WO2023063325A1 - Inhibiteur d'infection au nouveau coronavirus (sras-cov -2) - Google Patents

Inhibiteur d'infection au nouveau coronavirus (sras-cov -2) Download PDF

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WO2023063325A1
WO2023063325A1 PCT/JP2022/037941 JP2022037941W WO2023063325A1 WO 2023063325 A1 WO2023063325 A1 WO 2023063325A1 JP 2022037941 W JP2022037941 W JP 2022037941W WO 2023063325 A1 WO2023063325 A1 WO 2023063325A1
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cov
adam10
sars
cells
tmprss2
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純一郎 井上
寧 川口
仁 合田
徹 秋山
瑞生 山本
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国立大学法人 東京大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K31/396—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having three-membered rings, e.g. aziridine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K31/435—Heterocyclic 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4704—2-Quinolinones, e.g. carbostyril
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/541—Non-condensed thiazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/20—Elemental chlorine; Inorganic compounds releasing chlorine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the present invention relates to a therapeutic or prophylactic agent for novel coronavirus infectious disease (COVID-19, coronavirus disease 2019).
  • SARS-CoV-2 the virus that causes the new coronavirus infection, was recognized at the end of 2019 and became a global pandemic in 2020, posing a threat to civilization.
  • Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are also capable of causing fatal pneumonia and systemic symptoms, but the infectious capacity of SARS-CoV-2 Pathogenicity is further enhanced.
  • SARS-CoV Severe acute respiratory syndrome coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS-CoV-2 Middle East respiratory syndrome coronavirus
  • no drug has been developed that is sufficiently effective for the treatment of COVID-19, and although the vaccine currently inoculated has been successful in preventing the spread of infection, the emergence of virus mutations has reduced its effectiveness. has become uncertain. Further characterization of the virus and its interactions with host cells are needed to develop vaccines and therapeutics that reliably prevent SARS-CoV-2 infection.
  • the spike (S) protein present in the viral envelope also called viral membrane, outer membrane
  • ACE2 receptor binding domain
  • the S protein is then cleaved by the transmembrane serine protease 2 (TMPRSS2) present in the cell membrane, or by the endosomal protease cathepsin-B/L after the virus is endocytosed into the cell.
  • TMPRSS2 transmembrane serine protease 2
  • TMPRSS2 transmembrane serine protease 2
  • cathepsin-B/L endosomal protease cathepsin-B/L
  • cleavage exposes the fusion peptide within the S protein and attaches it to the cell or endosomal membrane.
  • fusion between the viral envelope and the cell membrane or endosomal membrane occurs, allowing viral RNA to enter the cytoplasm and establish infection.
  • Non-Patent Document 1 SARS-CoV-2 entry into cells can be inhibited by inhibiting TMPRSS2-dependent membrane fusion of SARS-CoV-2.
  • the purpose of the present invention is to provide therapeutic and preventive agents for COVID-19.
  • a therapeutic or preventive composition or a therapeutic or preventive agent for COVID-19 (coronavirus disease 2019), containing an ADAM10 inhibitor.
  • nucleic acid is siRNA.
  • nucleic acid is siRNA.
  • another agent preferably a therapeutic or preventive agent for COVID-19.
  • the other drug is one or more compounds selected from the group consisting of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine.
  • method of treatment or prevention of [12] The therapeutic or preventive method of [10] or [11] above, wherein the other drug is a TMPRSS2 inhibitor.
  • ADAM10 inhibitors for use in treating or preventing COVID-19 (coronavirus disease 2019).
  • the other drug is one or more compounds selected from the group consisting of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine. combination.
  • the other drug is a TMPRSS2 inhibitor.
  • a therapeutic or preventive composition or therapeutic or preventive agent for COVID-19 comprising one or two compounds selected from the group consisting of marimastat and prinomastat.
  • the other drug is one or more compounds selected from the group consisting of E-64d, ammonium chloride, chloroquine and hydroxychloroquine. Or a therapeutic or prophylactic agent.
  • the other drug is one or more compounds selected from the group consisting of E-64d, ammonium chloride, chloroquine and hydroxychloroquine.
  • FIG. 1 shows TMPRSS2-independent membrane fusion induced by SARS-CoV-2 S protein (Example 1).
  • Each effector cell expressing SARS-CoV S, SARS-CoV-2 S or MERS-CoV S
  • target cells as control (Cont), receptor (Receptor) or receptor + TMPRSS2 (Receptor + TMPRSS2)
  • FIG. 10 is a graph showing quantitative results of cell fusion assay using expressing cells.
  • each effector cell expressing SARS-CoV S, SARS-CoV-2 S or MERS-CoV S
  • target cells as control (Cont), receptor (Receptor) or receptor + TMPRSS2 (Receptor + TMPRSS2)
  • Cont Cont
  • Receptor receptor + TMPRSS2
  • TMPRSS2 Receptor + TMPRSS2
  • effector cells SARS-CoV-2, SARS-CoV-2, ; is a graph showing the quantitative results of a cell fusion assay using ACE2- or TMPRSS2+ACE2-expressing cells as control (C), spike protein (S)) and target cells.
  • FIG. 2 shows the suppression of TMPRSS2-independent membrane fusion induced by SARS-CoV-2 S protein by metalloprotease inhibitors (Example 2).
  • the compounds in the dotted box selectively inhibited TMPRSS2-independent membrane fusion.
  • the upper panel shows the effect of metalloprotease inhibitors on cell fusion in co-cultures of cells expressing SARS-CoV-2 S protein and cells expressing ACE2 alone or in combination with TMPRSS2 (each compound (Ilomastat ( Effector cells (Control (Cont), SARS-CoV-2 Spike) and target cells in the presence of ilomastat, CTS-1027, marimastat, prinomastat Quantitative results of a cell fusion assay using control (Cont), ACE2, or TMPRSS2+ACE2-expressing cells).
  • the vertical axis indicates the relative cell fusion rate (Relative Cell fusion (%)).
  • the lower row shows effector cells (SARS-CoV-2 S) and target cells in the presence of each compound (ilomastat, CTS-1027, marimastat, prinomastat), DSP1-7 and
  • FIG. 10 is a graph showing quantitative results of DSP assay using DSP8-11 co-expressing cells.
  • FIG. The vertical axis indicates relative DSP activity (%).
  • Figure 3 shows the presence of a SARS-CoV-2-specific metalloprotease-dependent viral entry pathway (Example 3).
  • the vertical axis indicates pseudovirus entry (% of control).
  • FIG. 4 shows that the pattern of entry pathways is conserved in various mutants of SARS-CoV-2 (entry of SARS-CoV-2 mutants into cells via the metalloprotease-dependent pathway) (Example 4). ).
  • (a) A diagram showing the expression of the S protein of each SARS-CoV-2 strain (control (Cont), wild (WT), each mutant strain). The S protein was detected using an anti-Flag tag antibody that binds to the Flag tag at the C-terminus of the S protein (top). Vesicular stomatitis virus matrix protein (VSV M) served as a control (bottom). S0 indicates the uncleaved S protein, S2 indicates the truncated S2 domain of the S protein.
  • VSV M Vesicular stomatitis virus matrix protein
  • each SARS- Fig. 10 is a graph showing quantitative results of infection assay using CoV-2 strains (control (Cont), wild (WT), each mutant strain). The vertical axis shows pseudovirus entry (% of DMSO).
  • FIG. 5 shows involvement of ADAM-10 in the metalloprotease-dependent entry pathway of SARS-CoV-2 (Example 5).
  • Each strain control (Cont, S FIG. 10 is a graph showing the quantitative results of infection assays using protein-free pseudovirus-infected cells), SARS-CoV-2 S, VSV-G).
  • the vertical axis shows pseudovirus entry (% of DMSO).
  • Figure 6 shows the effect of drug on cell viability (cell viability is not affected by metalloprotease inhibitors) (Example 5).
  • Cell viability (% of DMSO) in the presence of each compound and without E-64d (Without E-64d) or 25 ⁇ M E-64d (With 25 ⁇ M E-64d) for each cell line (VeroE6, HEC50B, A704) (Cell viability (% of DMSO)), each value represents the mean ⁇ SD (n 3/group).
  • FIG. 4 is a diagram showing; (a) Two types of control and three types of siRNA with different sequences against ADAM10 (transfected for 48 hours) suppress the expression of ADAM10 (mock, control (Cont), precursor ( precursor), active ADAM10 (active ADAM10), Tublin). (b) Graph showing the effect of each siRNA on invasion of each pseudovirus (SARS-CoV-2, SARS-CoV, MERS-CoV, VSV).
  • FIG. 8 shows inhibition of SARS-CoV-2 live virus infection growth by metalloprotease inhibitors or ADAM10 knockdown (Example 6).
  • the vertical axis shows the relative SARS-CoV-2 N expression (/rpl13a) in logarithm (Log10 [Relative SARS-CoV-2 N expression (/rpl13a)]) (marimastat, marima), prinomastat ( prinomastat), nafamostat).
  • composition and agent of the present invention comprise an ADAM10 inhibitor as an active ingredient.
  • ADAM10 is an abbreviation for A disintegrin and metalloproteinase domain-containing protein 10, which is a kind of metalloprotease called ADAM family.
  • the human ADAM10 gene is based on the nucleotide sequence published in Genbank/NCBI Gene ID: 102. Also in the present invention, the human ADAM10 protein is referenced to the amino acid sequences published at NCBI Reference Sequence: NP_001101.1 and NP_001307499.1. Further, in the present invention, human ADAM10 mRNA is based on NCBI Reference Sequence: NM_001110.4 and NM_001320570.2.
  • an ADAM10 inhibitor is any substance that can inhibit ADAM10, and is used in the sense of including substances that inhibit the expression of ADAM10 and substances that inhibit the function of ADAM10.
  • ADAM10 inhibitors that specifically inhibit ADAM10 can be used in the present invention.
  • Substances that inhibit the expression of ADAM10 include nucleic acids against ADAM10 (eg, antisense nucleic acids such as antisense DNA, nucleic acids targeting ADAM10 such as siRNA, shRNA, microRNA, gRNA, and ribozymes).
  • Substances that inhibit the function of ADAM10 include substances that interact with ADAM10 to inhibit the function, and examples thereof include small molecules, antibodies, peptides, nucleic acids, and aptamers.
  • modified bases with in vivo stability or artificial bases can also be used as the bases that constitute the nucleic acids.
  • the nucleic acid sequence may contain not only sequences that perfectly match with the target nucleic acid, but also mismatch sequences that do not match with the target sequence as long as the expression inhibitory activity is maintained.
  • An antisense nucleic acid is a nucleic acid complementary to a target sequence.
  • the antisense nucleic acid inhibits transcription initiation by triplex formation, inhibits transcription by hybridization with a site where an open loop structure is locally formed by RNA polymerase, inhibits transcription by hybridization with RNA that is being synthesized, Suppression of splicing by hybridization at junctions between introns and exons, suppression of splicing by hybridization with spliceosome-forming sites, suppression of translocation from the nucleus to the cytoplasm by hybridization with mRNA, capping sites and poly(A) addition sites Suppression of splicing by hybridization with , Suppression of translation initiation by hybridization with the translation initiation factor binding site, Translation suppression by hybridization with the ribosome binding site near the initiation codon, and Hybridization with the translational region of mRNA and the polysome binding site
  • the expression of the target gene can be suppressed by inhibiting elongation of the
  • the antisense nucleic acid against ADAM10 is, for example, a single-stranded nucleic acid complementary to a partial nucleotide sequence selected from the ADAM10 gene sequence described above, the nucleotide sequence encoding the ADAM10 amino acid sequence described above, and the ADAM10 mRNA sequence described above.
  • Such nucleic acids may be naturally occurring or artificial nucleic acids, and may be based on both DNA and RNA.
  • the length of the antisense nucleic acid is usually about 15 bases to the same length as the full-length mRNA, preferably about 15 to about 30 bases.
  • the complementarity of the antisense nucleic acid does not necessarily have to be 100%, and may be such that it can complementarily bind to ADAM10-encoding DNA or RNA in vivo.
  • siRNA small interfering RNA
  • siRNA is an artificially synthesized small double-stranded RNA used for gene silencing by RNA interference (mRNA degradation), and the double-stranded RNA is supplied in vivo. It shall be used in the sense of including an siRNA expression vector capable of siRNAs introduced into cells bind to the RNA-induced silencing complex (risc). This complex binds to and cleaves mRNA having a sequence complementary to siRNA, thereby suppressing gene expression in a sequence-specific manner.
  • siRNA is prepared by synthesizing sense strand and antisense strand oligonucleotides with an automatic DNA/RNA synthesizer. can be prepared by annealing for about 1 to 8 hours at . The length of the siRNA is preferably 19-27 base pairs, more preferably 21-25 base pairs or 21-23 base pairs.
  • siRNA against ADAM10 can be designed based on its base sequence to cause degradation (RNA interference) of mRNA transcribed from the ADAM10 gene.
  • Examples of siRNAs that inhibit the expression of ADAM10 include siRNAs whose target sequence is the above-mentioned ADAM10 mRNA sequence.
  • shRNA short hairpin RNA
  • shRNA is an artificially synthesized hairpin-shaped RNA sequence used for gene silencing by RNA interference (mRNA degradation).
  • shRNA may be introduced into cells by a vector and expressed with a U6 promoter or H1 promoter, or an oligonucleotide having a shRNA sequence may be synthesized by an automatic DNA/RNA synthesizer and self-annealed by a method similar to siRNA.
  • may be prepared by Hairpin structures of shRNAs introduced into cells are cleaved into siRNAs and bind to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA having a sequence complementary to siRNA, thereby suppressing gene expression in a sequence-specific manner.
  • RISC RNA-induced silencing complex
  • the shRNA against ADAM10 can be designed based on its base sequence to cause degradation (RNA interference) of mRNA transcribed from the ADAM10 gene.
  • shRNAs that inhibit the expression of ADAM10 include shRNAs whose target sequence is the above-mentioned ADAM10 mRNA sequence.
  • miRNA is a functional nucleic acid that is encoded on the genome and eventually becomes a micro RNA of about 20 bases through a multistep production process. miRNAs are classified as functional ncRNAs (non-coding RNAs: a generic term for RNAs that are not translated into proteins), and play an important role in life phenomena by regulating the expression of other genes.
  • ADAM10 gene expression can be suppressed by introducing miRNA having a specific nucleotide sequence into cells using a vector and administering the miRNA to a living body.
  • gRNA guide RNA
  • gRNA is an RNA molecule used in genome editing technology.
  • gRNA specifically recognizes the target sequence, guides the binding of Cas9 protein to the target sequence, and enables gene knockout and knockin.
  • ADAM10 gene expression can be suppressed in vivo by administering gRNA targeting the ADAM10 gene in vivo.
  • gRNA shall be used in the meaning including sgRNA (single guide RNA).
  • the design method of gRNA in genome editing technology is widely known, for example, Benchmarking CRISPR on-target sgRNA design, Yan et al., Brief Bioinform, 15 Feb 2017.
  • a ribozyme is an RNA with catalytic activity. Although some ribozymes have various activities, studies on ribozymes as RNA-cleaving enzymes have made it possible to design ribozymes for the purpose of site-specific cleavage of RNA.
  • the ribozyme may be group I intron type, M1 RNA contained in RNaseP, etc., having a size of 400 nucleotides or more, or hammerhead type, hairpin type, etc. having about 40 nucleotides.
  • Aptamers include nucleic acid aptamers and peptide aptamers.
  • Nucleic acid aptamers and peptide aptamers used in the present invention are represented by the SELEX method (Systematic Evolution of Ligands by Exponential enrichment) and the mRNA display method. They can be obtained using in vitro molecular evolution techniques that are formed and then selected on the basis of affinity.
  • Antisense nucleic acids, siRNAs, shRNAs, miRNAs, ribozymes, and nucleic acid aptamers may contain various chemical modifications to improve their stability and activity.
  • phosphate residues may be substituted with chemically modified phosphate residues such as phosphorothioates (PS), methylphosphonates, phosphorodithionates, etc., to prevent degradation by hydrolases such as nucleases.
  • PS phosphorothioates
  • methylphosphonates methylphosphonates
  • phosphorodithionates etc.
  • at least a part thereof may be composed of a nucleic acid analogue such as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • An antibody against ADAM10 is an antibody that specifically binds to ADAM10 and inhibits the function of ADAM10 by binding.
  • antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, mouse antibodies, rat antibodies, camelid antibodies, antibody fragments (e.g., Fab, Fv, Fab', F (ab') 2 , scFv) and the like may be used, and these can be prepared according to known techniques by those skilled in the art.
  • Antibodies against ADAM10 can be produced according to known antibody or antiserum production methods using the ADAM10 protein or a portion thereof as an antigen.
  • ADAM10 protein or portions thereof can be prepared by known protein expression and purification methods. Examples of the ADAM10 protein include, but are not limited to, human ADAM10 defined by the ADAM10 sequence information described above. ADAM10 proteins from various organisms may be used as immunogens.
  • Antibodies to ADAM10 that can be used in the present invention can also be generated via phage display technology (see, eg, FEBS Letter, 441:20-24 (1998)).
  • compositions and agents of the present invention also contain, as active ingredients, one or two compounds selected from the group consisting of marimastat and prinomastat.
  • ADAM10 is involved in the metalloprotease-dependent entry pathway of SARS-CoV-2, and that SARS-CoV-2 infection can be inhibited by inhibiting ADAM10. Therefore, ADAM10 inhibitors can be used as active ingredients for the treatment or prevention of COVID-19.
  • SARS-CoV-2 exists not only in the originally discovered virus strain, but also in variants thereof (e.g. strain B.1.1.7 (alpha strain), B.1.351 strain (beta strain), P.1 strain (gamma strain), B.1.617.2 strain (Delta strain), B.1.1.529 strain (Omicron strain)).
  • SARS-CoV-2 is synonymous with severe acute respiratory syndrome coronavirus-2.
  • compositions and agents of the present invention can be provided as pharmaceuticals or pharmaceutical compositions.
  • the drug and pharmaceutical composition of the present invention contain the active ingredient of the present invention and a pharmaceutically acceptable carrier.
  • the medicaments and pharmaceutical compositions of the present invention also include medicaments and pharmaceutical compositions intended for gene therapy.
  • Such pharmaceuticals and pharmaceutical compositions contain ADAM10-targeted nucleic acids such as antisense nucleic acids, siRNA, shRNA, microRNA, gRNA, and ribozymes as active ingredients.
  • compositions and agents of the present invention may be used in combination with other agents other than the active ingredient of the present invention. That is, the compositions and agents of the present invention may further contain drugs other than the active ingredient of the present invention, and in this case, the dosage form may be integrated as a combination drug.
  • the composition and agent of the present invention may be administered together with other drugs other than the active ingredient of the present invention as different formulations, in which case they may be administered simultaneously or at different times. good.
  • a combination of active ingredients of the invention and other agents are provided.
  • drugs other than ADAM10 inhibitors include SARS-CoV-2 infection inhibitors (especially COVID-19 therapeutic or preventive agents).
  • SARS-CoV-2 infection inhibitors especially COVID-19 therapeutic or preventive agents.
  • Such other agents include, for example, metalloprotease inhibitors such as marimastat and prinomastat, cathepsin-B/L inhibitors such as E-64d, ammonium chloride, chloroquine and hydroxychloroquine.
  • Such other agents also include TMPRSS2 inhibitors such as, for example, nafamostat, camostat.
  • drugs other than these compounds include SARS-CoV-2 infection inhibitors (especially COVID-19 therapeutic or preventive agents).
  • SARS-CoV-2 infection inhibitors especially COVID-19 therapeutic or preventive agents.
  • agents include cathepsin-B/L inhibitors such as E-64d, ammonium chloride, chloroquine and hydroxychloroquine.
  • agents also include TMPRSS2 inhibitors such as, for example, nafamostat, camostat.
  • the route of administration is not particularly limited as long as the therapeutic or preventive effect of COVID-19 is obtained, but oral administration or parenteral administration (e.g., intravenous administration, subcutaneous administration, intraperitoneal administration) can be selected.
  • oral administration or parenteral administration e.g., intravenous administration, subcutaneous administration, intraperitoneal administration
  • Orally administered drugs include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, liquids, jellies, emulsions, and suspensions.
  • an appropriate dosage form can be selected according to the specific dosage form, and examples thereof include injections and suppositories.
  • These formulations can be formulated using a pharmaceutically acceptable carrier by a method commonly practiced in the art (for example, a known method described in the 18th revision of the Japanese Pharmacopoeia General Rules for Formulations, etc.). can.
  • Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, perfumes, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc. is mentioned.
  • the dosage of the active ingredient in the present invention can be determined depending on the type of active ingredient, sex, age and body weight of the subject, symptoms, dosage form, route of administration, and the like.
  • the dosage per adult can be determined, for example, in the range of 0.0001 mg to 1000 mg / kg body weight. is not limited to
  • the above dosage of the active ingredient can be administered once a day or in 2 to 4 divided doses.
  • the compositions and agents of the present invention can be applied not only to humans in need thereof, but also to mammals other than humans (e.g., mice, rats, rabbits, dogs, cats, cows, horses, pigs, sheep, goats, monkeys). It can also be administered to
  • a method for treating or preventing COVID-19 comprising administering an ADAM10 inhibitor to a subject in need thereof.
  • the present invention also provides a method of treating or preventing COVID-19, comprising administering one or two compounds selected from the group consisting of marimastat and prinomastat to a subject in need thereof. be done.
  • the administration subject can typically be a COVID-19 patient or a person who may have COVID-19.
  • the therapeutic method and prophylactic method of the present invention can be carried out according to the description of the composition and agent of the present invention.
  • ADAM10 inhibitors for use in treating or preventing COVID-19 and combinations of ADAM10 inhibitors and other agents for use in treating or preventing COVID-19 are provided.
  • the present invention also provides one or two compounds selected from the group consisting of marimastat and prinomastat for use in the treatment or prevention of COVID-19 and Combinations of one or two compounds selected from the group consisting of , marimastat and prinomastat with other agents are provided.
  • ADAM10 inhibitors of the present invention, one or two compounds selected from the group consisting of marimastat and prinomastat, and combinations can be performed according to the description of the compositions and agents of the present invention.
  • an ADAM10 inhibitor for the manufacture of a composition for the treatment or prevention of COVID-19 or an agent for the treatment or prevention of COVID-19 and the composition for the treatment or prevention of COVID-19
  • a combination of an ADAM10 inhibitor and other agents for the manufacture of a therapeutic or prophylactic agent for COVID-19 is provided.
  • one or two compounds selected from the group consisting of marimastat and prinomastat for the manufacture of a composition for the treatment or prevention of COVID-19 or an agent for the treatment or prevention of COVID-19 and one or two compounds selected from the group consisting of marimastat and prinomastat for the manufacture of a composition for the treatment or prevention of COVID-19 or an agent for the treatment or prevention of COVID-19 , in combination with other agents is provided.
  • the uses of the invention can be carried out according to the description of the compositions and agents of the invention.
  • the present invention includes the following inventions.
  • a composition for treating COVID-19 coronavirus disease 2019
  • comprising an ADAM10 inhibitor comprising an ADAM10 inhibitor.
  • the therapeutic composition of claim 1 wherein the ADAM10 inhibitor is a nucleic acid.
  • the therapeutic composition of claim 2, wherein the nucleic acid is siRNA.
  • the therapeutic composition of any one of the above [101] to [103] which is used in combination with any one or a combination of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine .
  • the therapeutic composition of any one of the above [101] to [104] which is used in combination with a TMPRSS2 inhibitor.
  • the ADAM10 inhibitor is administered in combination with one or more compounds selected from the group consisting of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine, the above [ 106] to [108].
  • siRNAs and primers used in the Examples were as shown in Table 2.
  • the compounds (inhibitors) used in the examples were as shown in Table 3.
  • S spike protein
  • ACE2 ACE2, CD26, or TMPRSS2
  • pseudolentiviruses expressing one of the proteins were used as previously described (Yamamoto M. et al., 2020, Viruses, 12:629).
  • SARS-CoV-2 isolate (UT-NCGM02/Human/2020/Tokyo) (Imai M et al., 2020, Proc Natl Acad Sci USA 117:16587-16595) contains 5% fetal bovine serum (FBS) VeroE6-TMPRSS2 (JCRB1819) cells were grown in Dulbecco's Modified Eagle Medium (DMEM).
  • FBS fetal bovine serum
  • VeroE6-TMPRSS2 JCRB1819
  • siRNAs (Table 2) were transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific, MA, USA) according to the manufacturer's protocol. All protease inhibitors (Table 3) were dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mM.
  • DMSO dimethylsulfoxide
  • NC_045512.2) SARS-CoV-2 variants (B.1.1.7, EPI_ISL_601443; B.1.351 , MZ747297.1; B.1.617.1, EPI_ISL_1704611; B.1.617.2, EPI_ISL_3189054), SARS-CoV (NC_004718.3), WIV1-CoV (KF367457.1), HCoV-NL63 (NC_005831.2), Chimera S, and Flag-tagged 5′-GGA GGC GAT TAC AAG GAT GAC GAT GAC AAG TAA-3′ (underline indicates Flag tag at 3′ end) (SEQ ID NO: 10) are all from Integrated DNA Technologies (IA, USA) made by Synthesis with a Flag tag at the 3′ end corresponding to previously described codon-optimized MERS-CoV S (NC_019843.3) (Yamamoto M et al., 2016, Antimicrob Agents Chemother, 60:6532-65
  • DSP Assays to Monitor Membrane Fusion DSP assays were performed as previously described (Yamamoto M et al., 2020, Viruses, 12:629). Briefly, effector cells expressing S protein and target cells expressing CD26 or ACE2 alone or together with TMPRSS2 were seeded in 10 cm plates and incubated overnight. Cells were treated with 6 ⁇ M EnduRen (Promega), a substrate for Renilla luciferase (RL), for 2 hours. To test the effect of inhibitors, 0.25 ⁇ L of compound library or 1 ⁇ L of selected inhibitors dissolved in DMSO were added to 384-well plates (Greiner Bioscience, Frickenhausen, Germany).
  • VSV proteins pBS-N/pBS-P/pBS-L/pBS-G
  • Promoter-driven expression plasmids and p ⁇ G-Luci a plasmid lacking the G gene and encoding VSV genomic RNA encoding firefly luciferase
  • Supernatants were harvested 48 hours after transfection.
  • 293T cells were then transfected with S or VSV G expression plasmids by using calcium phosphate precipitation. Sixteen hours after transfection, cells were inoculated at a multiplicity of infection (MOI) of 1 with replication-deficient VSV. Two hours after infection, the cells were washed and incubated for an additional 16 hours before harvesting the pseudovirus-containing supernatant. For infection assays, cells were seeded in 96-well plates (2 x 104 cells/well) and incubated overnight. One hour prior to pseudovirus infection, cells were pretreated with inhibitors.
  • MOI multiplicity of infection
  • Luciferase activity was measured 16 hours after infection using the Bright-Glo luciferase assay system or the ONE-Glo luciferase assay system (Promega) and a Centro xS960 luminometer (Berthold).
  • RNA Cells were seeded in 96-well plates (5 x 104 cells/well) and incubated overnight. Cells were treated with inhibitors for 1 hour and SARS-CoV-2 was added at MOI. Cell lysis and cDNA synthesis were performed 24 h after infection using the SuperPrep II Cell Lysis and RT Kit for Quantitative PCR (qPCR) (SCQ-401; Toyobo, Osaka, Japan) according to the protocol.
  • qPCR Quantitative PCR
  • RT-PCR Quantitative real-time reverse transcription (RT)-PCR of SARS-CoV-2 N and ribosomal protein L13a (Rpl13a) was performed at 95 °C using the CFX Connect Real-Time PCR Detection System (Bio-Rad, CA, USA). 3 minutes followed by 50 cycles of 95°C for 10 seconds and 60°C for 1 minute. Data were normalized using the Rpl13a mRNA expression level of each sample.
  • Cytopathic Assay Cells were seeded in 24-well plates (1.5 ⁇ 10 5 cells/well) and incubated overnight. Cells were treated with inhibitors for 1 hour and then with MOI 1 of SARS-CoV-2. To maintain drug concentration, half of the culture supernatant was replaced daily with fresh medium containing drug. Three days after infection, cells were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. After washing with water four times, the wells were air-dried and crystal violet was dissolved in ethanol. Absorbance was measured at 595 nm using an iMark microplate reader (Bio-Rad).
  • Example 1 TMPRSS2-independent membrane fusion is induced by SARS-CoV-2 S protein.
  • a quantitative cell fusion assay between target cells co-expressing TMPRSS2 with a receptor such as SARS-CoV-2) or CD26 (for MERS-CoV) was used.
  • cell fusion kinetics induced by SARS-CoV, SARS-CoV-2, and MERS-CoV S proteins were determined using DSP assays.
  • Target cells expressing ACE2 alone or together with TMPRSS2 were used for co-culture with effector cells expressing SARS-CoV S and SARS-CoV-2 S, and cells expressing CD26 alone or together with TMPRSS2 were used for co-culture with MERS- Used for co-culture with effector cells expressing CoV S.
  • Relative cell fusion values were calculated by normalizing the RL activity of each co-culture to that of co-cultures with cells expressing both the receptor and TMPRSS2 at 240 min set at 100% (Fig. 1a).
  • target cells expressing ACE2 together with TMPRSS2 were also used for co-culture with effector cells expressing SARS-CoV S and SARS-CoV-2 S, and cells expressing CD26 together with TMPRSS2 were used for co-culture with MERS -used for co-culture with effector cells expressing CoV S.
  • Relative cell fusion values were calculated by normalizing the RL activity of each co-culture to that of co-cultures with cells expressing both the receptor and TMPRSS2 in the presence of DMSO set at 100% ( Fig. 1c).
  • target cells expressing ACE2 alone or together with TMPRSS2 were also used to co-culture with effector cells expressing SARS-CoV-2 S.
  • Relative cell fusion values were calculated by normalizing the RL activity of each co-culture to that of co-cultures with cells expressing both ACE2 and TMPRSS2 in the presence of DMSO set at 100% (Fig. 1d and Fig. 1e).
  • pepstatin A inhibitor of various aspartic protease
  • leupeptin leupeptin, inhibitor of various cysteine, serine, threonine protease
  • bestatin inhibitor of various amino acids peptidase inhibitor
  • Example 2 TMPRSS2-Independent Membrane Fusion Induced by SARS-CoV-2 S Protein is Suppressed by Various Metalloprotease Inhibitors
  • TMPRSS2-Independent Membrane Fusion Induced by SARS-CoV-2 S Protein is Suppressed by Various Metalloprotease Inhibitors
  • a validated compound library (1,630 compounds approved in clinical trials and 1,885 compounds with pharmacological activity) obtained from the University of Tokyo Drug Discovery Organization. ) were used to search for compounds that specifically inhibit TMPRSS2-independent membrane fusion but do not inhibit TMPRSS2-dependent membrane fusion.
  • relative cell fusion values were calculated by normalizing the RL activity of each compound to the RL activity of the control assay (DMSO alone; set at 100%). Each dot represents an individual compound. Dots within dotted boxes represent compounds that preferentially inhibit TMPRSS2-independent membrane fusion ( ⁇ 30% inhibition of relative cell fusion values using target cells expressing both TMPRSS2 and ACE2, and ACE2 only). >40% inhibition of relative cell fusion values using target cells expressing (Fig. 2a).
  • relative cell fusion values were obtained by normalizing the RL activity of each co-culture to that of co-cultures with cells expressing both ACE2 and TMPRSS2 in the presence of DMSO set at 100%. (Fig. 2b).
  • Example 3 The metalloprotease-dependent viral entry pathway is specific to SARS-CoV-2 and its presence depends on the cell type. Based on the findings from protein-mediated cell fusion experiments, we investigated whether a metalloprotease-dependent viral entry pathway to the cell surface exists in the SARS-CoV-2 S protein-enveloped vesicular stomatitis virus (VSV ) was confirmed using a pseudovirus (SARS-CoV-2 pseudovirus).
  • VSV vesicular stomatitis virus
  • VSV vesicular stomatitis virus
  • E-64d inhibited most of the entry routes in OVISE cells (Fig. 3c), suggesting that the endosomal route is almost exclusively in OVISE cells.
  • IGROV1 cells human ovary
  • OUMS-23 cells human colon
  • E-64d suppresses virus entry by about 80%, about 20% remains. This residual amount can be suppressed by combining E-64d with marimastat in IGROV1 cells and nafamostat in OUMS-23 cells (Fig. 3c).
  • IGROV1 cells the endosomal and metalloprotease-dependent invasion pathways coexist
  • OUMS-23 cells the endosomal and TMPRSS2-dependent pathways coexist.
  • HEC50B-TMPRSS2 cells HEC50B-TMPRSS2 cells
  • HEC50B-TMPRSS2 cells approximately 80% of the viral entry pathways were TMPRSS2-dependent, with most of the rest being marimastat-sensitive (Fig. 3e). This indicates the coexistence of metalloprotease-dependent and TMPRSS2-dependent invasion pathways. This result suggested the possibility that cells with both cell surface invasion pathways exist in vivo.
  • Example 4 Mutant strains of SARS-CoV-2 also enter cells via metalloprotease-dependent pathways like conventional strains. It is necessary to confirm whether the metalloprotease-dependent pathway is also utilized in mutant strains that continue to spread infection as the disease progresses. Therefore, we compared the effect of marimastat on the infection of pseudoviruses with the S protein of the conventional strain and the mutant strain.
  • ADAM-10 is involved in the SARS-CoV-2 metalloprotease-dependent entry pathway. Inhibition by metalloprotease inhibitors was analyzed.
  • VeroE6 a
  • HEC50B b
  • A704 c
  • G7570 CellTiter-Glo Luminescent Cell Viability Assay
  • Fig. 5 broad spectrum metalloprotease inhibitor.
  • VeroE6 cells Fig. 5a
  • HEC50B cells Fig. 5b
  • A704 cells Fig. 5c
  • SARS-CoV- 2 pseudovirus entry Fig. 5: broad spectrum metalloprotease inhibitor.
  • Fig. 5 selective metalloprotease inhibitor
  • VeroE6 and HEC50B cells have ⁇ 20-30% E-64d sensitive endosomal pathways (Fig. 3b)
  • Fig. 3b we selected in the presence of E-64d to readily recognize the reduction in metalloprotease-dependent pathways.
  • Fig. 5a, b we analyzed the effects of inhibitory agents (Fig. 5a, b).
  • A704 cells were mostly metalloprotease-dependent pathways (Fig. 3a, b), so selective inhibitors were used alone (Fig. 5c).
  • GW280264X (ADAM10/17 inhibitor) and GI1254023X (MMP9/ADAM10 inhibitor) significantly inhibited metalloprotease-dependent pathways
  • TAPI2 ADAM17 inhibitor
  • MMP2/9i MMP2/9 inhibitor
  • Fig. 5 A similar pattern of inhibition was observed in all three cell lines tested ( Figure 5), and cell viability was not affected by any of the metalloprotease inhibitors at the concentrations used in the experiments ( Figure 5). 6), metalloproteases involved in this pathway are common to the three cell lines, and the inhibitory effect of selective inhibitors strongly suggests the involvement of ADAM10.
  • ADAM10 expression was suppressed in HEC50B cells using 3 types of siRNA with different sequences.
  • HEC50B cells were transfected with two different control siRNAs or three different siRNAs against ADAM10 for 48 hours (Fig. 7a). HEC50B cells were transfected with siRNA for 48 hours and infected with pseudovirus. Relative pseudovirus entry was calculated by normalizing the FL activity of each condition to the FL activity of pseudovirus-infected cells in the absence of siRNA (mock), which was set at 100% (Fig. 7b).
  • SARS-CoV-2 virus metalloprotease-dependent entry pathway including ADAM10 could be a therapeutic target for COVID-19
  • pseudovirus it is necessary that the actual pathogenic SARS-CoV-2 virus infection is suppressed with metalloprotease inhibitors.
  • Marimastat and prinomastat have a blood concentration of approximately 600-900 nM in safe administration set in clinical trials, and in this experiment, they show an infection-inhibitory effect between 300 nM and 1000 nM. Therefore, these drugs have great potential to be used for the treatment of COVID-19. Furthermore, GW280264X (ADAM10/17 inhibitor) and GI1254023X (MMP9/ADAM10 inhibitor), but not TAPI2 (ADAM17 inhibitor), inhibited pathogenic SARS-CoV-2 virus infection in HEC50B cells ( Figure 8b). ).
  • ADAM10-targeted inhibitors suppress infection with the pathogenic SARS-CoV-2 virus and may be therapeutic agents for COVID-19.
  • the inhibitors that are actually administered for treatment include nucleic acid drugs similar to siRNA that suppress ADAM10 expression, as used in this study, ADAM10 enzymatic activity, and complex formation with proteins that functionally bind to ADAM10. A small molecule or antibody that inhibits is envisioned.
  • marimastat and E-64d Fig. 8d
  • marimastat and nafamostat Fig.
  • drugs that target the metalloprotease-dependent entry pathway and other drugs that can alleviate the symptoms of COVID-19 such as drugs that target the endosomal pathway and the TMPRSS2 pathway This is expected to lead to the development of more effective treatments.

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Abstract

L'objectif de la présente invention est de fournir des agents thérapeutiques et préventifs de la COVID-19. La présente invention fournit une composition thérapeutique ou préventive de la COVID-19, ladite composition contenant un Inhibiteur d'ADAM10. L'inhibiteur d'ADAM10 est, par exemple, une petite molécule, un acide nucléaire, un ARNsi ou analogue. La composition de la présente invention peut être utilisée en combinaison avec un médicament autre que l'inhibiteur d'ADAM10.
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WO2021076712A1 (fr) * 2019-10-15 2021-04-22 Ophirex, Inc. Gestion précoce, atténuation et prévention de la septicémie et de syndromes de type septicémie, y compris des syndromes de détresse respiratoire aiguë du nouveau-né par infection, lésion ou iatrogénèse

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