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WO2023195509A1 - Inhibiteur de défaillance de la barrière endothéliale vasculaire et son utilisation - Google Patents

Inhibiteur de défaillance de la barrière endothéliale vasculaire et son utilisation Download PDF

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Publication number
WO2023195509A1
WO2023195509A1 PCT/JP2023/014182 JP2023014182W WO2023195509A1 WO 2023195509 A1 WO2023195509 A1 WO 2023195509A1 JP 2023014182 W JP2023014182 W JP 2023014182W WO 2023195509 A1 WO2023195509 A1 WO 2023195509A1
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vascular endothelial
cldn5
inhibitor
sars
endothelial barrier
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Japanese (ja)
Inventor
和雄 高山
里菜 橋本
欣晃 岡田
慈 藤尾
理徳 尾花
潤也 高橋
靖雄 吉岡
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Kyoto University NUC
University of Osaka NUC
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Osaka University NUC
Kyoto University NUC
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
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Definitions

  • the present invention relates to an inhibitor of vascular endothelial barrier breakdown and its use. More specifically, the present invention relates to an inhibitor of vascular endothelial barrier breakdown, a pharmaceutical composition for treating respiratory infections, and a method for screening for an inhibitor of vascular endothelial barrier breakdown.
  • pathogenic bacteria or viruses overcome the defense mechanism and invade the body through the respiratory tract, multiply, and induce inflammation, causing symptoms such as coughing, phlegm, fever, chest pain, and difficulty breathing. Disruption of vascular endothelial barrier function is often observed in patients with severe respiratory infections.
  • SARS coronavirus-2 SARS coronavirus-2
  • SARS-CoV-2 SARS coronavirus-2
  • ACE2 angiotensin converting enzyme
  • vascular endothelial cells hardly express ACE2, SARS-CoV-2 hardly infects vascular endothelial cells (see, for example, Non-Patent Document 2).
  • Vascular endothelial cells that line the lumen of blood vessels firmly adhere to each other by forming tight junctions with multiple adhesion factors, and play a barrier function to prevent foreign substances such as pathogens from entering blood vessels from outside. .
  • breakdown of vascular endothelial barrier function is observed, as in patients with other respiratory infections (see, for example, Non-Patent Document 3).
  • Disruption of this barrier function facilitates the movement of SARS-CoV-2 from the respiratory tract to other organs via blood vessels.
  • failure of the barrier function induces infiltration of immune cells and blood components into the lungs, leading to respiratory failure including pneumonia and acute respiratory distress syndrome (ARDS) (see, for example, Non-Patent Document 4).
  • ARDS acute respiratory distress syndrome
  • the present invention has been made in view of the above circumstances, and provides an inhibitor of vascular endothelial barrier breakdown that can effectively prevent vascular endothelial barrier breakdown, and a screening method for an inhibitor of vascular endothelial barrier breakdown. do.
  • Claudin-5 (CLDN5) as a gene that plays an important role in vascular endothelial barrier function, and overexpressed CLDN5 or expressed CLDN5.
  • CLDN5 Claudin-5
  • the present inventors have discovered that vascular endothelial barrier breakdown can be prevented in pulmonary microvascular endothelial cells by applying a substance that induces this, and have completed the present invention.
  • the present invention includes the following aspects.
  • An inhibitor of vascular endothelial barrier breakdown which contains Claudin-5, a nucleic acid encoding Claudin-5, or a Claudin-5 expression inducer as an active ingredient.
  • the inhibitor of vascular endothelial barrier breakdown according to (1), wherein the Claudin-5 expression inducer is a statin compound.
  • the statin compound is at least one selected from the group consisting of fluvastatin, pitavastatin, atorvastatin, cerivastatin, simvastatin, and lovastatin.
  • the inhibitor of vascular endothelial barrier breakdown according to (1), wherein the Claudin-5 expression inducer is a TGF- ⁇ inhibitor.
  • the vascular endothelial barrier according to (5), wherein the TGF- ⁇ inhibitor is at least one selected from the group consisting of SB525334, R268712, EW-7197, RepSox, TP0427736, A83-01, and K02288. Inhibitor of bankruptcy.
  • the inhibitor of vascular endothelial barrier breakdown according to (7), wherein the virus is SARS coronavirus-2.
  • a pharmaceutical composition for treating respiratory infections comprising the inhibitor of vascular endothelial barrier breakdown according to any one of (1) to (8) and a pharmaceutically acceptable carrier.
  • the expression level of Claudin-5 in the pulmonary microvascular endothelial cells is measured after culturing the pulmonary microvascular endothelial cells, and the expression level of Claudin-5 is A method for screening for an inhibitor of vascular endothelial barrier breakdown, which indicates that the test substance is an inhibitor of vascular endothelial barrier breakdown when the test substance is significantly increased compared to the absence of the substance.
  • a first substrate including a first flow path configured to allow air to be injected; and a first substrate disposed below the first substrate and having a size that is permeable to the medium but not permeable to the cells.
  • the membrane has a plurality of through holes and forms the bottom surface of the first flow path, and is configured to be able to inject a culture medium, so that at least a portion thereof overlaps the first flow path in a plan view. using a cell culture container formed by laminating a second flow path having a top surface made of the membrane, and a second substrate disposed below the membrane.
  • airway epithelial cells infected with a virus or bacteria are seeded in the first flow path
  • pulmonary microvascular endothelial cells are seeded in the second flow path
  • the airway epithelium is infected in the presence of the test substance.
  • a method for screening for an inhibitor of vascular endothelial barrier breakdown which indicates that the test substance is an inhibitor of vascular endothelial barrier breakdown when the test substance is significantly reduced compared to the absence of the substance.
  • FIG. 1 is a schematic configuration diagram of an airway-on-a-chip used in Examples.
  • 2 is a graph showing changes over time in the virus copy number in the cell culture supernatant of the airway channel and vascular channel of the airway-on-a-chip in Experimental Example 1.
  • 1 is a volcano plot of genes showing differential expression between human lung microvascular endothelial cells (HMVEC-L) not infected with SARS-CoV-2 and HMVEC-L infected with SARS-CoV-2 in Experimental Example 1.
  • HMVEC-L human lung microvascular endothelial cells
  • Claudin-5 Claudin-5
  • VE-cadherin vascular endothelial cadherin
  • IL-6 interleukin-6
  • VCAM-1 VCAM-1 and intercellular adhesion molecule (ICAM-1). 2 is an image of immunofluorescence staining of CLDN5 and VE-cadherin in airway-on-a-chip HMVEC-L in Experimental Example 2.
  • 3 is a graph showing the expression level of CLDN5 when a medium containing 0.1 MOI and 1 MOI of SARS-CoV-2 is applied to HMVEC-L in Experimental Example 3.
  • 3 is a phase contrast microscopic image of SARS-CoV-2 infected and non-infected HMVEC-L in Experimental Example 3.
  • 3 is a graph showing the gene expression levels of CLDN5, VE-cadherin, IL-6, VCAM-1, and ICAM-1 in SARS-CoV-2-infected and uninfected HMVEC-L in Experimental Example 3.
  • TEER transendothelial electrical resistance
  • 3 is a graph showing the amount of Evans blue detected in each organ of hCLDN5-KI mice injected with anti-CLDN5 antibody or control IgG in Experimental Example 4.
  • 3 is a bright field image of each organ of a hCLDN5-KI mouse injected with anti-CLDN5 antibody or control IgG in Experimental Example 4.
  • 3 is a bright field image of the lungs (left) and a graph (right) showing the wet weight/dry weight ratio of the lungs of hCLDN5-KI mice injected with anti-CLDN5 antibody or control IgG in Experimental Example 4.
  • Spike protein in Experimental Example 5 SARS-CoV-2, or CLDN5, VE-cadherin, IL-6 in HMVEC-L treated with UV-irradiated SARS-CoV-2 (UV-SARS-CoV-2) , VCAM-1, and ICAM-1.
  • 2 is an immunofluorescence staining image of VE-cadherin in HMVEC-L treated with spike protein, SARS-CoV-2, or UV-SARS-CoV-2 in Experimental Example 5.
  • FIG. 6 is an image of immunofluorescence staining of VE-cadherin in HMVEC-L exposed to SARS-CoV-2 in the presence or absence of DOX in Experimental Example 6.
  • CLDN5 VE-cadherin
  • IL-6 VCAM-1
  • ICAM-1 interferon- ⁇
  • IFN- ⁇ interferon- ⁇
  • MxA Myxovirus resistance A
  • FIG. 7 is a graph showing virus copy numbers in cell culture supernatants of airway channels and vascular channels of airway-on-a-chip with or without fluvastatin in Experimental Example 7.
  • 2 is an image of immunofluorescence staining of VE-cadherin in HMVEC-L exposed to SARS-CoV-2 in the presence or absence of fluvastatin in Experimental Example 7.
  • CLDN5 VE-cadherin, IL-6, VCAM-1, ICAM-1, IFN- ⁇ , IFN- ⁇ in HMVEC-L exposed to SARS-CoV-2 with or without fluvastatin treatment in Experimental Example 7 , ISG15, ISG56, and MxA.
  • FIG. 12 is a graph showing the results of measuring the CLDN5 concentration in the serum of mild/moderate and severe COVID-19 patients within one week after onset of symptoms by ELISA in Experimental Example 8.
  • 3 is a graph showing the expression level of CLDN5 after the action of a statin drug on HMVEC-L in Experimental Example 9.
  • 3 is a graph showing the expression level of CLDN5 after the action of a TGF- ⁇ inhibitor on HMVEC-L in Experimental Example 10. It is a graph showing the virus genome copy number in the cell culture supernatant when SARS-CoV-2 is introduced into the airway channel after the action of any of six types of statin drugs on the blood vessel channel in Experimental Example 11. .
  • the inhibitor of vascular endothelial barrier breakdown of the present embodiment contains CLDN5, a nucleic acid encoding CLDN5, or a CLDN5 expression inducer as an active ingredient.
  • containing as an active ingredient means containing a therapeutically effective amount of CLDN5, a nucleic acid encoding CLDN5, or a CLDN5 expression inducer.
  • therapeutically effective amount means that when administered in accordance with the desired therapeutic regimen, the biological or medical amount required by a physician, clinician, veterinarian, researcher, or other appropriate professional It refers to the amount of CLDN5 or CLDN5 expression inducer, or the amount of CLDN5 or a nucleic acid encoding CLDN5 or a combination of CLDN5 expression inducer and one or more active agents that elicits an effect or response.
  • a preferred therapeutically effective amount is an amount that ameliorates the symptoms of respiratory infections, particularly COVID-19.
  • a "therapeutically effective amount” also includes a prophylactically effective amount, ie, an amount suitable for preventing a disease state.
  • the inhibitor of this embodiment vascular endothelial barrier breakdown can be effectively prevented. Therefore, the inhibitor of this embodiment is expected to be applied as a therapeutic agent for stopping the progression of symptoms of respiratory infections (especially COVID-19). Moreover, the inhibitor of this embodiment can also be called a vascular endothelial barrier function improving agent.
  • vascular endothelial barrier breakdown is caused directly by viruses, bacteria, etc., or indirectly by inflammatory reactions induced by them.
  • viruses that cause vascular endothelial barrier breakdown include RS virus (respiratory syncytial virus), SARS coronavirus (SARS-CoV), MERS coronavirus (MERS-CoV), SARS-CoV-2, etc. but not limited to.
  • bacteria that cause vascular endothelial barrier breakdown include, but are not limited to, Mycobacterium tuberculosis, Bacillus anthracis, and Streptococcus pneumoniae.
  • the inhibitor of this embodiment is preferably used as a therapeutic agent for respiratory infections accompanied by vascular endothelial barrier breakdown caused by the above-mentioned viruses, bacteria, or the like.
  • the inhibitor of the present embodiment is preferably applied to vascular endothelial barrier breakdown caused by viruses, and more preferably applied to vascular endothelial barrier breakdown caused by SARS-CoV-2.
  • CLDN5 is a four-transmembrane protein of approximately 23 kDa. Until now, it has been reported that it is involved in regulating the blood-brain barrier, but its functions in other organs were completely unknown. As shown in the Examples below, the inventors conducted a comprehensive gene expression analysis of pulmonary microvascular endothelial cells co-cultured with airway epithelial cells infected with SARS-CoV-2. We have identified CLDN5 as a gene that plays an important role, and have completed the present invention.
  • the amino acid sequence of human CLDN5 is disclosed in Genbank accession numbers NP_001124333.1 (SEQ ID NO: 1), NP_001349995.1 (SEQ ID NO: 2), NP_001349996.1 (SEQ ID NO: 3), NP_003268.2 (SEQ ID NO: 4), etc. ing.
  • CLDN5 includes both the full-length protein and its fragments.
  • a fragment is a polypeptide that includes any region of CLDN5 and has an activity of inhibiting vascular endothelial barrier breakdown. The presence or absence of inhibitory activity on vascular endothelial barrier breakdown can be evaluated using the screening method described below.
  • a protein having the same function as the CLDN protein a protein consisting of a sequence containing the amino acid sequence (a) or (b) below and having an activity of inhibiting vascular endothelial barrier breakdown can also be used.
  • the number of amino acids that may be deleted, substituted, or added is preferably 1 to 15, more preferably 1 to 10, particularly preferably 1 to 5.
  • nucleic acid encoding CLDN5 can be used instead of the CLDN5 protein.
  • the nucleic acid encoding CLDN5 is preferably used in the form of an expression vector containing the nucleic acid encoding CLDN5.
  • the base sequences of the nucleic acids encoding human CLDN5 have Genbank accession numbers NM_001130861.1 (SEQ ID NO: 5), NM_001363066.2 (SEQ ID NO: 6), NM_001363067.2 (SEQ ID NO: 7), and NM_003277.4 (SEQ ID NO: 8). etc. are disclosed.
  • the nucleotide sequence encoding human CLDN5 includes, for example, the amino acid sequence represented by SEQ ID NO: 9.
  • the amino acid sequence represented by SEQ ID NO: 9 is the sequence near the CDS extracted from the amino acid sequence (SEQ ID NO: 5) of Genbank accession number NM_001130861.1.
  • nucleic acid encoding CLDN5 a nucleic acid consisting of a sequence containing any of the following base sequences (c) to (e) and encoding a protein having inhibitory activity on vascular endothelial barrier breakdown can also be used.
  • nucleotide sequence having an identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more with the nucleotide sequence represented by any of SEQ ID NOS: 5 to 9;
  • the number of bases that may be deleted, substituted, or added is preferably 1 to 30, more preferably 1 to 15, particularly preferably 1 to 10, and 1 to 15. Most preferably, the number is 5 or more.
  • stringent conditions refers to, for example, Molecular Cloning-A LABORATORY MANUAL THIRD EDITION (Sambrook et al., Cold Spring Harbor Labor Examples include the method described in Ratory Press).
  • 5 ⁇ SSC composition of 20 ⁇ SSC: 3M sodium chloride, 0.3M citric acid solution, pH 7.0
  • 0.1% by mass N-lauroylsarcosine 0.2% by mass SDS
  • Examples of conditions for hybridization include incubation for several hours to overnight at 55° C. or higher and 70° C. or lower in a hybridization buffer consisting of a blocking reagent for nucleic acid hybridization and 50% formamide.
  • the washing buffer used for washing after incubation is preferably a 1 ⁇ SSC solution containing 0.1% by mass SDS, more preferably a 0.1 ⁇ SSC solution containing 0.1% by mass SDS.
  • the base sequence of the nucleic acid encoding CLDN5 may be codon-optimized for expression in the animal species to be introduced (mainly mammals including humans).
  • codon optimization is achieved by replacing at least one codon in the native sequence with a codon that is more frequently or most frequently used in the gene of the animal species into which it is introduced, while maintaining the native amino acid sequence.
  • Codon bias (differences in codon usage between animals) is often correlated with mRNA translation efficiency, which is thought to depend, among other things, on the characteristics of the codons being translated and the availability of specific tRNAs. There is. The predominance of selected tRNAs in a cell is generally a reflection of the codons most frequently used in peptide synthesis. Thus, genes can be personalized for optimal gene expression in a given organism based on codon optimization. Codon usage tables are easily available, for example, in the "Codon Usage Database" posted at www.kazusa.or.jp/codon/, and these tables can be used to optimize codons.
  • the expression vector may be a vector that expresses the protein in cultured cells derived from the recipient (mainly mammals including humans) or in cells of an individual recipient (mainly mammals including humans). It is not particularly limited, and may be a plasmid or a virus vector.
  • Examples of the plasmid include pRK5 (European Patent No. 307247), pSV16B (International Publication No. 91/08291), pCAGGS (Niwa K et al., “Efficient selection for high-expression transfectants with a novel eukaryotic vector ”, Gene, vol.108, no.2, p193-199, 1991.), pVL1392 (manufactured by Invitrogen), pBK-CMV, pZeoSV, pcDNA3, pcDNA3.1 (manufactured by Invitrogen and Stratagene), pVC0396 (Japanese Patent Publication No. 11-511009) and the like, but are not limited thereto.
  • the viral vector examples include adenovirus, adeno-associated virus (AAV), retrovirus, poxvirus, herpesvirus, herpes simplex virus, lentivirus (HIV), Sendai virus, Epstein-Barr virus (EBV), and vaccinia virus.
  • AAV adeno-associated virus
  • retrovirus poxvirus
  • herpesvirus herpes simplex virus
  • lentivirus HIV
  • Sendai virus Epstein-Barr virus
  • EBV Epstein-Barr virus
  • vaccinia virus examples include, but are not limited to, viral vectors derived from , poliovirus, Cymbivirus, SV40, and the like.
  • the viral vector is preferably a replication-defective viral vector that completely or almost completely deletes the viral genes.
  • the expression vector may contain a promoter, a marker gene operably linked in the cells of the animal to be introduced (mainly mammals including humans), various regulatory elements, and the like. Examples of regulatory elements include terminators and enhancers.
  • the type of expression vector, promoter, and regulatory element to be used may be selected as appropriate depending on the animal species to be introduced and the method of introduction.
  • the CLDN5 expression inducer may be any substance that increases the expression level of CLDN5 in the presence of the substance compared to its absence, and includes a substance that increases the expression of CLDN5 and a substance that suppresses the expression of CLDN5. Included are inhibitors of substances that inhibit
  • CLDN5 examples include statin compounds, glucocorticoids, estrogens, cyclic adenosine monophosphate (cAMP), adenomedullin, and the transcription factor SOX (SRY-related HMG-box)-18. can be mentioned.
  • statin compounds examples include fluvastatin (LESCOL (registered trademark); see US Pat. No. 5,354,772), simvastatin (ZOCOR (registered trademark); US Pat. No. 4, 444,784), atorvastatin (LIPITOR®; see US Pat. No. 5,273,995), lovastatin (JP 5-178841 (US Pat. 5,260,440)), pravastatin (PRAVACHOL®; see U.S. Pat. No. 4,346,227), rosuvastatin (MEVACOR®; U.S. Pat. No. 4,231,938), cerivastatin (also known as rivastatin; see US Pat. No.
  • mevastatin (compactin, US Pat. 3,983,140), pitavastatin (see JP-A-1-279866 (US Pat. No. 5,854,259 and US Pat. No. 5,856,336) ) etc.
  • mevastatin compactin, US Pat. 3,983,140
  • pitavastatin see JP-A-1-279866 (US Pat. No. 5,854,259 and US Pat. No. 5,856,336)
  • fluvastatin fluvastatin
  • atorvastatin atorvastatin
  • cerivastatin cerivastatin
  • simvastatin simvastatin
  • lovastatin lovastatin
  • glucocorticoids examples include dekitamethasone (CAS No. 50-02-2).
  • estrogen examples include 17 ⁇ -estradiol (CAS No. 50-28-2).
  • Substances that suppress the expression of CLDN5 include forkhead box protein O1 (FoxO1), vascular endothelial growth factor (VEGF) and factors involved in the VEGF signaling pathway, tumor necrosis factor- ⁇ (TNF- ⁇ ), transforming Examples include growth factor- ⁇ (TGF- ⁇ ), and these inhibitors can be used as inhibitors for substances that suppress the expression of CLDN5.
  • FoxO1 forkhead box protein O1
  • VEGF vascular endothelial growth factor
  • TGF- ⁇ tumor necrosis factor- ⁇
  • TGF- ⁇ growth factor- ⁇
  • FoxO1 inhibitors examples include AS1842856 (CAS No. 835520-48-5).
  • VEGF signal inhibitors examples include LY-317615 (PKC ⁇ inhibitor; CAS No. 170364-57-5).
  • TNF- ⁇ inhibitors examples include H-1152 (CAS No. 871543-07-6), Y-27632 (CAS No. 331752-47-7), and the like.
  • TGF- ⁇ inhibitors examples include SB525334 (CAS No. 356559-20-1), R268712 (CAS No. 879487-87-3), and EW-7197, which are inhibitors of TGF- ⁇ receptor (ALK5). (CAS No. 1352608-82-2), RepSox (CAS No. 446859-33-2), TP0427736 (CAS No. 2459963-17-6), A83-01 (CAS No. 909910-43-6), K02288 (CAS No. 1431985-92-0), etc.
  • CLDN5 expression inducer breakdown of the vascular endothelial barrier can be inhibited.
  • TGF- ⁇ inhibitors Bintrafusp Alfa (CAS No. 1918149-01-5), NIS793, Remedisc, HCW9218, AdAPT-001, BCA101, BNC-1021, GS-1423, PF-069522 29 (CAS No. .1801333-55-0), SRK-181, TST-005, YL-13027, Seprehvec, ZGGS-18, 6MW-3511, AGMB-129, AK-130, BA1201, ES014, GFH018, JS201, SHR-1701, CUE-401, NCE-401, PLN-75068, SON-3015, STP355, TrexTAM, ACE-1332, Galunisertib (CAS No. 700874-72-2), GSK3845097, IDL-2965, LY30228 59, LY3200882 (CAS No. 1898283 -02-7).
  • siRNA, shRNA, miRNA, ribozyme, antisense nucleic acid, etc. against the substance that suppresses the expression of CLDN5 can be used as an inhibitor of a substance that suppresses the expression of CLDN5 as an inhibitor of a substance that suppresses the expression of CLDN5.
  • siRNA small interfering RNA
  • siRNA introduced into cells binds to RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA having a sequence complementary to siRNA. This suppresses gene expression in a sequence-specific manner.
  • RISC RNA-induced silencing complex
  • siRNA is prepared by synthesizing sense strand and antisense strand oligonucleotides using an automatic DNA/RNA synthesizer, for example, after denaturing the oligonucleotides at 90°C or higher and 95°C or lower for about 1 minute in an appropriate annealing buffer. It can be prepared by annealing at a temperature of about 1 to 8 hours at a temperature of about 1 to 70 degrees Celsius.
  • siRNA, shRNA, miRNA, ribozyme, and antisense nucleic acid may contain various chemical modifications to improve stability and activity.
  • phosphoric acid residues may be substituted with chemically modified phosphoric acid residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithionate.
  • PS phosphorothioate
  • methylphosphonate methylphosphonate
  • phosphorodithionate phosphorodithionate
  • at least a portion thereof may be composed of a nucleic acid analog such as peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • a substance that specifically binds to a substance that suppresses the expression of CLDN5 can also be used as an inhibitor of a substance that suppresses the expression of CLDN5
  • Such substances include antibodies, antibody fragments, aptamers, and the like.
  • Antibodies can be produced, for example, by immunizing animals such as mice with FMO or a fragment thereof using FMO or a fragment thereof as an antigen. Alternatively, it can be produced, for example, by screening a phage library.
  • Antibody fragments include Fv, Fab, scFv, and the like.
  • the above-mentioned antibody is a monoclonal antibody. Alternatively, a commercially available antibody may be used.
  • An aptamer is a substance that has the ability to specifically bind to a target substance.
  • aptamers include nucleic acid aptamers, peptide aptamers, and the like.
  • Nucleic acid aptamers that have the ability to specifically bind to a target peptide can be selected, for example, by the systematic evolution of ligand by exponential enrichment (SELEX) method.
  • peptide aptamers that have the ability to specifically bind to a target peptide can be selected, for example, by the Two-hybrid method using yeast.
  • the pharmaceutical composition for treating respiratory infections of this embodiment contains the above-mentioned inhibitor and a pharmaceutically acceptable carrier. include.
  • the pharmaceutical composition of this embodiment is applied to respiratory infections accompanied by vascular endothelial barrier breakdown, and can effectively prevent vascular endothelial barrier breakdown. Therefore, the pharmaceutical composition of this embodiment is expected to be applied as a therapeutic agent for stopping the progression of symptoms of respiratory infections.
  • respiratory infections accompanied by vascular endothelial barrier breakdown include novel coronavirus infection-2019 (COVID-19), respiratory syncytial virus infection, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), etc. viral respiratory infections; bacterial respiratory infections such as tuberculosis, anthrax, and pneumococcal infections; and the like, but are not limited to these.
  • the pharmaceutical composition of this embodiment is preferably applied to COVID-19.
  • ⁇ Pharmaceutically acceptable carrier those commonly used in the formulation of pharmaceutical compositions can be used without particular limitation. More specifically, for example, binders such as gelatin, cornstarch, gum tragacanth, and gum arabic; excipients such as starch and crystalline cellulose; leavening agents such as alginic acid; solvents for injections such as water, ethanol, and glycerin; Examples include adhesives such as rubber adhesives and silicone adhesives.
  • the pharmaceutical composition of this embodiment may further contain an additive.
  • Additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavoring agents such as peppermint and red oil; stabilizers such as benzyl alcohol and phenol; phosphoric acid. Buffers such as salts and sodium acetate; solubilizing agents such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives and the like.
  • the pharmaceutical composition of this embodiment can be prepared by appropriately combining the above-mentioned inhibitor and the above-mentioned pharmaceutically acceptable carriers and excipients in a unit dosage form required for generally accepted pharmaceutical practice. It can be formulated into a formulation.
  • the pharmaceutical composition of this embodiment may be in a dosage form that can be used orally or parenterally, but a dosage form that can be used parenterally is preferable.
  • dosage forms for oral use include tablets, capsules, elixirs, and microcapsules.
  • dosage forms used parenterally include injections, sprays, ointments, and patches.
  • the pharmaceutical composition of this embodiment may be used in combination with other therapeutic agents.
  • vascular endothelial barrier function can be restored while alleviating the symptoms of COVID-19.
  • each formulation may be administered by the same administration route or by separate administration routes. Furthermore, each formulation may be administered simultaneously, sequentially, or separately at a certain time or period. In one embodiment, the inhibitor and other therapeutic agents may be provided in a kit containing them.
  • Subjects to be administered include, but are not limited to, humans, monkeys, dogs, cows, horses, sheep, pigs, rabbits, mice, rats, guinea pigs, hamsters, and cells thereof. Among these, mammals or mammalian cells are preferred, and humans or human cells are particularly preferred.
  • Administration to a patient can be carried out by any method known to those skilled in the art, for example, intrathecally, intraarterially, intravenously, subcutaneously, intranasally, transbronchially, intramuscularly, percutaneously, or orally. This can be done by
  • the dosage varies depending on the weight and age of the patient, the patient's symptoms, the method of administration, etc., but those skilled in the art can appropriately select an appropriate dosage.
  • the dosage of the pharmaceutical composition is generally about 0.2 mg or more and 3 g or less, preferably about 2 mg or more and 300 mg or less per day for an adult (assuming a body weight of 60 kg) as the amount of the above-mentioned inhibitor. It is considered appropriate to administer, more preferably about 20 mg or more and 30 mg or less, once a day or divided into several doses.
  • the dose When administered parenterally, for example in the form of an injection, for adults (assuming a body weight of 60 kg), the dose is generally about 0.3 mg or more and 100 g or less, preferably about 3 mg or more and 10 g or less, more preferably about 3 mg or more and 10 g or less per day. It is considered appropriate to administer approximately 30 mg or more and 1 g or less once a day or in divided doses.
  • the method for screening for inhibitors of vascular endothelial barrier breakdown according to the first embodiment of the present invention comprises: in the presence of a test substance; After culturing the pulmonary microvascular endothelial cells (hereinafter sometimes referred to as the "first culture step"), measuring the expression level of CLDN5 in the pulmonary microvascular endothelial cells (hereinafter referred to as the "first measuring step”). ), including When the expression level of CLDN5 is significantly increased compared to the absence of the test substance, it is indicated that the test substance is an inhibitor of vascular endothelial barrier breakdown (hereinafter referred to as "first evaluation step”). ).
  • the screening method according to the first embodiment of the present invention it is possible to easily screen for substances that inhibit vascular endothelial barrier breakdown.
  • First culture step In the first culture step, pulmonary microvascular endothelial cells are cultured in the presence of the test substance.
  • the test substance is not particularly limited, and includes, for example, a natural compound library, a synthetic compound library, an existing drug library, a metabolite library, and the like.
  • test substance By adding the test substance to the culture medium, it can be brought into contact with pulmonary microvascular endothelial cells.
  • Examples of the medium include those commonly used for culturing pulmonary microvascular endothelial cells, such as EGM-2-MV medium (manufactured by Lonza).
  • pulmonary microvascular endothelial cells that have been exposed to a high titer virus or bacteria of 1.0 MOI (Multiplicity of Infection) or higher may be used.
  • virus As the virus, SARS-CoV-2 is preferably used. As shown in the Examples below, SARS-CoV-2 does not infect pulmonary microvascular endothelial cells, but it reduces the expression level of CLDN5 produced by pulmonary microvascular endothelial cells, causing vascular endothelial barrier breakdown.
  • the culture can be carried out at a temperature of 30° C. or higher and 37° C. or lower, and under 5% CO 2 .
  • the expression level of CLDN5 can be measured at the gene level and protein level. Measurement at the gene level can be performed, for example, by real-time PCR. Measurement at the protein level can be performed, for example, by Western blotting, ELISA, immunostaining, etc.
  • the test substance is evaluated as an inhibitor of vascular endothelial barrier breakdown when the expression level of CLDN5 is significantly increased compared to the absence of the test substance.
  • the expression level of CLDN5 is at least the same as that under non-exposed virus or bacteria. , it indicates that the test substance is an inhibitor of vascular endothelial barrier breakdown.
  • the method for screening for inhibitors of vascular endothelial barrier breakdown according to the second embodiment of the present invention includes the following steps: a first substrate including a first flow path configured; a membrane that is disposed in a lower layer of the first substrate, has a plurality of through-holes of a size that allows the culture medium to pass through but does not allow the cells to pass through, and that forms the bottom surface of the first channel;
  • the second flow path is configured such that a culture medium can be injected therein, and is configured such that at least a portion thereof overlaps the first flow path in a plan view, and a second flow path whose top surface is formed of the membrane.
  • a second substrate comprising: a second substrate disposed below the membrane;
  • a cell culture container made by stacking Airway epithelial cells infected with a virus or bacteria are seeded in the first flow path, pulmonary microvascular endothelial cells are seeded in the second flow path, and the airway epithelial cells are infected in the presence of the test substance.
  • the second culture step After co-culturing the pulmonary microvascular endothelial cells and the pulmonary microvascular endothelial cells (hereinafter sometimes referred to as the "second culture step"), the number of the viruses or bacteria in the culture supernatant of the pulmonary microvascular endothelial cells is measured.
  • second measurement step If the number of viruses or bacteria is significantly reduced compared to the absence of the test substance, this indicates that the test substance is an inhibitor of vascular endothelial barrier breakdown (hereinafter referred to as “second evaluation”). (sometimes referred to as “process”).
  • the screening method according to the second embodiment of the present invention it is possible to easily screen for substances that inhibit vascular endothelial barrier breakdown. Furthermore, by screening the test substance selected in the screening method according to the first embodiment with the screening method according to the second embodiment, it is possible to screen for substances that inhibit vascular endothelial barrier breakdown with higher accuracy. can.
  • FIG. 1 is a schematic configuration diagram showing an example of a cell culture container used in the screening method according to the second embodiment of the present invention.
  • the structure of the cell culture container 10 will be described in detail with reference to FIG. 1.
  • the cell culture container 10 is a system in which airway epithelial cells A and pulmonary microvascular endothelial cells B are co-cultured via a membrane 3, and is a microfluidic device that reproduces the human lung.
  • the inventors named the cell culture container 10 "airway-on-a-chip.”
  • the cell culture container 10 is formed by laminating a second substrate 2, a membrane 3, and a first substrate 1 in this order.
  • the first substrate 1 has a first flow path 1a.
  • the first channel 1a is configured to allow air to be injected, and is used for culturing airway epithelial cells A.
  • the bottom surface of the first flow path 1a is made up of a membrane 3.
  • the cross-sectional size of the first channel 1a can be, for example, a width of 0.5 mm or more and 1.5 mm or less (preferably 1.0 mm), and a height of 200 ⁇ m or more and 400 ⁇ m or less (preferably 330 ⁇ m).
  • the second substrate 2 has a second flow path 2a.
  • the second channel 2a is configured so that a medium can be injected therein, and is used for culturing the pulmonary microvascular endothelial cells B.
  • the second flow path 2a is configured so that at least a portion thereof overlaps the first flow path 1a in a plan view, and the top surface is configured with the membrane 3.
  • the pulmonary microvascular endothelial cells B can be co-cultured with the airway epithelial cells A via the membrane 3, and the viruses or bacteria that have infected the airway epithelial cells A and proliferated are transferred to the pulmonary microvascular endothelial cells B. Can be moved.
  • the cross-sectional size of the second channel 2a can be, for example, a width of 0.5 mm or more and 1.5 mm or less (preferably 1.0 mm), and a height of 200 ⁇ m or more and 400 ⁇ m or less (preferably 330 ⁇ m).
  • the first substrate 1 and the second substrate 2 may be made of the same material or may be made of different materials. Among these, it is preferable that they be made of the same material because manufacturing is easy.
  • the material forming the first substrate 1 and the second substrate 2 is a material that has relatively low toxicity to cells that is normally used for cell culture, and has translucency for ease of observation. Examples of the material include transparent glass; various polymers such as acrylic resin, fluororesin, and silicone rubber (for example, poly(dimethylsiloxane): PDMS). These materials may be used alone or in combination.
  • the membrane 3 has a plurality of through holes that are sized to allow the medium to pass through but not to allow the cells to pass through. That is, the membrane 3 can also be called a semipermeable membrane.
  • the diameter of the through hole can be, for example, 0.1 ⁇ m or more and 10.0 ⁇ m or less, preferably 1.0 ⁇ m or more and 5.0 ⁇ m or less, and more preferably about 3.0 ⁇ m ⁇ 0.5 ⁇ m.
  • the material for the membrane 3 may be any material that has relatively low toxicity to cells, and may be a natural polymer compound or a synthetic polymer compound.
  • natural polymer compounds include components derived from extracellular matrix that gel, polysaccharides (e.g., alginate, cellulose, dextran, pullulane, polyhyaluronic acid, and derivatives thereof), chitin, poly(3 -hydroxyalkanoates) (especially poly( ⁇ -hydroxybutyrate), poly(3-hydroxyoctanoate)), poly(3-hydroxy fatty acids), fibrin, agar, agarose, etc., but are not limited to these. .
  • polysaccharides e.g., alginate, cellulose, dextran, pullulane, polyhyaluronic acid, and derivatives thereof
  • chitin e.g., alginate, cellulose, dextran, pullulane, polyhyaluronic acid, and derivatives thereof
  • poly(3 -hydroxyalkanoates) especially poly( ⁇ -hydroxybutyrate), poly(3-hydroxyoctanoate)
  • poly(3-hydroxy fatty acids) especially fibrin
  • Extracellular matrix-derived components to be gelled include, for example, collagen (type I, type II, type III, type V, type XI, etc.), mouse EHS tumor extract (type IV collagen, laminin, heparan sulfate proteoglycan, etc.) ) reconstituted basement membrane components (trade name: Matrigel), glycosaminoglycans, hyaluronic acid, proteoglycans, gelatin, etc., but are not limited to these.
  • Examples of synthetic polymer compounds include polyphosphazene, poly(vinyl alcohol), polyamide (such as nylon), polyesteramide, poly(amino acid), polyanhydride, polysulfone, polycarbonate, polyacrylate (acrylic resin), and polyamide.
  • Alkylene e.g., polyethylene, etc.
  • polyacrylamide polyalkylene glycol (e.g., polyethylene glycol, etc.), polyalkylene oxide (e.g., polyethylene oxide, etc.), polyalkylene terephthalate (e.g., polyethylene terephthalate, etc.), polyorthoester, polyvinyl ether , polyvinyl ester, polyvinyl halide, polyvinylpyrrolidone, polyester, polysiloxane, polyurethane, polyhydroxy acid (e.g., polylactide, polyglycolide, etc.), poly(hydroxybutyric acid), poly(hydroxyvaleric acid), poly[lactide-co-( Examples include, but are not limited to, poly[glycolide-co-( ⁇ -caprolactone)], poly(hydroxyalkanoate), and copolymers thereof.
  • the cell culture container 10 is produced, for example, by forming a first flow path 1a and a second flow path 2a on a first substrate 1 and a second substrate 2, respectively, using a known method such as lithography. It can be manufactured by bonding the second substrate 2, membrane 3, and first substrate 1 in this order using an adhesive or the like that has relatively low toxicity to cells.
  • ⁇ Second culture step> In the second culture step, using the cell culture container 10, airway epithelial cells A infected with a virus or bacteria are seeded in the first channel 1a, and pulmonary microvascular endothelial cells A are inoculated in the second channel 2a. Cells B are seeded, and airway epithelial cells A and pulmonary microvascular endothelial cells B are co-cultured in the presence of the test substance.
  • virus and bacteria the same ones as exemplified for the above-mentioned inhibitor of vascular endothelial barrier breakdown can be used, but among them, it is preferable to use a virus, and it is more preferable to use SARS-CoV-2.
  • airway epithelial cells A and pulmonary microvascular endothelial cells B are each filled with a medium, and after the start of co-culture, airway epithelial cells A are supplied with air from the end of the first channel 1a. is injected, and a medium is injected into the pulmonary microvascular endothelial cells B from the end of the second channel 2a to perform co-culture.
  • Examples of the medium for culturing airway epithelial cells A include those commonly used for culturing airway epithelial cells A, such as an airway organoid differentiation medium.
  • the titer of the virus to infect the airway epithelial cells A can be, for example, 0.1 MOI or more and 1.0 MOI or less.
  • airway epithelial cells A are cultured in a medium containing a virus or bacteria with a titer within the above range, and after 1 to 3 hours (preferably 2 hours) have elapsed from the start of co-culturing, the medium is incubated with virus or bacteria.
  • the medium may be replaced with a bacteria-free medium.
  • the medium for culturing the pulmonary microvascular endothelial cells B As the medium for culturing the pulmonary microvascular endothelial cells B, the same medium as described in the screening method according to the first embodiment can be used.
  • the test substance is used by being added to the medium for culturing pulmonary microvascular endothelial cells B at the start of co-culture.
  • Scaffolding materials for pulmonary microvascular endothelial cells B include, for example, collagen (type I, type II, type III, type V, type XI, etc.), gelatin, elastin, proteoglycan, glycosaminoglycan, fibronectin, vitronectin, laminin, pectin. , hyaluronic acid, chitin, chitosan, alginic acid, starch, polyethylene glycol, polydimethylsiloxane, polylactic acid, polyvinylpyrrolidone, polyvinyl alcohol, polyglutamic acid, polyglycolic acid, polycaprolactone, polyacrylic acid, etc., and polymer gels containing them as appropriate.
  • a modified polymer gel may be coated on the bottom surface of the second channel 2a.
  • Co-culture conditions can be, for example, at a temperature of 30° C. or higher and 37° C. or lower and 5% CO 2 .
  • ⁇ Second measurement process> the number of viruses or bacteria in the culture supernatant of pulmonary microvascular endothelial cells is measured.
  • the number of viruses or bacteria can be calculated, for example, by measuring their genome copy numbers by real-time PCR or the like.
  • test substance is evaluated to be an inhibitor of vascular endothelial barrier breakdown when the number of viruses or bacteria is significantly reduced compared to the absence of the test substance.
  • test substance screened by the screening method of the present embodiment can effectively prevent vascular endothelial barrier breakdown caused by viruses (preferably SARS-CoV-2) or bacteria. Therefore, the test substance is expected to be applied as a therapeutic agent to stop the progression of COVID-19 symptoms.
  • the invention provides a method for treating a respiratory tract infection, comprising administering to a patient in need thereof an effective amount of Claudin-5, a nucleic acid encoding Claudin-5, or an inducer of Claudin-5 expression.
  • a method for preventing or treating COVID-19 is provided.
  • the present invention provides the above three substances for use in the prevention or treatment of respiratory infections (preferably COVID-19).
  • the present invention provides the use of the above three substances for the manufacture of a pharmaceutical composition for the treatment of respiratory infections (preferably COVID-19).
  • Claudin-5 a nucleic acid encoding Claudin-5
  • Claudin-5 expression inducer a nucleic acid encoding Claudin-5
  • SARS-CoV-2 strain B. 1.1.214 and B. 1.617.2 was isolated from a nasopharyngeal swab sample of a novel coronavirus disease-2019 (COVID-19) patient. This study was approved by the Research Ethics Committee of Kyoto University. The virus was grown in TMPRSS2/Vero cells (JCRB1818, JCRB Cell Bank) and stored at -80°C. TMPRSS2/Vero cells were cultured in minimum essential medium (MEM, manufactured by Sigma-Aldrich) supplemented with 5 w/v% fetal bovine serum (FBS) and 1 w/v% penicillin/streptomycin. All virus infection experiments were performed in a biosafety level 3 facility at Kyoto University in strict accordance with regulations.
  • MEM minimum essential medium
  • HMVEC-L Human lung microvascular endothelial cells
  • EGM-2-MV medium Lonza
  • PDMS polydimethylsiloxane
  • FIG. 2A the lower channel of the polydimethylsiloxane (PDMS) device was first precoated with fibronectin (3 ⁇ g/mL, manufactured by Sigma). HMVEC-L were suspended in EGM2-MV medium at 5 ⁇ 10 6 cells/mL. 10 ⁇ L of cell suspension was injected into the fibronectin-coated bottom channel of the PDMS device. The PDMS device was then turned upside down and incubated for 1 hour.
  • fibronectin 3 ⁇ g/mL, manufactured by Sigma
  • EGM2-MV medium was added to the bottom channel of the device.
  • human airway organoids were isolated and seeded into the upper channel of the device. Specifically, airway organoids (AO) were separated into single cells and suspended in AO differentiation medium at 5 ⁇ 10 6 cells/mL. 10 ⁇ L of cell suspension was injected into the upper channel of the PDMS device. After 1 hour, AO differentiation medium was added to the upper channel. Cells were cultured at 37°C in a humidified atmosphere of 5v/v% CO2 .
  • SARS-CoV-2 In patients with COVID-19, SARS-CoV-2 is present in the blood, and SARS-CoV-2 protein is expressed in many organs, including the respiratory tract.
  • SARS-CoV-2 protein is hardly detected in organs other than the respiratory tract. This is thought to be due to species differences in the distribution of SARS-CoV-2 within the body. Therefore, we considered it desirable to use a human model to investigate the movement of SARS-CoV-2 from the respiratory tract to the blood vessels and the disruption of the endothelial barrier through SARS-CoV-2 infection.
  • the airway-on-a-chip was used to examine the effects of SARS-CoV-2 replicated in airway epithelial cells on vascular endothelial cells.
  • the airway-on-a-chip is a co-culture system for airway epithelial cells and endothelial cells that mimics the dynamic microenvironment in vivo by flowing air and culture medium.
  • organs-on-a-chip technology three-dimensional and dynamic in vitro models can be generated. It can also be used to study interactions between multiple organs (such as the respiratory tract and blood vessels).
  • AO Airway organoid
  • NHBE normal human bronchial epithelial cells
  • NHBE normal human bronchial epithelial cells
  • NHBE normal human bronchial epithelial cells
  • a 50 ⁇ L drop of the cell suspension was solidified in a preheated cell culture-treated multidish (24-well plate; manufactured by Thermo Fisher Scientific) at 37° C. for 10 minutes, and then 500 ⁇ L of growth medium was added to each well.
  • AO was cultured in AO growth medium for 10 days.
  • the expanded AO was cultured in AO differentiation medium for 5 days. Mature AOs were isolated and seeded into PDMS devices prior to SARS-CoV-2 infection experiments.
  • Microfluidic devices were prepared according to previous reports. Briefly, a microfluidic device consists of two layers of microchannels separated by a semipermeable membrane. The microchannel layer was fabricated from pPDMS using a soft lithography method. A PDMS prepolymer (SYLGARD 184, manufactured by Dow Corning) with a ratio of base material and curing agent of 10:1 was poured into a mold configured with an SU-8 2150 (manufactured by MicroChem) pattern formed on a silicon wafer. . The cross-sectional size of the microchannel was 1 mm in width and 330 ⁇ m in height.
  • SARS-CoV-2 infection AO differentiation medium containing SARS-CoV-2 (0.1 MOI) was injected into the upper channel (airway channel) of the airway-on-a-chip. After 2 hours, the medium containing SARS-CoV-2 was replaced with fresh medium.
  • HMVEC-L were infected with 0.1 or 1 MOI SARS-CoV-2 for 120 minutes and then cultured in EGM2-MV medium.
  • RNA sequencing Total RNA was prepared using Rneasy Mini Kit (manufactured by Qiagen). RNA integrity was evaluated using 2100 Bioanalyzer (manufactured by Agilent Technologies). Library preparation was performed using the TruSeq Strand mRNA Sample Prep Kit (Illumina) according to the manufacturer's instructions. Sequencing was performed on an Illumina NextSeq500. fastq files were generated using bcl2fastq-2.20. Adapter sequences and low quality bases were trimmed from raw data reads by Cutadapt ver v3.4.28.
  • the trimmed reads were mapped to the human reference genome sequence (hg38) using STAR ver 2.7.9a29 and GENCODE (Release 36, GRCh38.p13) 30gtf file.
  • Raw counts of protein-encoding genes can be found in the GENCODE gtf file, htseq-count ver. Calculated using 0.13.531.
  • Gene expression levels were determined using Deseq2 v1.30.132 as TPM.
  • RNA lysis buffer 0.U/ ⁇ L SUPERase InRnase Inhibitor (Thermo Fisher Scientific), 2v/v% Triton X-100, 50mM KCl, 100mM Tris-HCl (p H7. 4) and distilled water containing 40 v/v% glycerol) and incubated for 10 minutes at room temperature. The mixture was diluted 10 times with distilled water.
  • Viral RNA was collected using the QuantStudio 1 Real-Time PCR system (Thermo Fisher Scientific) using the One Step TB Green PrimeScript PLUS RT-PCR kit (Perfect Real-Time) (Takara Bio). Quantitated. The primers used in this experiment are shown in the table below. A standard curve was created using SARS-CoV-2 RNA (10 5 copies/ ⁇ L) purchased from Japan Gene Research Institute.
  • Relative quantification of target mRNA levels was performed using the 2- ⁇ CT method or by calculating the copy number of the target transcript from a standard curve generated using known amounts of plasmid containing the target sequence. . Values were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR primer sequences are shown in the table above.
  • PVDF polyvinylidene fluoride
  • HMVEC-L was seeded on a chamber slide (manufactured by Thermo Fisher Scientific) and cultured in EGM-2-MV medium containing SARS-CoV-2.
  • the resulting cells were fixed with 4v/v% paraformaldehyde, permeabilized with PBS containing 0.3v/v% Triton X-100, and blocked with PBS containing 1w/v% bovine serum albumin.
  • HMVEC-L 3 ⁇ 10 4 cells were seeded in cell culture inserts (for 24-well plates) with a pore size of 3.0 ⁇ m (manufactured by BD Falcon) and incubated for 72 hours.
  • the medium in the upper chamber was replaced with EGM-2-MV medium containing 1 MOI SARS-CoV-2, and transendothelial electrical resistance (TEER) was measured with a Millicell ERS-2 volt-ohmmeter (Merck Millipore).
  • the TEER value was calculated using the following formula.
  • TEER value ⁇ (resistance of experimental well) - (resistance of blank well) ⁇ x 0.32 (membrane area of cell culture insert)
  • HMVEC-L was electroporated with pPB-TRE3G-CLDN-5-V2 and pHL-EF1a-hcPBase-A33 vectors using a NEPA21 electroporator (Nepa Gene), and treated with 1 ⁇ g/mL puromycin ( (manufactured by InvivoGen).
  • the piggyBac-based CLDN5 expression plasmid pPB-TRE3G-CLDN-5-V2 contains human CLDN5 in the multiple cloning site of pPB-TRE3G-MCS(A)-P2AMCS(B)V2 (kindly provided by Dr.
  • Tsuyoshi Maruyama (Waseda University)). It was constructed by inserting a gene (SEQ ID NO: 9; the sequence near the CDS was extracted from the amino acid sequence (SEQ ID NO: 5) of Genbank accession number NM_001130861.1).
  • [Preparation of human CLDN5 knock-in mouse] A donor vector consisting of a homology arm sequence (2 kb) derived from the mouse genome ligated to both ends of human CLDN5 (GenBank: BC002404.2, SEQ ID NO: 50) was created. Cas9 protein, sgRNA, and donor vector were introduced into mouse ES cells for homologous recombination. ES cells containing human CLDN5 at the appropriate locus were selected by genotyping by PCR and DNA sequencing. ES cells with normal karyotype were introduced into early embryos. The resulting chimeric mouse was crossed with a C57BL/6N mouse to establish a human CLDN5 homozygous knock-in mouse (hCLDN5-KI mouse). For genotyping, DNA fragments were amplified using hCLDN5 knock-in mouse tail genomic DNA and primers (table above) and digested with EagI to confirm genomic DNA containing the human CLDN5 sequence.
  • mice Male, 7-8 weeks old.
  • mice were injected intravenously with 100 ⁇ L of PBS containing 1 g of Evans Blue.
  • mice were perfused with 2mM ethylenediaminetetraacetic acid in PBS, and organs were harvested, minced, and incubated in formamide at 55°C for 48 hours.
  • Eluted Evans blue was quantified by measuring optical density at 620 nm.
  • hCLDN5-KI mice male, 7-8 weeks old were injected with anti-CLDN5 antibody (R9) or control rat IgG (manufactured by Sigma-Aldrich).
  • R9 or control rat IgG manufactured by Sigma-Aldrich.
  • lungs were harvested from mice and weighed immediately (wet weight).
  • the lungs were dried in an incubator at 60°C for 48 hours, and the dry weight was measured. Pulmonary edema was assessed by calculating the wet weight/dry weight ratio.
  • a transparent flat-bottom non-sterile 96-well plate (Thermo Fisher Scientific) was plated with anti-CLDN5 antibody No. 1 (initial concentration 0.403 ⁇ g/mL diluted to final concentration 1.1 ⁇ g/mL), incubated for 1 hour at room temperature, and then coated with 1 w/v % bovine serum albumin in PBS. Blocked. Samples and standard CLDN5 protein were diluted 2-fold in dilution buffer (0.05 v/v % Tween 20 in PBS) and 100 ⁇ L of sample was added to each well and incubated for 1 hour at room temperature. After washing three times with washing buffer, 100 ⁇ L of anti-CLDN5 antibody No.
  • a solution containing 2 (initial concentration of 854 ⁇ g/mL was diluted to a final concentration of 0.2 ⁇ g/mL) was added to each well and incubated at room temperature for 1 hour. After washing three times with wash buffer, 100 ⁇ L of biotin-SP-AffiniPure donkey anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, 711-065-152) was added to each well and incubated for 1 hour at room temperature. After washing three times with washing buffer, 100 ⁇ L of Pierce High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific, 21130) was added to each well and incubated at room temperature for 30 minutes.
  • TMB substrate solution manufactured by R&D Systems
  • TMB substrate solution manufactured by R&D Systems
  • the reaction was stopped by adding 100 ⁇ L of stop solution to each well.
  • the optical density of each sample at 450 nm was measured using TriStar LB 941 (manufactured by Berthold). CLDN5 concentration was calculated from the calibration curve.
  • the virus genome copy number in the culture supernatant of cells infected with airway-on-a-chip SARS-CoV-2 was measured (see FIG. 2B).
  • the viral genome copy number in the airway channel increased and reached a maximum at 4 dpi, whereas the viral genome copy number in the vascular channel started increasing at 3 dpi and reached a maximum at 7 dpi. This suggested that SARS-CoV-2 inoculated from airway channels replicated in airway epithelial cells and entered vascular channels.
  • RNA-seq analysis was performed on HMVEC-L infected with SARS-CoV-2 airway-on-a-chip. HMVEC-L was cultured for 8 days under airway-on-a-chip infection, and HMVEC-L was collected for RNA-seq analysis.
  • SARS-CoV-2 infection up-regulated and down-regulated the 767 and 668 genes by more than two-fold, respectively (see Figure 2C).
  • upregulated genes included innate immune response, inflammation, and viral response related genes (not shown).
  • down-regulated genes included cell adhesion-related genes (see Figure 2D).
  • the top five down-regulated genes in the “plasma membrane adhesion molecule-mediated hemophilia cell adhesion” and “plasma membrane adhesion molecule-mediated cell-cell adhesion” categories were CLDN3, CLDN5, PCDHGB1, PCDHB8, and IGSF9B (see Figure 2E).
  • CLDN5 was the endogenous gene with the highest expression level in uninfected HMVEC-L in the above two categories (see FIG. 2F).
  • CLDN5 was suggested to be a potential causative gene for respiratory vascular barrier breakdown mediated by SARS-CoV-2 infection. Although SARS-CoV-2 does not infect HMVEC-L, the gene expression profile of HMVEC-L can be significantly altered upon contact with SARS-CoV-2.
  • HMVEC-Ls are continuously exposed to high titers of SARS-CoV-2, to induce CLDN5 downregulation, HMVEC-Ls are We expected that continuous exposure to high titers of SARS-CoV-2 secreted from infected airway epithelial cells would be necessary.
  • HMVEC-L were exposed to high titer (1 MOI) of SARS-CoV-2 for 4 consecutive days. This treatment destroyed the HMVEC-L monolayer, suggesting weakening of intercellular adhesion (see FIG. 3B), and a significant decrease in the expression level of CLDN5 was confirmed (see FIGS. 3A and 3C).
  • the medium containing SARS-CoV-2 was allowed to act on HMVEC-L for 4 consecutive days while changing the medium every day, and then the expression level of CLDN5 was measured by qRT-PCR. Determined by analysis.
  • SARS-CoV-2 significantly decreased the expression of CLDN5 at both mRNA and protein levels (see Figures 3C and 3D), whereas the downregulation of VE-cadherin was slight (see Figures 3C and 3D). See 3D. Furthermore, SARS-CoV-2 exposure increased VCAM-1 and ICAM-1 expression levels, but did not change the expression level of IL-6 (see Figure 3C).
  • Immunofluorescence staining showed decreased CLDN5 expression and impaired VE-cadherin-mediated adhesion (see Figure 3E). This was similar to the phenomenon observed in airway-on-a-chip infected with SARS-CoV-2. Immunofluorescence staining also showed that SARS-CoV-2 exposure disrupted and reduced ⁇ -catenin localization at cell junctions (see Figure 3E). Furthermore, phalloidin staining showed that SARS-CoV-2 exposure increased the localization of F-actin along the cell membrane and the distance between cells (see Figure 3E). Consistently, SARS-CoV-2 was able to penetrate the monolayer endothelium (see Figure 3F), and transendothelial electrical resistance was transiently reduced in the presence of SARS-CoV-2 (see Figure 3G).
  • FIG. 3H is a morphological observation image obtained by HE staining the airways and lungs of lung specimens from a healthy person and a severe COVID-19 patient.
  • FIG. 3I is a scatter plot comparing the expression levels of various genes between lung samples from healthy individuals and lung samples from severe COVID-19 patients through RNA-seq analysis.
  • CLDN5 The expression level of CLDN5 was decreased in the lungs of severe COVID-19 patients compared to the lungs of healthy individuals.
  • FIG. 3J shows the relative value of the expression level of each gene in severe COVID-19 patients, when the expression level of each gene in the lung sample of a healthy person is set to 1.
  • lung samples from healthy people and severe COVID-19 patients were tested for vascular endothelial markers VE-Cadherin and PECAM1, and markers specific for lung endothelial cells (aerocytes) that perform gas exchange, HPGD and TBX2.
  • vascular endothelial markers VE-Cadherin and PECAM1 markers specific for lung endothelial cells that perform gas exchange, HPGD and TBX2.
  • UV-SARS-CoV-2 and SARS-CoV-2 increased the expression of inflammatory genes including IL-6, ICAM-1, and VCAM-1 (see Figure 5A).
  • SARS-CoV-2 spike (S) protein did not change the expression of any genes (see Figure 5A). Consistent with these, UV-SARS-CoV-2, but not the spike protein, induced disordered localization of VE-cadherin (see Figure 5B).
  • fluvastatin treatment increases the expression of CLDN5 and suppresses respiratory vascular endothelial barrier breakdown mediated by SARS-CoV-2 exposure.
  • the amount of CLDN5 in serum was 484 pg/mL in mild and moderate COVID-19 patients, but 180 pg/mL in severe COVID-19 patients. Serum CLDN5 concentrations were significantly lower in severe patients than in mild/moderate patients.
  • Example 9 Upregulation of CLDN5 expression by various statin drugs
  • statin drugs fluvastatin, pitavastatin, atorvastatin, cerivastatin, simvastatin, and lovastatin were each allowed to act on HMVEC-L at 10 ⁇ M.
  • FIG. 9 shows the results of measuring the gene expression level of CLDN5 after 24 hours.
  • control means a drug non-effect group.
  • TGF- ⁇ 2 (5 ng/mL) was added, and the expression level of CLDN5 was measured 24 hours later.
  • HMVEC-L was cultured in a serum-free medium for 15 hours before the action of the TGF- ⁇ inhibitor in order to remove medium components such as growth factors that may affect the action of TGF- ⁇ 2. .
  • FIG. 10 shows the results of the expression level of CLDN5 under each inhibitor, when the expression level of CLDN5 in the "control" in which no TGF- ⁇ inhibitor or TGF- ⁇ 2 was applied was set to 1.
  • the "DMSO” group is a group in which TGF- ⁇ 2 was treated without the action of a TGF- ⁇ inhibitor.
  • statin drugs fluvastatin, pitavastatin, atorvastatin, cerivastatin, simvastatin, and lovastatin
  • TGF vascular endothelial growth factor
  • - ⁇ inhibitors SB525334, R268712, EW-7197, RepSox, TP0427736, A83-01, K02288, were applied at 10 ⁇ M each.
  • the airway channel of the airway-on-a-chip was infected with SARS-CoV-2 (B.1.1.214) at an MOI of 0.1.
  • Figures 11A and 11B show the results of measuring the virus genome copy number contained in the cell culture supernatant of the airway channel and blood vessel channel 8 days after infection.
  • statin drugs or TGF- ⁇ inhibitors when treated with statin drugs or TGF- ⁇ inhibitors, the virus genome contained in the cell culture supernatant was Copy number decreased.
  • SB525334 was applied at 10 ⁇ M to the airway-on-a-chip vascular channel. Thereafter, SARS-CoV-2 was introduced into the airway channel of the airway-on-a-chip.
  • CLDN5 VE-cadherin, inflammatory genes (IL-6, VCAM-1, ICAM-1) and interferon response-related genes (IFN- ⁇ , IFN- ⁇ , ISG15, ISG56, MxA) in cells of vascular channels 8 days after infection ) is shown in FIG. 12.
  • vascular endothelial barrier breakdown can be effectively prevented.
  • First substrate 1a First channel 2: Second substrate 2a: Second channel 3: Membrane 10: Cell culture container A: Airway epithelial cells B: Pulmonary microvascular endothelial cells

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Abstract

Inhibiteur de défaillance de la barrière endothéliale vasculaire contenant, comme principe actif, un inducteur d'expression de la claudine-5, un acide nucléique codant la claudine-5, ou la claudine-5.
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