点から、FoxO1作用型阻害剤、及び/又は、PI3K/PDK1/Akt作用型阻害剤は、各々に対して以下の様に作用することが好ましい。
・FoxO1に対して活性を阻害する作用
・PIP3を増加させる作用、
・PI3K、PDK1、Aktのうち少なくともいずれか1つに対して活性を促進する作用
From this point of view, the FoxO1-acting inhibitor and/or the PI3K/PDK1/Akt-acting inhibitor preferably acts on each in the following manner.
・Action to inhibit the activity of FoxO1 ・Action to increase PIP3 ,
・Action to promote the activity of at least one of PI3K, PDK1, and Akt
 好ましい実施形態において、FoxO1に対して活性を阻害する作用を有する薬剤としては、例えば、以下の式で表される化合物又はその塩が挙げられる。
Figure JPOXMLDOC01-appb-C000005
{ただし、
1は、NH又はCH2であり、
2は、N-R8又はCH-R8 (R8:C1~C3までのアルキル基)であり、
3は、F、Cl、Br、又はIであり、
4は、それぞれ独立して、H又はCH3であり、
5は、S又はOであり、
6は、カルボキシル基であり、
7は、NH2、又はCH3であり、
nは、1-11である。
In a preferred embodiment, the drug having an activity-inhibiting effect on FoxO1 includes, for example, compounds represented by the following formulas or salts thereof.
Figure JPOXMLDOC01-appb-C000005
{however,
R1 is NH or CH2 ;
R 2 is NR 8 or CH-R 8 (R 8 : C1-C3 alkyl group),
R3 is F , Cl, Br, or I;
each R4 is independently H or CH3 ;
R5 is S or O;
R6 is a carboxyl group,
R7 is NH2 or CH3 ,
n is 1-11.
}
 更に、好ましい実施形態において、FoxO1に直接結合して阻害する剤の例として、以下の式で表される化合物又はその塩が挙げられる。
Figure JPOXMLDOC01-appb-C000006
{ただし、
1は、NHであり、
2は、N-R8 (R8:C1~C3までのアルキル基)であり、
3は、F、Cl、Br、又はIであり、
4は、それぞれ独立して、H又はCH3であり、
5は、Oであり、
6は、カルボキシル基であり、
7は、NH2であり、
nは、1-11である。
Furthermore, in preferred embodiments, examples of agents that directly bind to and inhibit FoxO1 include compounds represented by the following formulas or salts thereof.
Figure JPOXMLDOC01-appb-C000006
{however,
R 1 is NH;
R 2 is NR 8 (R 8 is an alkyl group of C1 to C3);
R3 is F , Cl, Br, or I;
each R4 is independently H or CH3 ;
R5 is O;
R6 is a carboxyl group,
R7 is NH2 ;
n is 1-11.
}
 上記式で表される化合物又はその塩の具体例として、AS1842856(CAS No. 836620-48-5)が挙げられる。
Figure JPOXMLDOC01-appb-C000007
A specific example of the compound represented by the above formula or a salt thereof is AS1842856 (CAS No. 836620-48-5).
Figure JPOXMLDOC01-appb-C000007
 AS1842856以外にFoxO1に対して活性を阻害する作用を有する薬剤の例としてAS1708727が挙げられる。 In addition to AS1842856, AS1708727 is an example of a drug that has the effect of inhibiting the activity of FoxO1.
 また、FoxO1に対して活性を阻害する作用を有する薬剤としては、KIS-154、カルコン類、トリテルペン類、フラボン類、モノテルペノイド類、リグナン類、クマリン類、及びフィトケミカル類からなるFOXO1活性阻害剤(再表2018/079715)などが挙げられる。 Further, agents having an activity-inhibiting effect on FoxO1 include KIS-154, chalcones, triterpenes, flavones, monoterpenoids, lignans, coumarins, and FOXO1 activity inhibitors consisting of phytochemicals. (Re-Table 2018/079715) and the like.
 具体例としては、再表2018/079715の0066及び0067に記載の通りである。例えば、以下の化合物が挙げられる。 Specific examples are as described in 0066 and 0067 of Table 2018/079715. Examples include the following compounds.
 Tranchelogenin, β-Naphthoflavone, 6,7-Dihydroxyflavone, 3,4-Dihydrocoumarin, 2-tert-butyl-p-quinone, Amarogentin, (±)Isomenthone, Saponarin, 4-Hydroxychalcone, trans-Chalcone, 2,3-Dimethoxy-2’-hydroxychalcone, Corosolic Acid, 6-Methylflavone, 5-Methyoxyflavone, 7,8-Dihydroxyflavone, Ipriflavone, Chrysin, Enterodiol Tranchelogenin, β-Naphthoflavone, 6,7-Dihydroxyflavone, 3,4-Dihydrocoumarin, 2-tert-butyl-p-quinone, Amarogentin, (±)Isomenthone, Saponarin, 4-Hydroxychalcone, trans-Chalcone, 2,3-Dimethoxy -2'-hydroxychalcone, Corosolic Acid, 6-Methylflavone, 5-Methyoxyflavone, 7,8-Dihydroxyflavone, Ipriflavone, Chrysin, Enterodiol
 一方で、PI3K/PDK1/Aktに対して活性を促進する作用を有する薬剤としては、例えば、以下の化合物が挙げられる。
 VO-Ohpic trihydrate
 SF1670
 bpV(HOpic)
 これらの化合物は、PI3Kと逆の作用をするPTENを阻害することで、間接的にPI3K/PDK1/Aktに対して活性を促進する効果があることが知られている。
On the other hand, examples of drugs having an activity-promoting effect on PI3K/PDK1/Akt include the following compounds.
VO-Ohpic trihydrate
SF1670
bpV (HOpic)
These compounds are known to have the effect of indirectly promoting the activity of PI3K/PDK1/Akt by inhibiting PTEN, which acts oppositely to PI3K.
2-4.FoxO1遺伝子発現抑制剤を用いたアンジオテンシン転換酵素2(ACE2)及び/又はTMPRSS2の発現量を抑制するための組成物
 一実施形態においては、組成物は、FoxO1遺伝子発現抑制剤を含む。これにより、FoxO1自体の発現量が減少する。そして、ACE2及び/又はTMPRSS2の遺伝子の転写が減少し、mRNA量が減少する。典型的には、FoxO1遺伝子発現抑制剤の例として、siRNA(small interference RNA)、あるいはmiRNA(microRNA)などを用いたFoxO1遺伝子発現の阻害が挙げられる。
2-4. Composition for Suppressing the Expression Level of Angiotensin Converting Enzyme 2 (ACE2) and/or TMPRSS2 Using FoxO1 Gene Expression Inhibitor In one embodiment, the composition comprises a FoxO1 gene expression inhibitor. This reduces the expression level of FoxO1 itself. Then, the transcription of the ACE2 and/or TMPRSS2 gene is decreased, and the amount of mRNA is decreased. Typical examples of FoxO1 gene expression inhibitors include inhibition of FoxO1 gene expression using siRNA (small interference RNA), miRNA (microRNA), or the like.
3.用途
 一実施形態において、本開示の組成物は、アンジオテンシン転換酵素2(ACE2)及び/又はTMPRSS2が関与する疾患の治療及び/又は予防に利用することができる。具体的には、これらが関与する感染症の治療及び/又は予防に利用することができる。感染症の例としては、コロナウィルス感染症、例えば、SARS-CoV又はSARS-CoV2などのウイルスによる感染症が挙げられる。好ましくは、SARS-CoV2による感染症(COVID19)である。これらのウイルスは、ヒト細胞のACE2受容体に、自身のスパイクタンパク質を結合させ、これを足掛かりとして細胞内に侵入する。また、SARS-CoV2は、ヒト細胞の表面のACE2と結合した後、ヒト細胞のTMPRSS2の酵素処理によって、細胞内への侵入効率が増強される。従って、ACE2及び/又はTMPRSS2の発現を抑制することで、こうしたウイルスによる細胞の侵入を抑制することができる。これによって感染を抑制することができる。
3. Uses In one embodiment, the compositions of the present disclosure can be used to treat and/or prevent diseases involving angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2. Specifically, they can be used for treatment and/or prevention of infectious diseases in which they are involved. Examples of infectious diseases include coronavirus infections, eg infections by viruses such as SARS-CoV or SARS-CoV2. Preferably, it is an infection caused by SARS-CoV2 (COVID19). These viruses bind their spike proteins to the ACE2 receptors of human cells and use this as a springboard to enter cells. In addition, after SARS-CoV2 binds to ACE2 on the surface of human cells, enzymatic treatment of TMPRSS2 of human cells enhances the efficiency of entry into cells. Therefore, cell invasion by such viruses can be suppressed by suppressing the expression of ACE2 and/or TMPRSS2. Infection can be suppressed by this.
 こうしたアプローチのメリットは、ウイルス側の変異による影響を受けにくい点にある。一般にSARS-CV2をはじめとするRNA型ウイルスは、変異を起こしやすい。そして、ウイルス表面のタンパク質(例えば、スパイクタンパク質)は、ワクチンのターゲットとされるものの、このタンパク質に変異が起こることにより、ワクチンによる効果が回避されてしまう問題があった。しかし、このアプローチでは、変異による直接的な影響は受けにくいという点で、有利である。 The advantage of this approach is that it is less susceptible to mutations on the virus side. In general, RNA viruses including SARS-CV2 are prone to mutation. Although proteins on the surface of viruses (for example, spike proteins) are targeted by vaccines, there is a problem that the effects of vaccines can be avoided due to mutations in these proteins. However, this approach has the advantage that it is less directly affected by mutations.
 また、特筆すべきは、本開示の組成物は、感染の治療のみならず、感染の予防にも寄与することができる。例えば、感染の治療には、抗炎症作用などに基づいたアプローチがあるが、こうしたアプローチは、感染が起こった後での対処法となるため、予防には寄与できない。しかし、本開示の組成物は、いったん感染が起こってウイルスが増殖した後でも、他の細胞への更なる感染を防ぐという意味では、治療薬としても寄与できる。しかし、それだけではなく、感染する前の状態であっても、予め投与することで、ウイルスが感染する際の足掛かりとなる遺伝子の発現を抑制することで、細胞への感染を防ぐという意味で予防薬としても寄与できる。 Also, it should be noted that the composition of the present disclosure can contribute not only to the treatment of infection but also to the prevention of infection. For example, there are approaches to treatment of infection based on anti-inflammatory effects, but these approaches are countermeasures after infection has occurred and cannot contribute to prevention. However, the compositions of the present disclosure can also serve as therapeutic agents in the sense that, once infection has occurred and the virus has propagated, it prevents further infection of other cells. However, not only that, but even before infection, by administering it in advance, it suppresses the expression of genes that are a stepping stone for virus infection, thereby preventing cell infection. It can also be used as a medicine.
 従って、一実施形態において、本開示の組成物は、投与形態に応じて、適切な追加成分を配合してもよい。例えば、本開示の組成物は、医薬品に一般的に含まれる成分を含んでもよい。当該成分の例として、保存料、防腐剤、香料、pH調整剤、酸化防止剤、防カビ剤、賦形剤、結合剤、崩壊剤、滑沢剤などが挙げられる。 Therefore, in one embodiment, the composition of the present disclosure may be formulated with suitable additional ingredients depending on the dosage form. For example, compositions of the present disclosure may include ingredients commonly included in pharmaceuticals. Examples of such ingredients include preservatives, preservatives, fragrances, pH adjusters, antioxidants, antifungal agents, excipients, binders, disintegrants, lubricants, and the like.
 好ましい実施形態においては、本明細書で記載されるFoxO1阻害剤は、糖尿病患者への投与は対象外とする。 In preferred embodiments, the FoxO1 inhibitors described herein are not intended for administration to diabetic patients.
4.実施例
4-1.実施例1(HepG2細胞の培養)
 HepG2細胞はATCC(登録商標)(American Type Culture Collection、HB-8065)から購入した。当該細胞はDMEM(SIGMA,D8900)+10%FBS(SIGMA,F7524)の血清培地を用いて継代・維持した。
4. Example
4-1. Example 1 (HepG2 cell culture)
HepG2 cells were purchased from ATCC® (American Type Culture Collection, HB-8065). The cells were subcultured and maintained using a serum medium of DMEM (SIGMA, D8900) + 10% FBS (SIGMA, F7524).
4-2.実施例2(FoxO1阻害剤AS1842856の効果)
4-2-1.RNAの調製 
 HepG2細胞を12 well plate(Corning(登録商標),3336)の各ウェルに50,000cells/cm2で播種し、一晩培養した。翌日(播種後16~24時間)、FoxO1阻害剤AS1842856を複数の濃度で添加(1nM、10nM、50nM、100nM、1μM)し、又はその溶媒として用いたDMSOをコントロールとして加え、1日(約24時間)培養した。培養後、ReliaPrep RNA Miniprep system(Promega(登録商標), Z6012)を用いてHepG2細胞からTotal RNAを抽出した。抽出後、500ngのRNAを用いてcDNA合成(PrimeScript RT Master Mix; Takara(登録商標), RR036A)を行い、更に、定量的PCR(Thunderbird(登録商標) Sybr qPCR Mix; TOYOBO(登録商標), QPS-201X5)を行った。
4-2. Example 2 (Effect of FoxO1 inhibitor AS1842856)
4-2-1. Preparation of RNA
HepG2 cells were seeded into each well of a 12-well plate (Corning (registered trademark), 3336) at 50,000 cells/cm 2 and cultured overnight. The next day (16-24 hours after seeding), the FoxO1 inhibitor AS1842856 was added at multiple concentrations (1 nM, 10 nM, 50 nM, 100 nM, 1 μM) or DMSO, which was used as its solvent, as a control. time) cultured. After culturing, total RNA was extracted from HepG2 cells using ReliaPrep RNA Miniprep system (Promega (registered trademark), Z6012). After extraction, cDNA synthesis (PrimeScript RT Master Mix; Takara (registered trademark), RR036A) was performed using 500 ng of RNA, and quantitative PCR (Thunderbird (registered trademark) Sybr qPCR Mix; TOYOBO (registered trademark), QPS -201X5) was performed.
 cDNA合成のためのミクスチャは以下の組成に従って調製した。
 5xPrimeScript RT Master Mix 2 μl(最終濃度 1 x)
 Total RNA                  500 ng
 RNase free H20            合計10 μlに調整
A mixture for cDNA synthesis was prepared according to the following composition.
2 μl of 5x PrimeScript RT Master Mix (final concentration 1x)
Total RNA 500 ng
Adjusted to 10 μl total of RNase free H 2 O
 上記ミクスチャを、Applied Biosystems(登録商標)社のVeriti 96 well Thermal Cyclerを用いて、以下の条件で処理した。
 37℃ 15分
 ↓
 85℃ 5秒
 ↓
 4℃ ∞
The above mixture was processed using a Veriti 96 well Thermal Cycler manufactured by Applied Biosystems (registered trademark) under the following conditions.
37°C 15 minutes ↓
85°C 5 seconds ↓
4°C ∞
 合成したcDNA(10 μl)は90μlのTE(10mM Tris-HCl pH8.0 + 1mM EDTA pH8.0)を用いて10倍に希釈した。当該希釈物を、定量PCRに供した。 The synthesized cDNA (10 µl) was diluted 10-fold with 90 µl of TE (10 mM Tris-HCl pH 8.0 + 1 mM EDTA pH 8.0). The dilutions were subjected to quantitative PCR.
4-2-3.HepG2細胞におけるACE2及びTMPRSS2遺伝子発現の解析
 より具体的には、当該希釈物を、以下の条件で混合した。
2×THUNDERBIRD(登録商標) Probe qPCR Mix                          10μl
5mM Forward Primer           0.4μl
5mM Reverse Primer           0.4μl
20                            8.2μl
cDNA(10倍希釈)                   1 μl
4-2-3. More specifically, for the analysis of ACE2 and TMPRSS2 gene expression in HepG2 cells , the dilutions were mixed under the following conditions.
10 μl of 2×THUNDERBIRD® Probe qPCR Mix
0.4 μl of 5 mM Forward Primer
0.4 μl of 5 mM Reverse Primer
8.2 μl of H 2 O
cDNA (10-fold dilution) 1 μl
 上記混合物を、Bio-Rad(登録商標)社のCFX-Connectを用いて、cDNAを増幅させた。PCRサイクルの条件は以下の通りであった。
1.95℃ 1分  (初期変性) 
2.95℃ 15秒 (変性)
3.60℃ 30秒 (伸長)
(2~3のステップを40回繰り返し、ステップ3が終わるたびに蛍光シグナルを検出)
4.65℃~95℃まで0.5℃刻みで温度を上昇させ、5秒ずつ温度を保持してから蛍光シグナルを検出
cDNA was amplified from the above mixture using Bio-Rad (registered trademark) CFX-Connect. The PCR cycle conditions were as follows.
1.95°C 1 minute (initial denaturation)
2.95°C 15 seconds (denaturation)
3.60°C 30 seconds (elongation)
(Repeat steps 2-3 40 times, detect fluorescence signal each time step 3 is completed)
4. Raise the temperature from 65°C to 95°C in increments of 0.5°C, hold the temperature for 5 seconds, and then detect the fluorescence signal.
 上記サイクルにて、PCR産物の検出を行い、併せて、Melting curveによるPCR産物の単一性の確認を行った。 In the above cycle, the PCR product was detected, and at the same time, the unity of the PCR product was confirmed by Melting curve.
 内部標準(すべての細胞・条件において同じ発現をしているハウスキーピング遺伝子)として、GAPDH(lycerldehyde 3-hosphate ydrogenase)を用いた。
各遺伝子を検出するためのプライマー配列は以下の通りであった。
GAPDH Forward primer: 5’ - agccacatcgctcagacac - 3’(配列番号1)
GAPDH Reverse primer: 5’ - gcctaatacgaccaaatcc - 3’(配列番号2)
ACE2 Forward primer: 5’ - ttctgtcacccgattttcaa - 3’(配列番号3)
ACE2 Reverse primer: 5’ - tcccaacaatcgtgagtgc - 3’(配列番号4)
TMPRSS2 Forward primer: 5’ - cgctggcctactctggaa - 3’(配列番号5)
TMPRSS2 Reverse primer: 5’ - ctgaggagtcgcactctatcc - 3’(配列番号6)
GAPDH ( Glyceraldehyde 3- phosphate dehydrogenase) was used as an internal standard ( a housekeeping gene expressing the same expression in all cells and conditions ) .
The primer sequences for detecting each gene were as follows.
GAPDH Forward primer: 5'-agccacatcgctcagacac-3' (SEQ ID NO: 1)
GAPDH Reverse primer: 5'-gcctaatacgaccaaatcc-3' (SEQ ID NO: 2)
ACE2 Forward primer: 5'-ttctgtcacccgattttcaa-3' (SEQ ID NO: 3)
ACE2 Reverse primer: 5'-tcccaacaatcgtgagtgc-3' (SEQ ID NO: 4)
TMPRSS2 Forward primer: 5'-cgctggcctactctggaa-3' (SEQ ID NO: 5)
TMPRSS2 Reverse primer: 5'-ctgaggagtcgcactctatcc-3' (SEQ ID NO: 6)
 全てのプライマーは株式会社ファスマック(登録商標)から購入した(逆相カラム精製グレード)。
各遺伝子の発現量は、定量PCRで得られた各遺伝子の発現量を、さらにGAPDHの発現量で割って標準化したものを示している。さらに、FoxO1阻害剤で処理をしていないコントロール条件(DMSO処理)での発現量を「1」になるように標準化している。以上のプライマーを用いて増幅したPCR産物は全て単一のものであること(つまり、同一のプライマーで、複数種類の配列が増幅されていないこと)をMelting curveによって確認した。
All primers were purchased from Fasmac (registered trademark), Inc. (reverse-phase column purification grade).
The expression level of each gene indicates the standardized value obtained by dividing the expression level of each gene obtained by quantitative PCR by the expression level of GAPDH. Furthermore, the expression level under the control condition (DMSO treatment) without FoxO1 inhibitor treatment is normalized to "1". It was confirmed by a melting curve that the PCR products amplified using the above primers were all single (that is, multiple types of sequences were not amplified with the same primers).
 結果を図1に示す。コントロールのDMSO処理と比較すると、FoxO1阻害剤AS1842856を加えると、その濃度依存的にACE2及びTMPRSS2のmRNAの発現が減少することが観察された(多重比較検定(Tukey-Kramer法))。この結果は、FoxO1がACE2及びTMPRSS2のmRNAの発現に必要であることを示している。 The results are shown in Figure 1. When the FoxO1 inhibitor AS1842856 was added, it was observed that the expression of ACE2 and TMPRSS2 mRNAs decreased in a concentration-dependent manner (multiple comparison test (Tukey-Kramer method)) when compared with DMSO-treated controls. This result indicates that FoxO1 is required for the expression of ACE2 and TMPRSS2 mRNAs.
4-2-4.スパイクタンパク質の細胞内への取り込みへの影響
 次に、FoxO1阻害剤によるACE2の発現抑制が、SARS-CoV2とホスト細胞の結合及び細胞内への取り込みを阻害できるか検討した。この目的で、SARS-CoV2のスパイクタンパク質のS1の中でACE2に結合するために必要十分な領域RBD(eceptor inding omain、R319~F541)にマウスIgGのFc領域が融合されたリコンビナントタンパク質RBD-mFc(Sino Biological、40592-V05H2)を準備した。実験の模式図を図2に示す。
4-2-4. Effect of Spike Protein on Intracellular Uptake Next, we examined whether suppression of ACE2 expression by a FoxO1 inhibitor could inhibit the binding and intracellular uptake of SARS-CoV2 to host cells. For this purpose, a recombinant protein in which the Fc region of mouse IgG is fused to the region RBD (Receptor Binding Domain, R319 to F541 ) necessary and sufficient for binding to ACE2 in S1 of SARS- CoV2 spike protein RBD-mFc (Sino Biological, 40592-V05H2) was prepared. A schematic diagram of the experiment is shown in FIG.
 HepG2細胞を6 well plate(CellBIND(登録商標); Corning(登録商標), 3335)の各ウェルに100,000 個を播種し、一晩培養した。翌日(播種後16~24時間)、FoxO1阻害剤AS1842856を1μMの濃度で添加、又はその溶媒として用いたDMSOをコントロールとして加え、3日間培養した(約72時間)。培養後、最終濃度が100nMとなるようにRBD-mFc、またはコントロールとして何も融合していないマウスIgG由来Fc領域(mFc;Abcam(登録商標),ab179982)を添加し、さらに3時間培養した。その後、PBS(-)で細胞を洗浄後、剥離し細胞を回収した。回収した細胞は4% パラホルムアルデヒド(PFA)(富士フィルム和光純薬、 163-204115)で15分間室温で固定した。固定後、PBS(-)で洗浄し、マウスIgGを認識する抗体にAlexa Fluor(登録商標) 488蛍光色素が共有結合した二次抗体(JacksonImmunoResearch,715-545-150)で細胞表面や細胞内に取り込まれたmFcを染色し、フローサイトメトリー(Beckman Coulter(登録商標),FC500)を用いて一細胞あたりのAlexa Fluor(登録商標) 488の蛍光強度を測定し、定量した。解析にはKaluza Analysis 2.1(Beckman Coulter(登録商標))を用いた。典型的な細胞の写真は、蛍光顕微鏡(Keyence、BX-700)を用いて撮影した。 100,000 HepG2 cells were seeded in each well of a 6-well plate (CellBIND (registered trademark); Corning (registered trademark), 3335) and cultured overnight. On the next day (16 to 24 hours after seeding), the FoxO1 inhibitor AS1842856 was added at a concentration of 1 μM, or DMSO used as its solvent was added as a control, and cultured for 3 days (about 72 hours). After culturing, RBD-mFc was added to a final concentration of 100 nM, or mouse IgG-derived Fc region (mFc; Abcam (registered trademark), ab179982) with nothing fused as a control was added, and the cells were further cultured for 3 hours. Then, after washing the cells with PBS(-), the cells were collected by detachment. The recovered cells were fixed with 4% paraformaldehyde (PFA) (Fuji Film Wako Pure Chemical Industries, Ltd., 163-204115) at room temperature for 15 minutes. After fixation, it was washed with PBS (-), and a secondary antibody (Jackson ImmunoResearch, 715-545-150), in which Alexa Fluor (registered trademark) 488 fluorescent dye was covalently bound to an antibody that recognizes mouse IgG, was applied to the cell surface and intracellularly. The incorporated mFc was stained, and the fluorescence intensity of Alexa Fluor (registered trademark) 488 per cell was measured and quantified using flow cytometry (Beckman Coulter (registered trademark), FC500). Kaluza Analysis 2.1 (Beckman Coulter (registered trademark)) was used for the analysis. Photographs of typical cells were taken using a fluorescence microscope (Keyence, BX-700).
 結果を図3に示す。まず、RBDがHepG2細胞に取り込まれ得るのか、免疫染色法及び蛍光顕微鏡を用いて観察した。コントロールのmFcを添加した場合には、全くほとんど蛍光シグナルが検出されなかったのに対し、RBD-mFcは50%近くの細胞で観察された。これらの結果から、HepG2細胞はRBDを取り込むことが示唆された。 The results are shown in Figure 3. First, whether RBD could be taken up into HepG2 cells was observed using an immunostaining method and a fluorescence microscope. Almost no fluorescence signal was detected when control mFc was added, whereas RBD-mFc was observed in nearly 50% of the cells. These results suggested that HepG2 cells take up RBD.
 次に、FoxO1阻害剤AS1842856を用いて、あらかじめACE2の発現を抑制した時、取り込まれるRBDの量が抑制されるかフローサイトメトリーを用いて検討した。蛍光強度の違いを図4に示す。まず、ネガティブコントロールであるmFcを添加した際の細胞の示す蛍光強度は、ACE2を介さずにmFcのみで取り込まれてしまったもの、二次抗体が非特異的に結合してしまったもの、及び細胞のもつ自家蛍光、の3つに由来し、これはバックグラウンドシグナルと考えられる。従って、mFcでの蛍光強度を引くことで、より正確に取り込まれたRBD由来のシグナルの差を正確に比較することが可能になる。すると、コントロールのDMSOを添加した際のRBDの蛍光シグナルと比較すると、FoxO1阻害剤AS1842856で予め処理することで、RBD由来の蛍光シグナルの強度が有意に減少した(T検定;p<0.0005)。すなわち、この結果はFoxO1阻害剤によってRBDのHepG2細胞への取り込みが抑制されたことを示しており、FoxO1阻害剤によるACE2等のmRNA発現量の抑制が、RBDのHepG2細胞への取り込みを抑制できるレベルまで抑えていることを示唆している。 Next, flow cytometry was used to examine whether the amount of RBD taken up was suppressed when the expression of ACE2 was previously suppressed using the FoxO1 inhibitor AS1842856. FIG. 4 shows the difference in fluorescence intensity. First, the fluorescence intensity exhibited by the cells when mFc, which is a negative control, was added, was taken up only by mFc without mediated by ACE2, was nonspecifically bound by the secondary antibody, and autofluorescence of cells, which is considered to be a background signal. Subtracting the fluorescence intensity at the mFc therefore allows an accurate comparison of signal differences from more accurately incorporated RBDs. Then, pretreatment with the FoxO1 inhibitor AS1842856 significantly decreased the intensity of the fluorescence signal from the RBD when compared to the fluorescence signal from the RBD upon addition of control DMSO (T-test; p<0.0005 ). That is, this result indicates that the FoxO1 inhibitor inhibited the uptake of RBD into HepG2 cells, and the suppression of the expression level of mRNA such as ACE2 by the FoxO1 inhibitor can suppress the uptake of RBD into HepG2 cells. It suggests that it is suppressed to the level.
 さらに、この結果はFoxO1阻害剤によって、SARS-CoV2がホスト細胞に結合し感染する(取り込まれる)ことが抑制されることを示唆している。 Furthermore, this result suggests that FoxO1 inhibitors suppress SARS-CoV2 binding and infecting (uptake) host cells.
 以上の実験結果から示される、一実施形態における本開示のFoxO1阻害剤AS1842856の新型コロナウィルスの細胞内への取り込みが阻害される作用機序を図5にまとめた。AS1842856は細胞内のFoxO1に作用して、ACE2及び/又はTMPRSS2の遺伝子の発現を抑制する。これによって、細胞表面のACE2及び/又はTMPRSS2の量が減少する。そして、ウイルス表面に存在するスパイクタンパク質とACE2との結合が阻害される。また、TMPRSS2によるウイルスの細胞内への侵入の促進効果も抑制される。そしてその結果として、ACE2及び/又はTMPRSS2によって介在される感染症、特にコロナウィルスや、その他の疾患の予防又は治療効果をもたらす。 FIG. 5 summarizes the mechanism of action of the FoxO1 inhibitor AS1842856 of the present disclosure, which is shown by the above experimental results, to inhibit the uptake of the novel coronavirus into cells. AS1842856 acts on intracellular FoxO1 to suppress gene expression of ACE2 and/or TMPRSS2. This reduces the amount of ACE2 and/or TMPRSS2 on the cell surface. Then, the binding between the spike protein present on the virus surface and ACE2 is inhibited. In addition, the effect of TMPRSS2 to promote virus entry into cells is also suppressed. As a result, it provides preventive or therapeutic effects for infectious diseases, particularly coronaviruses, and other diseases mediated by ACE2 and/or TMPRSS2.
4-3.実施例3(インスリンの効果)
 次に、インスリンの効果について検討するため、HepG2細胞を12 well plate(Corning(登録商標),3336)の各ウェルに50,000cells/cm2で播種し、一晩培養した。翌日(播種後16~24時間)、野生型および改変型インスリンを10μg/mlで添加し、又はコントロールとしてPBS(-)を加え、3日(約72時間)培養した。培養後、上記の実施例2に記載の通りに、Total RNA抽出、cDNA合成、定量的PCRを行った。
4-3. Example 3 (effect of insulin)
Next, in order to examine the effect of insulin, HepG2 cells were seeded in each well of a 12-well plate (Corning (registered trademark), 3336) at 50,000 cells/cm 2 and cultured overnight. The next day (16-24 hours after seeding), wild-type and modified insulin were added at 10 μg/ml, or PBS(−) was added as a control, and cultured for 3 days (about 72 hours). After culturing, total RNA extraction, cDNA synthesis, and quantitative PCR were performed as described in Example 2 above.
 結果を図6に示す。コントロールのPBS(-)処理と比較すると、野生型インスリンを加えると、ACE2及びTMPRSS2のmRNAの発現が減少することが観察された(多重比較検定(Tukey-Kramer法))。この結果は、インスリンとその下流のシグナル伝達経路がACE2及びTMPRSS2のmRNAの発現を抑制することを示している。 The results are shown in Figure 6. A decrease in the expression of ACE2 and TMPRSS2 mRNAs was observed when wild-type insulin was added compared to the PBS(−)-treated control (multiple comparison test (Tukey-Kramer method)). This result indicates that insulin and its downstream signaling pathway suppress the expression of ACE2 and TMPRSS2 mRNAs.
 インスリンは、遺伝子工学によってアミノ酸に変異が導入され、その効果が投与後いつ現れるか、そしてどの程度持続するか、によって、超速効型インスリン、速効型インスリン、中間型インスリン、などの改変型インスリンが製造され、糖尿病患者の病態によって使い分けがなされている。そこで、これらの改変型インスリンを用いて、野生型のインスリンによるACE2及びTMPRSS2のmRNAの発現の減少が観察されるか、検討した。 Insulin is categorized into modified insulins such as fast-acting insulin, rapid-acting insulin, intermediate-acting insulin, etc., depending on when mutations are introduced into amino acids by genetic engineering and how long the effect appears after administration and how long it lasts. It is manufactured and used properly according to the condition of the diabetic patient. Therefore, using these modified insulins, it was examined whether or not reduction in expression of ACE2 and TMPRSS2 mRNAs by wild-type insulin was observed.
 具体的には、以下の改変型インスリンを使用した。
 商品名:リスプロ(インスリン リスプロ)(超速効型)
 商品名:ノボラピッド(登録商標)(インスリンアスパルト)(超速効型)
 商品名:アピドラ(登録商標)(インスリン グルリジン)(超速効型)
 商品名:ヒューマリン(登録商標)R(レギュラーインスリン)(速効型)
 商品名:ヒューマリン(登録商標)N(イソフェンインスリン)(中間型)
 商品名:ノボリン(登録商標)N(イソフェンインスリン)(中間型)
 商品名:ノボラピッド(登録商標)30ミックス(インスリンアスパルト(超速効型)とプロタミン結晶性インスリンアスパルト(中間型)を3:7で混合したもの)(混合型)
 商品名:レベミル(登録商標)(インスリン デテミル)(持効型溶解)
 商品名:ライゾデグ(登録商標)(インスリンアスパルト(超速効型)とインスリンデグルデク(持効型)を3:7で混合したもの)(配合溶解性)
Specifically, the following modified insulin was used.
Product name: Lispro (insulin lispro) (rapidly acting)
Product name: NovoRapid (registered trademark) (insulin aspart) (rapidly acting)
Product name: Apidra (registered trademark) (insulin glulisine) (rapidly acting)
Product name: Humarin (registered trademark) R (regular insulin) (rapid-acting)
Trade name: Humalin® N (Isophane insulin) (intermediate-acting)
Trade name: Novolin® N (isophane insulin) (intermediate-acting)
Trade name: Novorapid (registered trademark) 30 mix (3:7 mixture of insulin aspart (ultra-rapid-acting type) and protamine crystalline insulin aspart (intermediate type)) (mixed type)
Brand name: Levemir® (insulin detemir) (long-acting dissolution)
Trade name: Ryzodeg (registered trademark) (mixture of insulin aspart (rapid-acting) and insulin degludec (long-acting) at a ratio of 3:7) (blended solubility)
 野生型インスリンは、A鎖とB鎖が結合した合計51個のアミノ酸配列から構成される。以下に、上述したインスリンのアミノ酸配列を示す。 Wild-type insulin consists of a total of 51 amino acid sequences in which the A chain and B chain are linked. The amino acid sequence of insulin mentioned above is shown below.
A鎖(全て共通)
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn  (配列番号7)
A chain (all common)
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn (SEQ ID NO: 7)
B鎖(下線部は、相互に相違するアミノ酸部分を示す)
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr(ヒト野生型、レギュラーインスリン) (配列番号8)
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Lys Thr(アスパルト) (配列番号9)
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Lys Pro Thr(リスプロ) (配列番号10)
Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Glu Thr(グルリジン) (配列番号11)
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys -Myristic Acid(デテミル) (配列番号12)
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys -γ-Glu-Nε-hexadecandioyl(デグルデク) (配列番号13)
B chain (underlined parts indicate mutually different amino acid moieties)
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr (human wild type, regular insulin) (SEQ ID NO: 8)
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Lys Thr (Aspart) (SEQ ID NO: 9)
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Lys Pro Thr (lispro) (SEQ ID NO: 10)
Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Glu Thr (Glulisine) (SEQ ID NO: 11)
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys -Myristic Acid (SEQ ID NO: 1 detemir)
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys -γ-Glu-Nε-hdexadel3 )
イソフェンインスリンは別名NPHインスリンとも呼ばれ、野生型(レギュラー)インスリンとプロタミンが結合・結晶化したものである。プロタミン結晶性インスリンアスパルトはプロタミンとインスリンアスパルトが結合・結晶化したものである。 Isophane insulin, also known as NPH insulin, is a product obtained by binding and crystallizing wild-type (regular) insulin and protamine. Protamine crystalline insulin aspart is a combination and crystallization of protamine and insulin aspart.
 結果を図7に示す。コントロールのPBS(-)処理と比較すると、どの改変型インスリンを加えても、野生型インスリンと同程度のACE2及びTMPRSS2のmRNAの発現減少が観察された。この結果は、どのタイプのインスリン製剤であっても、インスリンの下流のシグナル伝達経路を活性化する限りは、ACE2及びTMPRSS2のmRNAの発現を抑制できることを示している。 The results are shown in Figure 7. When compared with the PBS(−)-treated control, ACE2 and TMPRSS2 mRNA expression levels were reduced to the same extent as wild-type insulin when any of the modified insulins were added. This result indicates that any type of insulin preparation can suppress the expression of ACE2 and TMPRSS2 mRNA as long as it activates downstream signaling pathways of insulin.
4-4.実施例4(インスリンによるスパイクタンパク質の細胞内への取り込みへの影響)
 次に、インスリンによるACE2の発現抑制が、SARS-CoV2とホスト細胞の結合及び細胞内への取り込みを阻害できるか検討した。この目的で、SARS-CoV2のスパイクタンパク質のS1ドメイン、さらにS1の中でACE2に結合するために必要十分な領域RBD(eceptor inding omain、R319~F541)にマウスIgGのFc領域が融合されたリコンビナントタンパク質S1-mFc(Sino Biological、40592-V05H1)、及びRBD-mFc(Sino Biological、40592-V05H2)を準備した。
4-4. Example 4 (Effect of insulin on spike protein uptake into cells)
Next, we investigated whether suppression of ACE2 expression by insulin could inhibit the binding of SARS-CoV2 to host cells and their uptake into cells. For this purpose, the Fc region of mouse IgG is fused to the S1 domain of the spike protein of SARS- CoV2 , and to the region RBD (Receptor Binding Domain, R319 to F541 ) necessary and sufficient for binding to ACE2 in S1. Recombinant protein S1-mFc (Sino Biological, 40592-V05H1) and RBD-mFc (Sino Biological, 40592-V05H2) were prepared.
 HepG2細胞を6 well plate(CellBIND(登録商標);Corning(登録商標),3335)の各ウェルに100,000個を播種し、一晩培養した。翌日(播種後16~24時間)、野生型インスリンを10μg/mlの濃度で添加、又はPBS(-)をコントロールとして加え、3日間培養した(約72時間)。培養後、最終濃度が100nMとなるようにS1m-FC、RBD-mFc、またはコントロールとして何も融合していないマウスIgG由来Fc領域(mFc;Abcam(登録商標),ab179982)を添加し、さらに3時間培養した。その後、上記の実施例2で記載した通りに、細胞内に取り込まれたS1及びRBDの量を定量した。 100,000 HepG2 cells were seeded in each well of a 6-well plate (CellBIND (registered trademark); Corning (registered trademark), 3335) and cultured overnight. The next day (16 to 24 hours after seeding), wild-type insulin was added at a concentration of 10 μg/ml, or PBS(−) was added as a control, and cultured for 3 days (about 72 hours). After culturing, S1m-FC, RBD-mFc, or an unfused mouse IgG-derived Fc region (mFc; Abcam (registered trademark), ab179982) was added as a control to a final concentration of 100 nM. cultured for hours. The amount of S1 and RBD taken up into cells was then quantified as described in Example 2 above.
 まず、ネガティブコントロールであるmFcを添加した際の細胞の示す蛍光強度は、ACE2を介さずにmFcのみで取り込まれてしまったもの、二次抗体が非特異的に結合してしまったもの、及び細胞のもつ自家蛍光、の3つに由来し、これはバックグラウンドシグナルと考えられる。従って、mFcでの蛍光強度を引くことで、より正確に取り込まれたS1、及びRBD由来のシグナルの差を正確に比較することが可能になる。そこで、mFcでの蛍光強度を引いたうえで、コントロールのPBS(-)を添加した際のS1、及びRBDの蛍光シグナルと比較すると、インスリンで予め処理することで、S1、及びRBD由来の蛍光シグナルの強度が有意に減少した(図8参照、多重比較検定(Tukey-Kramer法))。すなわち、この結果はインスリンによってS1、及びRBDのHepG2細胞への取り込みが抑制されたことを示しており、インスリンによるACE2等のmRNA発現量の抑制が、S1、及びRBDのHepG2細胞への取り込みを抑制できるレベルまで抑えていることを示唆している。 First, the fluorescence intensity exhibited by the cells when mFc, which is a negative control, was added, was taken up only by mFc without mediated by ACE2, was nonspecifically bound by the secondary antibody, and autofluorescence of cells, which is considered to be a background signal. Subtracting the fluorescence intensity at the mFc therefore allows for an accurate comparison of the signal differences from more accurately incorporated S1 and RBD. Therefore, after subtracting the fluorescence intensity of mFc, when compared with the fluorescence signal of S1 and RBD when PBS (-) was added as a control, pretreatment with insulin showed that S1 and RBD-derived fluorescence The signal intensity was significantly reduced (see FIG. 8, multiple comparison test (Tukey-Kramer method)). That is, this result indicates that insulin inhibited the uptake of S1 and RBD into HepG2 cells, and that insulin-induced suppression of mRNA expression levels such as ACE2 inhibited the uptake of S1 and RBD into HepG2 cells. It suggests that it is suppressed to a level that can be suppressed.
 さらに、この結果はインスリンによって、SARS-CoV2がホスト細胞に結合し感染する(取り込まれる)ことが抑制されることを示唆している。 Furthermore, this result suggests that insulin suppresses SARS-CoV2 from binding and infecting (uptake) host cells.
 以上の実験結果から示される、一実施形態における本開示のインスリン系化合物の新型コロナウィルスの細胞内への取り込みが阻害される作用機序を図9にまとめた。インスリン系化合物は細胞に作用して、ACE2及び/又はTMPRSS2の遺伝子の発現を抑制する。これによって、細胞表面のACE2及び/又はTMPRSS2の量が減少する。そして、ウイルス表面に存在するスパイクタンパク質とACE2との結合が阻害される。また、TMPRSS2によるウイルスの細胞内への侵入の促進効果も抑制される。そしてその結果として、ACE2及び/又はTMPRSS2によって介在される感染症、特にコロナウィルスや、その他の疾患の予防又は治療効果をもたらす。 FIG. 9 summarizes the mechanism of action by which the insulin-based compound of the present disclosure inhibits the cellular uptake of the novel coronavirus in one embodiment, as shown by the above experimental results. Insulin-based compounds act on cells to suppress the expression of the ACE2 and/or TMPRSS2 genes. This reduces the amount of ACE2 and/or TMPRSS2 on the cell surface. Then, the binding between the spike protein present on the virus surface and ACE2 is inhibited. In addition, the effect of TMPRSS2 to promote virus entry into cells is also suppressed. As a result, it provides preventive or therapeutic effects for infectious diseases, particularly coronaviruses, and other diseases mediated by ACE2 and/or TMPRSS2.
4-5.実施例5(成長因子による効果)
 実施例1~4と同様の実験を行った。ただし、各種インスリン化合物等に代えて、以下の成長因子を10ng/mlの濃度で使用した。これらは、シグナル経路の下流として、FoxO1を抑制することが知られている。
IGF1(PeproTech社、製品番号AF-100-11)、
IGF2(PeproTech社、製品番号AF-100-12)、
EGF(PeproTech社、製品番号AF-100-15)、
FGF2(PeproTech社、製品番号AF-100-18B)、
FGF14(BioVision(登録商標)社、製品番号7347)、
VEGF165(PeproTech社、製品番号AF-100-20)、
4-5. Example 5 (Effects of growth factors)
Experiments similar to Examples 1-4 were conducted. However, instead of various insulin compounds, the following growth factors were used at a concentration of 10 ng/ml. These are known to suppress FoxO1 as a downstream signal pathway.
IGF1 (PeproTech, product number AF-100-11),
IGF2 (PeproTech, product number AF-100-12),
EGF (PeproTech, product number AF-100-15),
FGF2 (PeproTech, product number AF-100-18B),
FGF14 (BioVision®, product number 7347),
VEGF165 (PeproTech, product number AF-100-20),
上記の成長因子のアミノ酸配列は以下のとおりである。 The amino acid sequences of the above growth factors are as follows.
IGF1
https://www.peprotech.com/gb/recombinant-human-igf-i-2
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu Lys Pro Ala Lys Ser Ala(配列番号14)
IGF1
https://www.peprotech.com/gb/recombinant-human-igf-i-2
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu Lys Pro Ala Lys Ser Ala (SEQ ID NO: 14)
IGF2
https://www.peprotech.com/en/recombinant-human-igf-ii-2
Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala Lys Ser Glu(配列番号15)
IGF2
https://www.peprotech.com/en/recombinant-human-igf-ii-2
Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala Lys Ser Glu (SEQ ID NO: 15)
EGF
https://www.peprotech.com/en/recombinant-human-eg
fAsn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg(配列番号16)
EGF
https://www.peprotech.com/en/recombinant-human-eg
fAsn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp GluLeuArg (SEQ ID NO: 16)
FGF2
https://www.peprotech.com/en/recombinant-human-fgf-basic-154-aa-2
Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser(配列番号17)
FGF2
https://www.peprotech.com/en/recombinant-human-fgf-basic-154-aa-2
Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser (SEQ ID NO: 17)
FGF14
https://www.biovision.com/fgf14-human-recombinant.html
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser His Met Gly Ser His Met Ala Ala Ala Ile Ala Ser Gly Leu Ile Arg Gln Lys Arg Gln Ala Arg Glu Gln His Trp Asp Arg Pro Ser Ala Ser Arg Arg Arg Ser Ser Pro Ser Lys Asn Arg Gly Leu Cys Asn Gly Asn Leu Val Asp Ile Phe Ser Lys Val Arg Ile Phe Gly Leu Lys Lys Arg Arg Leu Arg Arg Gln Asp Pro Gln Leu Lys Gly Ile Val Thr Arg Leu Tyr Cys Arg Gln Gly Tyr Tyr Leu Gln Met His Pro Asp Gly Ala Leu Asp Gly Thr Lys Asp Asp Ser Thr Asn Ser Thr Leu Phe Asn Leu Ile Pro Val Gly Leu Arg Val Val Ala Ile Gln Gly Val Lys Thr Gly Leu Tyr Ile Ala Met Asn Gly Glu Gly Tyr Leu Tyr Pro Ser Glu Leu Phe Thr Pro Glu Cys Lys Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile Tyr Ser Ser Met Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp Phe Leu Gly Leu Asn Lys Glu Gly Gln Ala Met Lys Gly Asn Arg Val Lys Lys Thr Lys Pro Ala Ala His Phe Leu Pro Lys Pro Leu Glu Val Ala Met Tyr Arg Glu(配列番号18)
FGF14
https://www.biovision.com/fgf14-human-recombinant.html
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser His Met Gly Ser His Met Ala Ala Ala Ile Ala Ser Gly Leu Ile Arg Gln Lys Arg Gln Ala Arg Glu Gln His Trp Asp Arg Pro Ser Ala Ser Arg Arg Arg Ser Ser Pro Ser Lys Asn Arg Gly Leu Cys Asn Gly Asn Leu Val Asp Ile Phe Ser Lys Val Arg Ile Phe Gly Leu Lys Lys Arg Arg Leu Arg Arg Gln Asp Pro Gln Leu Lys Gly Ile Val Thr Arg Leu Tyr Cys Arg Gln Gly Tyr Tyr Leu Gln Met His Pro Asp Gly Ala Leu Asp Gly Thr Lys Asp Asp Ser Thr Asn Ser Thr Leu Phe Asn Leu Ile Pro Val Gly Leu Arg Val Val Ala Ile Gln Gly Val Lys Thr Gly Leu Tyr Ile Ala Met Asn Gly Glu Gly Tyr Leu Tyr Pro Ser Glu Leu Phe Thr Pro Glu Cys Lys Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile Tyr Ser Ser Met Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp Phe Leu Gly Leu Asn Lys Glu Gly Gln Ala Met Lys Gly Asn Arg Val Lys Lys Thr Lys Pro Ala Ala His Phe Leu Pro Lys Pro Leu Glu Val Ala Met Tyr Arg Glu (SEQ ID NO: 18)
VEGF 165
https://www.peprotech.com/en/recombinant-human-vegfsub165sub-2
Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg(配列番号19)
VEGF 165
https://www.peprotech.com/en/recombinant-human-vegfsub165sub-2
Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg (SEQ ID NO: 19)
 結果を図10に示す。いずれの成長因子もコントロールと比べて、ACE2及び/又はTMPRSS2の発現量について有意な差をもたらす結果となった。このことは、上記インスリン系化合物と同様に、成長因子がACE2及び/又はTMPRSS2によって介在される感染症、特にコロナウィルスや、その他の疾患の予防又は治療効果をもたらすことを示す。 The results are shown in Figure 10. All growth factors resulted in a significant difference in the expression level of ACE2 and/or TMPRSS2 compared to the control. This indicates that growth factors, like the above insulin-based compounds, provide prophylactic or therapeutic effects on infections, particularly coronaviruses, and other diseases mediated by ACE2 and/or TMPRSS2.
4-6.実施例6(HGFによる効果)
 実施例1~4と同様の実験を行った。ただし、各種インスリン化合物等に代えて、HGF(R&D systems社、製品番号294-HGN-005/CF)を使用した。HGFは、受容体に結合後、細胞内シグナル経路の下流として、FoxO1を抑制することは広く知られている。
4-6. Example 6 (Effect of HGF)
Experiments similar to Examples 1-4 were conducted. However, HGF (R&D systems, product number 294-HGN-005/CF) was used instead of various insulin compounds. HGF is widely known to repress FoxO1 as a downstream intracellular signaling pathway after binding to its receptor.
 結果を図11に示す。コントロールと比べて、HGFが1ng/ml以上の濃度において、ACE2及び/又はTMPRSS2の発現量について有意な差をもたらす結果となった。しかも、HGFによる抑制度合いは、10ng/ml以上では、上記インスリン系化合物、他の成長因子と比べて、優れていた。このことは、HGFが、ACE2及び/又はTMPRSS2によって介在される感染症、特にコロナウィルスや、その他の疾患の予防又は治療に対してきわめてすぐれた効果をもたらすことを示す。 The results are shown in Fig. 11. Compared to the control, HGF at a concentration of 1 ng/ml or higher resulted in a significant difference in the expression levels of ACE2 and/or TMPRSS2. Moreover, the degree of inhibition by HGF was superior at 10 ng/ml or more as compared with the insulin compounds and other growth factors. This indicates that HGF is highly effective in preventing or treating infections, especially coronaviruses, and other diseases mediated by ACE2 and/or TMPRSS2.
 HGFのアミノ酸配列は以下のとおりである。
https://www.uniprot.org/uniprot/P14210
Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp Glu Lys Cys Lys Gln Val Leu Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val Leu Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile Leu Thr Tyr Lys Val Pro Gln Ser(配列番号20)
The amino acid sequence of HGF is as follows.
https://www.uniprot.org/uniprot/P14210
Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp Glu Lys Cys Lys Gln Val Leu Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val Leu Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile Leu Thr Tyr Lys Val Pro Gln Ser (SEQ ID NO: 20)
4-7.実施例7(siRNAによる効果)
 実施例1~4と同様の実験を行った。ただし、FoxO1の遺伝子発現抑制のために、siRNAを使用した。siRNAはコントロールとして何も抑制しないもの(Cell Signaling(登録商標)社,製品番号6568S)、及びヒトFoxO1遺伝子をターゲットとしたもの(#1;Cell Signaling(登録商標)社,製品番号6242S、及び#2;Cell Signaling(登録商標)社,製品番号6256S、配列情報はコントロールも含め非公開)を用いた。siRNAの細胞内への導入にはトランスフェクション試薬(Horizon Discovery社,DharmaFECT1,製品番号T2001-01)を使用した。各種siRNAを導入し、3日後(約72時間後)に細胞を回収し、上記のようにqPCRで定量した。
4-7. Example 7 (Effect of siRNA)
Experiments similar to Examples 1-4 were conducted. However, siRNA was used to suppress the gene expression of FoxO1. As a control, siRNAs that do not suppress anything (Cell Signaling (registered trademark), product number 6568S) and those targeting the human FoxO1 gene (#1; Cell Signaling (registered trademark), product number 6242S, and # 2; Cell Signaling (registered trademark), product number 6256S, sequence information including controls not disclosed) was used. A transfection reagent (Horizon Discovery, DharmaFECT1, product number T2001-01) was used to introduce siRNA into cells. Three days (approximately 72 hours) after introduction of various siRNAs, cells were harvested and quantified by qPCR as described above.
 結果を図12に示す。コントロールのsiRNAを導入した細胞と比べて、FoxO1をターゲットとするsiRNAを導入した細胞では、確かにFoxO1 mRNAの発現が減少している。この時、同時にACE2及び/又はTMPRSS2の発現量についても有意な差をもたらす結果となった。この結果は、FoxO1が確かにACE2及び/又はTMPRSS2の遺伝子発現に必要であることを示している。言い換えると、FoxO1の活性や遺伝子発現量を抑制することで、確かにACE2及び/又はTMPRSS2の遺伝子発現を阻害できる、というこれまでの実験結果を支持するものである。 The results are shown in Fig. 12. The expression of FoxO1 mRNA certainly decreased in the cells into which the siRNA targeting FoxO1 was introduced, compared with the cells into which the control siRNA was introduced. At the same time, there was also a significant difference in the expression levels of ACE2 and/or TMPRSS2. This result indicates that FoxO1 is indeed required for gene expression of ACE2 and/or TMPRSS2. In other words, it supports the experimental results so far that the gene expression of ACE2 and/or TMPRSS2 can be inhibited by suppressing the activity and gene expression level of FoxO1.
 以上の実験結果から、FoxO1阻害剤一般について、シグナル伝達経路を介する場合、あるいは直接阻害の場合(阻害剤あるいはsiRNAなど)など、その阻害方法が大きく異なるといえども、FoxO1阻害剤として機能する化合物の含有量に応じて、ACE2又はTMPRSS2の発現阻害剤又はコロナウィルス感染抑制剤として有効であることが示されている。 From the above experimental results, for FoxO1 inhibitors in general, compounds that function as FoxO1 inhibitors, even though the method of inhibition is greatly different, such as via a signal transduction pathway or in the case of direct inhibition (inhibitors, siRNA, etc.) It has been shown that it is effective as an ACE2 or TMPRSS2 expression inhibitor or a coronavirus infection inhibitor depending on the content of .
 以上、具体的な実施形態について説明してきた。上記実施形態は、具体例に過ぎず、本発明は上記実施形態に限定されない。例えば、上述の実施形態の1つに開示された技術的特徴は、他の実施形態に適用することができる。また、特記しない限り、特定の方法については、一部の工程を他の工程の順序と入れ替えることも可能であり、特定の2つの工程の間に更なる工程を追加してもよい。本発明の範囲は、特許請求の範囲によって規定される。 Specific embodiments have been described above. The above embodiments are only specific examples, and the present invention is not limited to the above embodiments. For example, technical features disclosed in one of the above embodiments may be applied to other embodiments. Also, unless otherwise stated, for a particular method, some steps may be interchanged with other steps, and additional steps may be added between two particular steps. The scope of the invention is defined by the claims.

Claims (20)

  1.  アンジオテンシン転換酵素2(ACE2)及び/又はTMPRSS2の発現を阻害するための組成物であって、
     前記組成物は、FoxO1阻害剤を含む、組成物。
    A composition for inhibiting the expression of angiotensin converting enzyme 2 (ACE2) and/or TMPRSS2, comprising:
    A composition, wherein the composition comprises a FoxO1 inhibitor.
  2.  前記FoxO1阻害剤は、以下の式で表される化合物又はその塩である、請求項1の組成物。
    Figure JPOXMLDOC01-appb-C000001
    {ただし、
    1は、NH又はCH2であり、
    2は、N-R8又はCH-R8 (R8:C1~C3までのアルキル基)であり、
    3は、F、Cl、Br、又はIであり、
    4は、それぞれ独立して、H又はCH3であり、
    5は、S又はOであり、
    6は、カルボキシル基であり、
    7は、NH2、又はCH3であり、
    nは、1-11である。
    2. The composition of claim 1, wherein the FoxO1 inhibitor is a compound represented by the following formula or a salt thereof.
    Figure JPOXMLDOC01-appb-C000001
    {however,
    R1 is NH or CH2 ;
    R 2 is NR 8 or CH-R 8 (R 8 : C1-C3 alkyl group),
    R3 is F , Cl, Br, or I;
    each R4 is independently H or CH3 ;
    R5 is S or O;
    R6 is a carboxyl group,
    R7 is NH2 or CH3 ,
    n is 1-11.
    }
  3.  前記阻害剤が、以下の式で表される化合物又はその塩である、請求項1の組成物。
    Figure JPOXMLDOC01-appb-C000002
    {ただし、
    1は、NHであり、
    2は、N-R8 (R8:C1~C3までのアルキル基)であり、
    3は、F、Cl、Br、又はIであり、
    4は、それぞれ独立して、H又はCH3であり、
    5は、Oであり、
    6は、カルボキシル基であり、
    7は、NH2であり、
    nは、1-11である。
    2. The composition of claim 1, wherein said inhibitor is a compound represented by the following formula or a salt thereof.
    Figure JPOXMLDOC01-appb-C000002
    {however,
    R 1 is NH;
    R 2 is NR 8 (R 8 is an alkyl group of C1 to C3);
    R3 is F , Cl, Br, or I;
    each R4 is independently H or CH3 ;
    R5 is O;
    R6 is a carboxyl group,
    R7 is NH2 ;
    n is 1-11.
    }
  4.  前記阻害剤が、AS1842856又はAS1708727である、請求項1の組成物。 The composition of claim 1, wherein the inhibitor is AS1842856 or AS1708727.
  5.  前記FoxO1阻害剤は、インスリン系化合物である、請求項1の組成物。 The composition of claim 1, wherein said FoxO1 inhibitor is an insulin-based compound.
  6.  前記インスリン系化合物が、野生型インスリンである、請求項5の組成物。 The composition of claim 5, wherein said insulin-based compound is wild-type insulin.
  7.  前記インスリン系化合物が、改変型インスリンである、請求項5の組成物。 The composition of claim 5, wherein said insulin-based compound is modified insulin.
  8.  前記改変型インスリンが、超速効型インスリン、速効型インスリン、中間型インスリン、持効型溶解性インスリン、混合型インスリン、及び配合溶解性インスリンから選択される1種以上である、請求項7の組成物。 8. The composition of claim 7, wherein the modified insulin is one or more selected from ultra-rapid-acting insulin, rapid-acting insulin, intermediate-acting insulin, long-acting soluble insulin, mixed-acting insulin, and combined soluble insulin. thing.
  9.  前記改変型インスリンが、ヒト野生型インスリンと90%以上同一のアミノ酸配列を有する、請求項7の組成物。 The composition of claim 7, wherein said modified insulin has an amino acid sequence that is 90% or more identical to human wild-type insulin.
  10.  前記改変型インスリンが、ヒト野生型インスリンと95%以上同一のアミノ酸配列を有する、請求項7の組成物。 The composition of claim 7, wherein said modified insulin has an amino acid sequence that is 95% or more identical to human wild-type insulin.
  11.  前記改変型インスリンのA鎖が、ヒト野生型インスリンのA鎖と100%同一のアミノ酸配列を有する、請求項9又は10の組成物。 The composition of claim 9 or 10, wherein the modified insulin A chain has an amino acid sequence that is 100% identical to that of human wild-type insulin.
  12.  前記FoxO1阻害剤は、成長因子及び/又はこれをコードする核酸である、請求項1の組成物。 The composition of claim 1, wherein the FoxO1 inhibitor is a growth factor and/or a nucleic acid encoding the same.
  13.  前記成長因子が、以下から選択されるいずれかと、90%以上同一のアミノ酸配列を有する、請求項12の組成物:
     IGF1、IGF2、EGF、FGF2、FGF14、VEGF、HGF。
    13. The composition of claim 12, wherein said growth factor has 90% or more amino acid sequence identity with any selected from:
    IGF1, IGF2, EGF, FGF2, FGF14, VEGF, HGF.
  14.  前記成長因子が、以下から選択されるいずれかと、95%以上同一のアミノ酸配列を有する、請求項13の組成物:
     IGF1、IGF2、EGF、FGF2、FGF14、VEGF、HGF。
    14. The composition of claim 13, wherein said growth factor has 95% or more amino acid sequence identity with any selected from:
    IGF1, IGF2, EGF, FGF2, FGF14, VEGF, HGF.
  15.  前記成長因子が、以下から選択されるいずれかと、100%以上同一のアミノ酸配列を有する、請求項14の組成物:
     IGF1、IGF2、EGF、FGF2、FGF14、VEGF、HGF。
    15. The composition of claim 14, wherein said growth factor has 100% or more amino acid sequence identity with any selected from:
    IGF1, IGF2, EGF, FGF2, FGF14, VEGF, HGF.
  16.  前記FoxO1阻害剤は、FoxO1作用型阻害剤である、請求項1の組成物。 The composition of claim 1, wherein the FoxO1 inhibitor is a FoxO1-acting inhibitor.
  17.  前記FoxO1阻害剤は、PI3K/PDK1/Akt作用型阻害剤である、請求項1の組成物。 The composition of claim 1, wherein said FoxO1 inhibitor is a PI3K/PDK1/Akt acting inhibitor.
  18.  前記FoxO1阻害剤は、FoxO1の遺伝子発現抑制剤である、請求項1の組成物。 The composition of claim 1, wherein the FoxO1 inhibitor is a FoxO1 gene expression inhibitor.
  19.  コロナウィルス感染症を予防又は治療するための組成物である、請求項1~18いずれか1つに記載の組成物(投与対象として、糖尿病患者を除く)。 The composition according to any one of claims 1 to 18, which is a composition for preventing or treating coronavirus infections (excluding diabetics as the administration target).
  20.  コロナウィルス感染症を予防又は治療するための組成物であって、FoxO1阻害剤を含む、組成物(投与対象として、糖尿病患者を除く)。 A composition for the prevention or treatment of a coronavirus infection, which contains a FoxO1 inhibitor (excluding diabetic patients).
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