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WO2011135734A1 - Complexe de virus, agent thérapeutique le comprenant, et procédé thérapeutique - Google Patents

Complexe de virus, agent thérapeutique le comprenant, et procédé thérapeutique Download PDF

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Publication number
WO2011135734A1
WO2011135734A1 PCT/JP2010/058236 JP2010058236W WO2011135734A1 WO 2011135734 A1 WO2011135734 A1 WO 2011135734A1 JP 2010058236 W JP2010058236 W JP 2010058236W WO 2011135734 A1 WO2011135734 A1 WO 2011135734A1
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Prior art keywords
virus
group
polymer
cationic
complex
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Japanese (ja)
Inventor
義之 小山
智恵子 芳原
雄行 濱田
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ALPHA-NANO-MEDICA KK
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ALPHA-NANO-MEDICA KK
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10332—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the present invention relates to a virus complex, a therapeutic agent such as cancer containing the virus, and a treatment method.
  • Patent Documents 1 to 3 As gene therapy for diseases such as cancer, gene therapy using viruses that incorporate various therapeutic genes and viruses that selectively grow in tumor cells has been tried, but its effects are limited. Have been reported (Patent Documents 1 to 3). However, even if the virus is administered in vivo as it is, the expression of the introduced therapeutic gene and the antitumor activity of the virus are greatly suppressed due to neutralization by the antibody. In particular, for adenoviruses often used for gene therapy, patients often have antibodies from the beginning, and the effect is greatly suppressed. Even in patients who do not have antibodies, antibodies are produced during multiple administrations, and sufficient effects cannot be obtained. In order to avoid neutralization by antibodies, gene therapy using carrier cells into which viruses have been introduced has been widely studied.
  • the effective route of administration is due to the poor diffusibility of the carrier cells in vivo and tissues and the high toxicity of the naked virus itself from the carrier cells.
  • treatment targeting a wide range of sites by intraperitoneal, arterial, or intravenous administration is required. is there.
  • side effects may occur due to its toxicity.
  • administration into the bloodstream does not provide sufficient delivery to target tissues / cells due to the hepatic accumulation of the virus.
  • the effective administration site is limited to the local area of the tumor, and there is no report showing a high healing effect by intraperitoneal or intravenous administration.
  • the virus used for gene therapy is not effective as it is due to neutralization by the antibody as it is, and has a strong side effect. Therefore, the administration route is limited, and it is extremely small by systemic administration or intraperitoneal administration. Small tumors, tumors that cannot be locally administered, or metastatic lesions cannot be widely targeted.
  • a method of binding a polymer Non-patent Document 1
  • mixing a cationic lipid with a virus to prevent blocking of an antibody Non-patent Document 2
  • the present inventors have found that when a virus is complexed with a cationic polymer or a cationic lipid or an aggregate and an anionic polymer, systemic administration or A therapeutic agent for diseases such as cancer is obtained that can be administered in a wide range such as intraperitoneal administration, exhibits high therapeutic effects, has few side effects, and is effective in suppressing cancer metastasis when used in cancer. As a result, the present invention has been completed.
  • the present invention relates to a complex comprising a virus; a cationic polymer or a cationic lipid or an assembly containing the same; and an anionic polymer.
  • the present invention also provides a method for producing the above-described complex, which comprises the step of forming a complex by sequentially mixing a therapeutic virus; a cationic polymer or a cationic lipid or an assembly containing the same; and an anionic polymer. Relates to a method comprising:
  • the complex of the present invention is not easily neutralized by antiviral antibodies and has a high therapeutic effect in vivo.
  • various administrations such as local administration, intraperitoneal administration, intraarterial administration, intravenous administration and intracranial administration are possible.
  • preparation and handling are simple and storage is excellent.
  • the efficiency and selectivity of uptake into a target cell can be improved by introducing a ligand for a target cell into the complex.
  • the zeta potential of the complex obtained when PEI (polyethyleneimine) is added to adenovirus is shown.
  • the time-dependent change of zeta potential of the complex obtained when 0.2 microgram of PEI is added to adenovirus is shown.
  • the zeta potential of the complex obtained when HA (hyaluronic acid) is added to the adenovirus / PEI complex is shown.
  • the zeta potential of the complex obtained when PEI is added to the adenovirus / PEI / HA complex is shown.
  • the zeta potential of the complex obtained when HA is added to the adenovirus / PEI / HA / PEI complex is shown.
  • the zeta potential of the complex obtained when PEI is added to the adenovirus / PEI / HA / PEI / HA complex is shown.
  • the zeta potential of the complex obtained when HA is added to the adenovirus / PEI / HA / PEI / HA / PEI complex is shown.
  • the zeta potential of the complex obtained by alternately adding PEI and CS to adenovirus is shown.
  • the infectivity to the cell in the presence of the antibody of the polymer-coated virus complex is shown.
  • the cytotoxicity of the polymer-coated virus complex in the presence of antibody is shown.
  • the zeta potential of the complex obtained when PRT (protamine) is added to adenovirus is shown.
  • the zeta potential of the complex obtained when HA is added to the adenovirus / PRT complex is shown.
  • the zeta potential of the complex obtained when superfect is added to adenovirus is shown.
  • the zeta potential of the complex obtained when HA is added to the adenovirus / superfect complex is shown.
  • the infectivity to a cell in the presence of an antibody of a virus complex coated with a polyanion having a ligand is shown.
  • the complex of the present invention includes a virus; a layer containing one or more cationic polymers or cationic lipids or an assembly containing the same (cationic layer); and a layer containing one or more anionic polymers (anionic layer) ) Containing two or more coating layers.
  • the virus is coated with a cation layer and further coated with an anion layer (two-layer complex).
  • the virus is coated with a cation layer, further coated with an anion layer, and further coated with a cation layer (three-layer complex).
  • a complex including two or more coating layers in which a virus is coated with a cation layer and an anion layer and a cation layer are alternately coated can be preferably exemplified.
  • a complex for example, a two-layer complex in which a virus is coated in the order of a cation layer and an anion layer, a three-layer complex in which this two-layer complex is further coated with a cation layer, and this three-layer complex
  • Examples include a layer composite and a seven-layer composite in which the six-layer composite is further coated with a cationic layer.
  • a complex including two or more coating layers is preferable from the viewpoint of increasing the infection rate to cultured cells, and a complex including three or more coating layers is preferable from the viewpoint of increasing the infection rate to cells in vivo.
  • the upper limit of the number of layers is not particularly limited, but is preferably 19 layers or less, more preferably 14 layers or less, and particularly preferably 11 layers or less from the viewpoint of increasing the production efficiency of the composite.
  • one or two or more cationic polymers or cationic lipids or aggregates or anionic polymers containing them are contained. May be coated simultaneously or sequentially.
  • the virus can be coated with RGD-polyethyleneimine into which the RGD peptide has been introduced, then coated with polyethyleneimine, and then with hyaluronic acid.
  • the types of the cationic polymer or cationic lipid contained in each layer or the aggregate containing the cationic polymer and the anionic polymer are not necessarily the same, and different types. These polymers, cationic lipids, aggregates containing them, or combinations thereof can be modified for each layer.
  • the above-mentioned layer may cover all or part of the surface of the virus or multilayer complex, but it is preferable to cover the entire surface from the viewpoint of increasing the infection rate to cells.
  • the virus forms a complex by ionic bonding with a cationic polymer or a cationic lipid or an aggregate containing the cationic polymer, or a cationic lipid or an aggregate containing the cationic lipid or an anionic polymer.
  • a cationic polymer or a cationic polymer having a specific adhesion ability to a target cell for example, RGD or the like
  • the virus forms a complex by ionic bonding with a cationic polymer or a cationic lipid or an aggregate containing the cationic polymer, or a cationic lipid or an aggregate containing the cationic lipid or an anionic polymer.
  • a cationic polymer or a cationic polymer having a specific adhesion ability to a target cell for example, RGD or the like
  • the virus forms a complex by ionic bonding with a cationic polymer or a cationic lipid or an aggregate containing the cationic polymer, or
  • the surface is a cationic complex
  • the surface is an anionic complex
  • the surface charge is almost neutral. Any of these complexes can be used.
  • the viruses are adenovirus, herpes simplex virus, adeno-associated virus, vaccinia virus, measles virus, Sendai virus, reovirus, foamy virus, Newcastle disease virus, lentivirus, Rabies Virus, Pox virus,
  • DNA viruses such as myxoma virus or RNA viruses introduced with a therapeutic gene, or their restricted-growth oncolytic viruses, etc., which can be appropriately selected according to the animal species and diseases to be treated it can.
  • a virus that can be safely used in humans such as adenovirus or its restricted-growth oncolytic virus, can be appropriately used.
  • Mutant viruses and recombinant viruses introduced with therapeutic genes can also be used.
  • the therapeutic gene that can be introduced into the virus can be appropriately selected according to the disease that is the target of gene therapy. For example, when cancer diseases are targeted for gene therapy, genes such as p53, thymidine kinase, and GM-CSF can be introduced into the virus. When a muscular dystrophy disease is targeted for gene therapy, a gene such as dystrophin can be introduced into the virus.
  • the type of virus, the type of gene to be introduced, and the gene introduction method can be appropriately selected according to the type of virus and the gene to be introduced.
  • the cationic polymer is a naturally-derived or synthetic polymer having a positively charged molecular weight of about 1,000 to 3,000,000.
  • a polymer having a plurality of functional groups, preferably 5 or more, in a molecule that can form a complex with a virus in a solution can be used.
  • Examples of such a functional group include an optionally substituted amino group or ammonium group or a salt thereof (these groups may be mono- or poly-substituted with, for example, an alkyl group having 1 to 6 carbon atoms or a phenyl group). And an organic amino group such as an imino group, an imidazolyl group, and a guanidino group.
  • Examples of such cationic polymers include positively charged proteins and polypeptides; positively charged dendrimers; positively charged synthetic polymers; and positively charged polysaccharide derivatives, and salts thereof. As well as combinations thereof.
  • the molecular weight of the positively charged protein or positively charged polypeptide that can be used as the cationic polymer in the complex of the present invention is preferably about 1,000 to 500,000.
  • proteins and polypeptides include proteins and polypeptides such as protamine, histone, Hel ⁇ 1, and gelatin.
  • Polyamino acids containing positively charged amino acid residues are also exemplified. It can be illustrated.
  • Specific examples of such a polyamino acid containing a positively charged amino acid residue include poly-L-lysine, polyarginine, polyornithine and the like.
  • salts of these proteins and polypeptides include hydrochlorides, sulfates, phosphates, borates and the like.
  • the positively charged dendrimer having a functional group as described above that can be used as a cationic polymer is an amino group or ammonium group which may be substituted at the end or inside of a branched molecular chain or a salt thereof
  • These groups are, for example, dendrimers having an organic amino group such as an alkyl group having 1 to 6 carbon atoms, a phenyl group or the like, which may be mono- or polysubstituted), an imino group, an imidazolyl group, a guanidino group,
  • the molecular weight is preferably about 1,000 to 500,000.
  • Specific examples of dendrimers include polyamidoamine dendrimers and polylysine dendrimers.
  • Examples of the dendrimer salt include hydrochloride, sulfate, phosphate, borate and the like.
  • a positively charged synthetic polymer that can be used as a cationic polymer is a synthetic polymer having a plurality of functional groups, preferably 5 or more, in one molecule, which can form a complex with a virus in solution as described above.
  • Specific examples of synthetic polymers include polyethyleneimine (including linear polyethyleneimine or polybranched polyethyleneimine), 2-dimethylaminoethyl methacrylate polymer or copolymer, and 2-trimethylaminoethyl methacrylate polymer.
  • Examples thereof include a polymer or a copolymer and a salt thereof.
  • the molecular weight of polyethyleneimine which is an example of a synthetic polymer, is preferably about 1,000 to 500,000, more preferably about 5,000 to 200,000, and most preferably about 10,000 to 100,000.
  • Examples of the polyethyleneimine salt include hydrochloride, sulfate, phosphate, borate and the like.
  • the positively charged polysaccharide derivative that can be used as a cationic polymer has a plurality of functional groups, preferably 5 or more, in a molecule that can form a complex with a virus in solution, and has a molecular weight of preferably 1000.
  • polysaccharide derivative having a viscosity of ⁇ 3 million, more preferably 5,000 to 500,000.
  • polysaccharides include chitosan, dextran derivatives introduced with the above functional groups, and salts thereof.
  • the molecular weight of chitosan is preferably about 1,000 to 500,000, more preferably about 5,000 to 200,000, and most preferably about 10,000 to 100,000.
  • the salt of chitosan include hydrochloride and acetate.
  • the molecular weight of the dextran derivative is preferably 3,000 to 1,000,000.
  • specific examples of such dextran derivatives include diethylaminoethyl-dextran.
  • the cationic polymer may be a polymer that is positively charged by introducing a functional group such as an amino group into a conventionally non-positively charged polymer. Moreover, even if it is not normally positively charged, it can be used as long as it is positively charged at the time of complex formation, and if necessary, it can be further modified with a sugar chain, oligopeptide, antibody or the like. It may be.
  • Cationic lipids that can be used in the complex of the present invention include DC-Chol (3 ⁇ - (N- (N ′, N′-dimethylaminoethane) carbamoyl) cholesterol), DDAB (N, N-distearyl-N, N-dimethylammonium bromide), DMRI (N- (1,2-dimyristyloxyprop-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide), DODAC (N, N-dioleyl-N, N-dimethylammonium chloride), DOGS (diheptadecylamidoglycylspermidine), DOSPA (N- (1- (2,3-dioleyloxy) propyl) -N- ( 2- (sperminecarboxamido) ethyl) -N, N-dimethylammonium trifluoro Cetate), DOTAP (N- (1- (1-
  • aggregates containing cationic lipids include lipofectamine (3: 1 w / w mixture liposome of DOSPA and DOPE), lipofectin (1: 1 w / w mixture liposome of DOTMA and DOPE), or a mixture thereof. Can be preferably mentioned.
  • a polymer or copolymer of aminoethyl methacrylate can be preferably used, and polyethyleneimine, polyamidoamine dendrimer, polylysine dendrimer, chitosan, and protamine, particularly polyethyleneimine, polyamidoamine dendrimer, and protamine are particularly preferably used. it can.
  • lipofectamine (the above-mentioned 3: 1 w / w mixture liposome of DOSPA and DOPE) can be preferably used.
  • the anionic polymer used in the complex of the present invention is a negatively charged, naturally-occurring or synthetic polymer having a molecular weight of about 5 to 4 million containing an anionic group in the molecule, A polymer having a plurality of functional groups, preferably 5 or more, in one molecule can form a complex with a cation can be used. Examples of such a functional group include a carboxyl group and -OSO.
  • H group, -SO 3 Mention may be made of H groups, phosphate groups, and salts thereof.
  • anionic polymers include amphoteric polymers.
  • the anionic polymer is more specifically a carboxyl group, -OSO.
  • glucosaminoglycan can be preferably used as the polysaccharide having a functional group as described above or a derivative thereof which can be used as an anionic polymer in the complex of the present invention.
  • the molecular weight of such a glucosaminoglycan is preferably 1,000 to 4,000,000, more preferably 4,000 to 3,000,000.
  • Specific examples of such glucosaminoglycans include hyaluronic acid, chondroitin, chondroitin sulfate, keratan sulfate, heparin, dermatan sulfate, and salts thereof. Of these, hyaluronic acid can be preferably used.
  • Hyaluronic acid can also be used as its salt or a negatively charged derivative.
  • the molecular weight may be 5,000 or more, preferably 10,000 or more, and more preferably 100,000 to 3,000,000.
  • Examples of the salt of hyaluronic acid include sodium salt, potassium salt, ammonium salt and the like.
  • Examples of the derivatives of hyaluronic acid include those obtained by introducing polyethylene glycol, peptides, sugars, proteins, iodic acid, antibodies or a part thereof into hyaluronic acid, and spermine, spermidine, etc. are introduced. And zwitterionic derivatives having a positively charged moiety.
  • the polyamino acid containing an amino acid residue having a negatively charged side chain that can be used as an anionic polymer in the complex of the present invention is a carboxyl group, -O-SO. 3 H group, -SO 3
  • Specific examples of such polyamino acids include polyglutamic acid and polyaspartic acid.
  • the salt of H group and phosphate group include sodium salt, potassium salt and ammonium salt.
  • the PEG derivative having a carboxyl side chain that can be used as an anionic polymer in the complex of the present invention has a plurality of carboxyl side chains per PEG molecule, preferably 5 or more, 500 or more, preferably 2,000 or more, A PEG derivative having a molecular weight of 4,000 to 40,000 or a salt thereof is more preferable.
  • a PEG derivative having a carboxyl side chain can also be used as a salt thereof or a negatively charged derivative. Examples of these salts include sodium salts, potassium salts, ammonium salts and the like. Specific examples of such PEG derivatives include those described in J. Org. Biometer. Sci. Polymer Edn. Vol. 14, pp 515-531 (2003).
  • a carboxyl group that can be used as an anionic polymer in the complex of the present invention -OSO 3 H group, -SO 3
  • the synthetic polymer having a functional group selected from H group, phosphate group, and salts thereof includes a plurality of, preferably 5 or more, carboxyl groups, —O—SO per molecule.
  • 3 H group, -SO 3 A polymer or copolymer having a functional group selected from an H group, a phosphate group, and a salt thereof, and preferably a polymer or copolymer having a molecular weight of 5 to 4 million.
  • Such a polymer or copolymer include a polymer or copolymer of acrylic acid or methacrylic acid having a molecular weight of 1,000 to 3,000,000, a sulfate ester of polyvinyl alcohol, or succinimidylated poly-L-lysine. Etc. can be illustrated.
  • carboxyl group, -O-SO 3 H group, -SO 3 Examples of the salt of H group and phosphate group include sodium salt, potassium salt and ammonium salt.
  • a carboxyl group that can be used as an anionic polymer in the complex of the present invention -OSO 3 H group, -SO 3 A functional group selected from an H group, a phosphate group, and a salt thereof, and an optionally substituted amino group or ammonium group or a salt thereof (for example, these groups include an alkyl group having 1 to 6 carbon atoms, phenyl group, And a polymer having a mono- or poly-substituted group such as a carboxyl group, -OSO per molecule.
  • H group, -SO 3 A plurality of, preferably 5 or more, functional groups selected from H groups, phosphate groups, and salts thereof, and optionally substituted amino groups or ammonium groups or salts thereof (these groups have, for example, carbon number PEG derivative having a molecular weight of 500 or more, preferably 2,000 or more, more preferably 4,000 to 40,000, which may be mono- or polysubstituted by 1 to 6 alkyl groups, phenyl groups, etc. And other polymers.
  • the salt of H group and phosphate group include sodium salt, potassium salt and ammonium salt.
  • amino group or ammonium group salt examples include hydrochloride, sulfate and acetate.
  • a polymer is preferably a carboxyl side chain and an amino group or ammonium group having an equivalent amount or less or a salt thereof (these groups are, for example, alkyl groups having 1 to 6 carbon atoms, phenyl groups, etc.).
  • PEG derivatives having (which may be substituted) can be mentioned, specifically, Macromol. Biosci. Vol. 2, pp 251-256 (2002), PEG derivatives that can be prepared, hyaluronic acid in which acetamide groups are partially hydrolyzed, hyaluronic acid in which hydrazine is introduced into some carboxyl groups, etc. Can be illustrated.
  • the anionic polymer that can be used in the composite of the present invention may be one that has been negatively charged by introducing a functional group such as a carboxyl group into a conventionally non-negatively charged one. Even if it is not negatively charged normally, it can be used as long as it is negatively charged at the time of complex formation, and if necessary, it may be further modified with sugar chain, oligopeptide, antibody, etc. Good.
  • a functional group such as a carboxyl group into a conventionally non-negatively charged one. Even if it is not negatively charged normally, it can be used as long as it is negatively charged at the time of complex formation, and if necessary, it may be further modified with sugar chain, oligopeptide, antibody, etc. Good.
  • an anionic polymer such as polyacrylic acid, or a salt thereof can be preferably used.
  • a PEG derivative having a salt thereof or a salt thereof, particularly hyaluronic acid can be particularly preferably used.
  • cationic polymers or cationic lipids or aggregates containing them, or anionic polymers that have specific adhesion to target cells the introduction of therapeutic genes specifically to target cells Can be done.
  • hyaluronic acid is used as the anionic polymer, cells having cell surface molecules such as CD44 that specifically bind to hyaluronic acid can be targeted.
  • an anionic polymer or cationic polymer into which RGD peptide is introduced preferably PEG derivative or polyethyleneimine into which RGD peptide is introduced
  • many types of tumor cells can be targeted, and galactose side chain Hepatocytes or liver-derived cells can be targeted by using a cationic polymer or an anionic polymer into which is introduced.
  • the combination of the cationic polymer or the cationic lipid or the assembly containing the cationic polymer and the anionic polymer includes polyethyleneimine and hyaluronic acid; protamine and hyaluronic acid; cationic dendrimer and hyaluronic acid; A PEG derivative having a carboxyl side chain; an assembly containing DOSPA (eg, lipofectamine (3: 1 w / w mixture liposome of DOSPA and DOPE)) and hyaluronic acid; an assembly containing DOSPA (eg, lipofectamine) and a carboxyl side chain Preferred examples include PEG derivatives possessed.
  • DOSPA eg, lipofectamine (3: 1 w / w mixture liposome of DOSPA and DOPE
  • the mixing ratio between the virus used in the complex of the present invention and the cationic polymer or cationic lipid or an assembly containing the virus is the type of the virus, the type of the cationic polymer or the cationic lipid or the assembly containing the same, or the production process.
  • virus 1 ⁇ 10 8 Preferably, 0.001 to 500 ⁇ g, more preferably 0.01 to 50 ⁇ g, particularly preferably 0.1 to 5 ⁇ g of a cationic polymer or cationic lipid or an aggregate containing the same can be mixed with pfu. .
  • adenovirus when used as the virus and polyethyleneimine is used as the cationic polymer, adenovirus 1 ⁇ 10 8
  • the amount of polyethyleneimine added in each step with respect to pfu is preferably 0.001 to 500 ⁇ g, more preferably 0.01 to 50 ⁇ g, and particularly preferably 0.1 to 5 ⁇ g.
  • the mixing ratio between the virus used in the complex of the present invention and the anionic polymer can be appropriately selected according to the type of virus, the type of anionic polymer, and the step in the production process.
  • 0.005 to 2000 ⁇ g, more preferably 0.05 to 200 ⁇ g, and particularly preferably 0.5 to 20 ⁇ g of an anionic polymer can be mixed with respect to pfu.
  • adenovirus 1 ⁇ 10 8 The amount of hyaluronic acid added in each step with respect to pfu is preferably 0.005 to 2000 ⁇ g, more preferably 0.05 to 200 ⁇ g, and particularly preferably 0.5 to 20 ⁇ g.
  • the preferred mixing ratio of the virus contained in the complex of the present invention; the cationic polymer or the cationic lipid or the aggregate containing the same; and the anionic polymer is as described above. Since the optimum conditions vary depending on the disease and disease, the compounding ratio can be determined appropriately by those skilled in the art.
  • the complex of the present invention may be prepared by the step of forming a complex by sequentially mixing the virus described above; a cationic polymer or a cationic lipid or an assembly containing the same; and an anionic polymer in this order. it can.
  • the step of mixing the cationic polymer or the cationic lipid or the aggregate containing the same; and the step of mixing the anionic polymer can be alternately performed as necessary.
  • the virus can be mixed with a cationic polymer or cationic lipid or an assembly comprising it to obtain a one-layer complex, and then mixed with an anionic polymer to form a complex of the present invention that is a two-layer. obtain. Further, this two-layer composite is mixed with a cationic polymer or a cationic lipid or an assembly containing the same to obtain a three-layer composite, and further mixed with an anionic polymer to form a four-layer composite. Can be obtained.
  • this four-layer composite is mixed with a cationic polymer or a cationic lipid or an assembly containing the same to obtain a five-layer composite, and further mixed with an anionic polymer to form a six-layer composite.
  • a cationic polymer or a cationic lipid or an assembly containing the same to obtain a five-layer composite
  • an anionic polymer to form a six-layer composite.
  • one or two or more cationic polymers or cationic lipids or aggregates or anionic polymers containing them are mixed. When two or more are mixed, they are mixed at the same time. Or may be mixed sequentially.
  • the virus can be mixed with RGD-polyethyleneimine into which the RGD peptide has been introduced, then mixed with polyethyleneimine, and then mixed with hyaluronic acid.
  • the type of the cationic polymer or cationic lipid added or the aggregate containing the cationic polymer and the anionic polymer need not be the same, and different types of polymers and cationic lipids may be used. Or it is also possible to change and add the aggregate
  • a cationic polymer or a cationic lipid or an aggregate containing it; and an anionic polymer can be prepared by adding the above amounts at each step.
  • the virus forms a complex by ionic bonding with the cationic polymer or cationic lipid or an aggregate containing the cationic polymer, or the cationic polymer or cationic lipid or the same.
  • the assemblage and the anionic polymer are also ionically bonded.
  • the surface can be a complex with a cationic surface, an anionic surface with a complex, or a complex with an almost neutral surface charge.
  • the complex of the present invention prepared as described above can be used for various gene therapy, immunotherapy for humans and animals, or creation of experimental animals and cells into which each gene has been introduced.
  • the application amount of the complex of the present invention varies depending on the administration route, the administration site, the type of disease, and the type of virus to be introduced.
  • the amount of virus contained in intraperitoneal administration is, for example, 10 8 ⁇ 10 12 pfu / individual.
  • herpes simplex virus for example, 1 ⁇ 10 4 ⁇ 5 ⁇ 10 5
  • the pfu / individual can be administered into a subcutaneous tumor or a breast cancer tumor mass.
  • administration routes include intratumoral, intraperitoneal, hepatic artery, intravenous, intracranial and the like.
  • an adenovirus or oncolytic herpes simplex virus introduced with the p53 gene it can be administered intratumorally, intraperitoneally, intrahepatic artery, intravenously, or intracranially.
  • the complex of the present invention is used for preparation of a gene-transferred cell, a complex of 1 to 8000 pfu in terms of viral load can be applied per cell.
  • the virus used in the present invention can be freely selected from adenovirus, herpes simplex virus, adeno-associated virus, and the like depending on the animal species and diseases to be used.
  • additives usually used for injections may be appropriately added and used.
  • the present invention will be described more specifically with reference to the following examples. In addition, these examples are for demonstrating this invention, Comprising: This invention is not limited at all.
  • PEI Polyethyleneimine
  • MA Hydrochloride
  • Mw 40000
  • RGD-added PEI polyPE-translation jetPEI TM -RGD was used.
  • HA hyaluronic acid
  • CS chondroitin sulfate
  • shark-derived chondroitin sulfate molecular weight of about 10,000
  • Protamine (hereinafter referred to as PRT) was Wako Pure Chemical Protamine Sulfate (made by salmon).
  • Superfect which is a cationic dendrimer (polyamideamine dendrimer)
  • the wild type human adenovirus (type V) used was obtained from Microbix. Based on this, human adenovirus (AD-GFP) and oncolytic virus (ADE3-IAI.3B) incorporating the GFP gene were prepared by the same method as Patent Document 1.
  • a human adenovirus (AD-GM-CSF) into which a GM-CSF gene was incorporated was prepared by inserting a mouse GM-CSF gene in place of the GFP gene in the same manner as AD-GFP.
  • zeta potential (data is shown in mV unless otherwise stated) is obtained by using MALVERN Zetasizer Nano ZS. Measurements were made by These are the same in the following examples unless otherwise specified. (Example 1) The zeta potential of the resulting complex when PEI was added to adenovirus was measured.
  • Example 4 The zeta potential of the resulting complex when PEI was added to the adenovirus / PEI / HA complex was measured.
  • Human adenovirus (AD-GFP) incorporating the GFP gene was suspended in a 5% aqueous glucose solution at a concentration of 1.5 ⁇ 10 8 pfu / ml. Take 800 microliters of this solution, add 0.2 micrograms of PEI, stir well, let stand for 30 minutes, add 2 micrograms of HA, leave it for 30 minutes, add a predetermined amount of PEI, and The zeta potential was measured. [result] The results are shown in FIG.
  • the zeta potential of the adenovirus / PEI / HA complex was increased again by adding PEI, resulting in a complex with a positive surface charge. (Example 5)
  • the zeta potential of the resulting complex was measured when HA was added to the adenovirus / PEI / HA / PEI complex.
  • Human adenovirus (AD-GFP) incorporating the GFP gene was suspended in a 5% aqueous glucose solution at a concentration of 1.5 ⁇ 10 8 pfu / ml.
  • the zeta potential of the adenovirus / PEI / HA / PEI / HA complex increased again by adding PEI, resulting in a complex with a positive surface charge.
  • the zeta potential of the resulting complex was measured when HA was added to the adenovirus / PEI / HA / PEI / HA / PEI complex.
  • Human adenovirus (AD-GFP) incorporating the GFP gene was suspended in a 5% aqueous glucose solution at a concentration of 1.5 ⁇ 10 8 pfu / ml.
  • Example 8 Similarly, the zeta potential of the complex obtained by alternately adding PEI and CS to adenovirus was measured using CS instead of HA.
  • Human adenovirus (AD-GFP) incorporating the GFP gene was suspended in 8 mM phosphate buffer (pH 7.4) at a concentration of 1 ⁇ 10 11 pfu / ml. 200 microliters of this liquid was taken and 100 micrograms of PEI and 200 micrograms of CS were added alternately and stirred well. A small amount was sampled at each step, diluted 40-fold with water, and the zeta potential of the complex was measured. [result] The results are shown in FIG.
  • the zeta potential of adenovirus changes alternately with positive and negative values by alternately adding PEI and CS, and when PEI is added last, a complex with a positive surface charge is formed. In addition, when CS was added last, a composite having a negative surface charge was obtained. (Example 9) The ability of the polymer-coated virus complex to infect cells in the presence of antibody was examined. [Operating procedure] [1] One day before adding the virus and its complex, A549 cells (obtained from JCRB Cell Bank) were seeded in a 24-well multiplate (50,000 cells / well) and incubated overnight (temperature: 37 ° C .; humidity 99%) Medium: RPMI medium containing 10% FCS).
  • a complex with a polymer was prepared in the same manner as in Examples 3 to 7, using human adenovirus (AD-GFP) incorporating a GFP gene.
  • AD-GFP human adenovirus
  • 200 microliters of human immunoglobulin drip solution obtained from Nippon Pharmaceutical Co., Ltd .; titer: ⁇ 6000 was added to half of the wells in advance.
  • the complex prepared in [2] was added to the well (addition amount: 2 ⁇ 10 8 pfu / well).
  • the cells were incubated for 8 days at 37 ° C. and 5% CO 2 -95% air.
  • Cells were observed with a fluorescence microscope on a daily basis, and the number of infected cells was counted.
  • the complex consisting of oncolytic virus, PEI and CS showed a much higher cell killing ability in the presence of the antibody than the original virus alone.
  • Example 11 The curative effect on OVHM cell transplanted mice by intraperitoneal injection of a complex consisting of oncolytic virus, PEI and HA was examined. [Operating procedure] [1] Oncolytic virus (ADE3-IAI.3B) was suspended in 5% aqueous glucose solution at a concentration of 5 ⁇ 10 9 pfu / ml.
  • mice administered intraperitoneally with the oncolytic virus complex survived after 70 days or more without accumulation of ascites (number of cases: 3 each).
  • Example 12 The curative effect on mice transplanted with OVHM cells by intraperitoneal injection of a complex consisting of a virus incorporating the GM-CSF gene, PEI and HA was examined.
  • [Operating procedure] [1] Human adenovirus (AD-GM-CSF) incorporating the GM-CSF gene was suspended in 5% aqueous glucose solution at a concentration of 5 ⁇ 10 9 pfu / ml. Take 5 ml of this solution, add 41.7 micrograms of PEI, leave for 30 minutes, add 417 micrograms of HA, leave for 30 minutes, add 417 micrograms of PEI, and add virus / PEI / HA / PEI. A complex was prepared.
  • AD-GM-CSF Human adenovirus
  • OVHM cells ("OV2944-HM-1" described in Jpn. J. Cancer Res. 80, 459-463 (1989)) prepared by the same method as in [1] of Example 9 10 6 percentage per animal, female (C57BL / 6 ⁇ C3 / He ) F1 mice: were administered intraperitoneally (obtained from CLEA Japan) (example: 9).
  • the virus complex having the GM-CSF gene prepared in [1] is intraperitoneally injected into the mouse, and the amount of virus is converted to 2 per mouse. 5 ⁇ 10 10 pfu were administered and the number of days of survival was examined. In the control group, the complex was not administered (number of cases: 3). [result] The control group to which nothing was administered died within 29 days after tumor transplantation (number of cases: 3). On the other hand, mice administered with the virus complex having the GM-CSF gene intraperitoneally survived after 70 days or more without accumulation of ascites (number of cases: 3 each).
  • Example 13 The zeta potential of the resulting complex when PRT was added to adenovirus was measured.
  • Human adenovirus (AD-GFP) incorporating the same GFP gene as used in Example 1 was suspended in a 5% aqueous glucose solution at a concentration of 1.5 ⁇ 10 8 pfu / ml. 800 microliters of this liquid was taken, a predetermined amount of PRT was added and stirred well, and the zeta potential immediately after that was measured.
  • the results are shown in FIG. The zeta potential of the virus gradually increased with the addition of PRT, resulting in a complex with a positive surface charge.
  • Example 14 The zeta potential of the resulting complex when HA was added to the adenovirus / PRT complex was measured.
  • Human adenovirus (AD-GFP) incorporating the same GFP gene as used in Example 1 was suspended in a 5% aqueous glucose solution at a concentration of 1.5 ⁇ 10 8 pfu / ml. 800 microliters of this liquid was added, 1.5 micrograms of PRT was added and stirred well, allowed to stand for 30 minutes, a predetermined amount of HA was added, and the zeta potential immediately after that was measured. [result] The results are shown in FIG.
  • the zeta potential of the virus / PRT complex decreased again by adding HA, resulting in a complex with a negative surface charge.
  • the zeta potential of the resulting complex was measured when the superfect of cationic dendrimer was added to adenovirus.
  • Human adenovirus (AD-GFP) incorporating the GFP gene was suspended in a 5% aqueous glucose solution at a concentration of 1.5 ⁇ 10 8 pfu / ml. 800 microliters of this liquid was taken, a predetermined amount of Superfect was added, and the mixture was well stirred, and the zeta potential immediately after that was measured. [result] The results are shown in FIG.
  • the zeta potential of the virus gradually increased with the addition of Superfect, resulting in a complex with a positive surface charge.
  • the zeta potential of the resulting complex was measured when HA was added to the adenovirus / superfect complex.
  • Human adenovirus (AD-GFP) incorporating the same GFP gene as used in Example 1 was suspended in a 5% aqueous glucose solution at a concentration of 1.5 ⁇ 10 8 pfu / ml. 800 microliters of this solution was added, 0.5 microgram of Superfect was added and stirred well, allowed to stand for 30 minutes, a predetermined amount of HA was added, and the zeta potential immediately after that was measured.
  • a complex with PEI was prepared in the same manner as in Example 1 using human adenovirus (AD-GFP) incorporating the GFP gene. Further, a complex with PRT was prepared in the same manner as in Example 11.
  • RGD-PEG-Suc (synthesized by the method described in Biomedicine & Pharmacotherapy 62 (2008) 448), which is an anionic PEG derivative having an RGD side chain, is added to the obtained complex (addition amount: 10). Microgram), a virus complex carrying RGD was prepared.
  • 200 microliters of human immunoglobulin drip solution obtained from Nippon Pharmaceutical Co., Ltd .; titer: x6000 was added to half of the wells in advance.
  • the complex composed of adenovirus, PRT, and PEG derivative having RGD side chain also showed significantly higher infectivity in the presence of the antibody compared to the original virus alone.
  • Example 18 The infectivity of cells in the presence of antibodies of the virus complex polymer-coated using PEI with RGD side chain was examined. [Operating procedure] [1] One day before adding the virus and its complex, A549 cells (obtained from JCRB Cell Bank) were seeded in a 24-well multiplate (50,000 cells / well) and incubated overnight (temperature: 37 ° C .; humidity 99%) Medium: RPMI medium containing 10% FCS).
  • a complex with a polymer was prepared in the same manner as in Examples 6 and 7, using human adenovirus (AD-GFP) incorporating a GFP gene. However, instead of the last PEI added, 3.6 microliters of jetPEI TM -RGD was added, and a virus complex having an RGD ligand, ie, virus / PEI / HA / PEI / HA / RGD-PEI complex (5-layer complex) Body) and virus / PEI / HA / PEI / HA / RGD-PEI / HA complex (6-layer complex).
  • the complex consisting of adenovirus, PEI, HA and RGD-PEI shows much higher infectivity than the original virus alone in the presence of antibody, with 40% for the 5 layer complex and 6 layer complex. In the body, 30% of the cells were infected.

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Abstract

La présente invention concerne un agent thérapeutique pour des maladies comprenant le cancer, qui peut être administré par différentes voies, telles qu'une voie systémique et une voie intrapéritonéale, qui présente un effet thérapeutique marqué, qui a peu d'effets secondaires, et qui est en outre efficace pour l'inhibition de la métastase du cancer lorsqu'il est utilisé pour le cancer. La présente invention concerne en outre : un complexe comprenant un virus, au moins une couche contenant au moins un polymère cationique, un lipide cationique ou un agrégat de ceux-ci et une couche contenant au moins un polymère anionique ; un procédé de production du complexe, qui implique une étape de mélange d'un virus, d'un polymère cationique, d'un lipide cationique ou d'un agrégat de ceux-ci, et d'un polymère anionique séquentiellement dans cet ordre pour former le complexe. La présente invention concerne également un procédé thérapeutique utilisant le complexe.
PCT/JP2010/058236 2010-04-28 2010-04-28 Complexe de virus, agent thérapeutique le comprenant, et procédé thérapeutique Ceased WO2011135734A1 (fr)

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CN110923209A (zh) * 2019-10-11 2020-03-27 中国农业大学 一种细胞培养物中添加脂质体提高有囊膜病毒滴度的方法
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US20230285296A1 (en) * 2020-07-24 2023-09-14 Nanyang Technological University Layer-by-layer coated nanoliposome for oral delivery of insulin

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013039098A (ja) * 2011-08-19 2013-02-28 National Institute For Materials Science 被覆ｈｖｊ−ｅ及び被覆ｈｖｊ−ｅの製造方法
JP2018517778A (ja) * 2015-06-19 2018-07-05 シルラジェン，インコーポレイテッド ウイルス塞栓術のための組成物および方法
US11752220B2 (en) 2018-08-03 2023-09-12 The United States Government as represented by the Department of Veterans Affair Method of delivering genes and drugs to a posterior segment of an eye
CN110923209A (zh) * 2019-10-11 2020-03-27 中国农业大学 一种细胞培养物中添加脂质体提高有囊膜病毒滴度的方法
US20230285296A1 (en) * 2020-07-24 2023-09-14 Nanyang Technological University Layer-by-layer coated nanoliposome for oral delivery of insulin

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